WO2024024929A1 - Method for producing three-dimensional muscle tissue - Google Patents
Method for producing three-dimensional muscle tissue Download PDFInfo
- Publication number
- WO2024024929A1 WO2024024929A1 PCT/JP2023/027702 JP2023027702W WO2024024929A1 WO 2024024929 A1 WO2024024929 A1 WO 2024024929A1 JP 2023027702 W JP2023027702 W JP 2023027702W WO 2024024929 A1 WO2024024929 A1 WO 2024024929A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- muscle tissue
- culture
- culture container
- dimensional
- myoblasts
- Prior art date
Links
- 210000003205 muscle Anatomy 0.000 title claims abstract description 86
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 33
- 239000000017 hydrogel Substances 0.000 claims abstract description 51
- 210000003098 myoblast Anatomy 0.000 claims abstract description 43
- 230000001939 inductive effect Effects 0.000 claims abstract description 8
- 230000004069 differentiation Effects 0.000 claims description 10
- 238000000034 method Methods 0.000 abstract description 12
- 210000004027 cell Anatomy 0.000 description 34
- 235000013372 meat Nutrition 0.000 description 21
- 210000004369 blood Anatomy 0.000 description 17
- 239000008280 blood Substances 0.000 description 16
- 210000002381 plasma Anatomy 0.000 description 15
- 239000000306 component Substances 0.000 description 13
- 241000283690 Bos taurus Species 0.000 description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 12
- 230000008602 contraction Effects 0.000 description 12
- 210000002235 sarcomere Anatomy 0.000 description 10
- 230000000638 stimulation Effects 0.000 description 9
- 239000003146 anticoagulant agent Substances 0.000 description 8
- 229940127219 anticoagulant drug Drugs 0.000 description 8
- 239000000701 coagulant Substances 0.000 description 8
- 239000000499 gel Substances 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 244000144972 livestock Species 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 108010049003 Fibrinogen Proteins 0.000 description 6
- 102000008946 Fibrinogen Human genes 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 229940012952 fibrinogen Drugs 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 210000000663 muscle cell Anatomy 0.000 description 6
- 210000004623 platelet-rich plasma Anatomy 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 108010035532 Collagen Proteins 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 229920001436 collagen Polymers 0.000 description 5
- 210000003743 erythrocyte Anatomy 0.000 description 5
- 238000012744 immunostaining Methods 0.000 description 5
- 108010082117 matrigel Proteins 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 210000004683 skeletal myoblast Anatomy 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 239000000853 adhesive Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 210000001087 myotubule Anatomy 0.000 description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 3
- 241000283073 Equus caballus Species 0.000 description 3
- 241000270322 Lepidosauria Species 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 230000001070 adhesive effect Effects 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 229920000052 poly(p-xylylene) Polymers 0.000 description 3
- -1 polypropylene Polymers 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 210000002363 skeletal muscle cell Anatomy 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- 102000010825 Actinin Human genes 0.000 description 2
- 108010063503 Actinin Proteins 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 241000283086 Equidae Species 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 102000016359 Fibronectins Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 108010085895 Laminin Proteins 0.000 description 2
- 102000007547 Laminin Human genes 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 102000003505 Myosin Human genes 0.000 description 2
- 108060008487 Myosin Proteins 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000023555 blood coagulation Effects 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000004205 dimethyl polysiloxane Substances 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 238000001879 gelation Methods 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000012533 medium component Substances 0.000 description 2
- 210000003365 myofibril Anatomy 0.000 description 2
- 230000032696 parturition Effects 0.000 description 2
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 2
- 229920005992 thermoplastic resin Polymers 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- QDGAVODICPCDMU-UHFFFAOYSA-N 2-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]propanoic acid Chemical compound OC(=O)C(N)CC1=CC=CC(N(CCCl)CCCl)=C1 QDGAVODICPCDMU-UHFFFAOYSA-N 0.000 description 1
- 239000004925 Acrylic resin Substances 0.000 description 1
- 229920000178 Acrylic resin Polymers 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000272517 Anseriformes Species 0.000 description 1
- 241000269350 Anura Species 0.000 description 1
- 241000473391 Archosargus rhomboidalis Species 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000269333 Caudata Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000270722 Crocodylidae Species 0.000 description 1
- 241000252233 Cyprinus carpio Species 0.000 description 1
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 208000009331 Experimental Sarcoma Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 239000004158 L-cystine Substances 0.000 description 1
- 235000019393 L-cystine Nutrition 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- 229930182844 L-isoleucine Natural products 0.000 description 1
- 239000004395 L-leucine Substances 0.000 description 1
- 235000019454 L-leucine Nutrition 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 241000287127 Passeridae Species 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000269622 Salamandridae Species 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 241000270295 Serpentes Species 0.000 description 1
- 241000271567 Struthioniformes Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 108090000054 Syndecan-2 Proteins 0.000 description 1
- 241000270666 Testudines Species 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 229930003779 Vitamin B12 Natural products 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229960002713 calcium chloride Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 230000001112 coagulating effect Effects 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 235000006694 eating habits Nutrition 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229960003284 iron Drugs 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000003632 microfilament Anatomy 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 230000036640 muscle relaxation Effects 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 229920001225 polyester resin Polymers 0.000 description 1
- 239000004645 polyester resin Substances 0.000 description 1
- 229920000139 polyethylene terephthalate Polymers 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 229920005672 polyolefin resin Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 1
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 1
- 229960001327 pyridoxal phosphate Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 229920002050 silicone resin Polymers 0.000 description 1
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- 210000001988 somatic stem cell Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 210000002948 striated muscle cell Anatomy 0.000 description 1
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 229920002725 thermoplastic elastomer Polymers 0.000 description 1
- 229920001187 thermosetting polymer Polymers 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 229960002898 threonine Drugs 0.000 description 1
- 229960004441 tyrosine Drugs 0.000 description 1
- 229960004295 valine Drugs 0.000 description 1
- 238000007740 vapor deposition Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- the present invention relates to a method for producing three-dimensional muscle tissue and a culture vessel for producing three-dimensional muscle tissue.
- cultured minced meat is a collection of disparate muscle cells and has an inferior texture
- cultured steak meat reproduces the three-dimensional structure of muscle tissue, making it an alternative meat that allows you to experience the texture of meat. It is.
- Cited Document 1 describes a method for producing a three-dimensional muscle tissue particularly suitable for edible food, in which a hydrogel contains skeletal myoblasts and a substantially rectangular first cell module having a plurality of mutually parallel substantially rectangular holes is disclosed. , and creating a substantially rectangular second cell module containing skeletal myoblasts in the hydrogel and having a plurality of substantially rectangular holes parallel to each other at positions different from the first cell module in the vertical direction. step, a step of alternately stacking the obtained first cell module and the second cell module to obtain a laminate, a step of proliferating and culturing skeletal myoblasts contained in the obtained laminate, and proliferation.
- a method for producing three-dimensional muscle tissue is disclosed, which includes a step of inducing differentiation of skeletal myoblasts into myotubes.
- Cited Document 1 produces three-dimensional muscle tissue that is especially suitable for human consumption, and more specifically, has a sarcomere structure similar to that of living body-derived muscle, and is similar to livestock meat when used for human consumption.
- a method for producing three-dimensional muscle tissue with a promising texture is provided.
- further improvements are needed regarding the simple production of three-dimensional muscle tissue that is large enough to be eaten.
- the present invention aims to provide a method for producing three-dimensional muscle tissue that can easily produce three-dimensional muscle tissue having a sufficient size suitable for human consumption, and a culture container for producing three-dimensional muscle tissue. Take it as a challenge.
- Method for producing three-dimensional muscle tissue including the following steps (A) and (B): (A) A step of supplying a hydrogel containing myoblasts to a culture container, a step in which the culture container is a culture container including a pair of anchor portions facing each other at ends of the container, and a plurality of convex portions arranged between the pair of anchor portions, and (B) A step of inducing differentiation of myoblasts into myotubes in the culture vessel.
- the length (X) of the protrusion in the longitudinal direction is 1 cm or more
- the length (Y) of the protrusion in the transverse direction is 1 cm or more.
- the ratio [(Y)/(X)] of the length (Y) of the convex part in the transverse direction to the length (X) of the convex part in the longitudinal direction is 0.7 or more.
- the distance between the convex portions is 10 ⁇ m to 1 mm.
- a culture container for producing three-dimensional muscle tissue comprising a pair of anchor parts facing each other at the ends of the container, and a plurality of convex parts arranged between the pair of anchor parts.
- the present invention provides a method for producing three-dimensional muscle tissue that is particularly suitable for human consumption.
- the three-dimensional muscle tissue produced by the production method of the present invention has a sarcomere structure, similar to muscles of biological origin. Moreover, three-dimensional muscle tissue having a sufficient size suitable for consumption can be produced. Therefore, the three-dimensional muscle tissue produced by the production method of the present invention can be expected to have a texture similar to that of livestock meat when used for human consumption.
- FIG. 1 is a perspective view of a culture container 100 according to an embodiment of the present invention.
- FIG. 2 is a cross-sectional view taken along line A-A' in FIG.
- FIG. 3 is an enlarged view of the anchor portion in FIG. 1.
- FIG. 4 is a perspective view of a culture container 200 according to an embodiment of the present invention.
- FIG. 5 is (a) a plan view, (b) a front view, (c) a right side view, and (d) a bottom view of a culture container 200 according to an embodiment of the present invention.
- FIG. 6 is a perspective view of a culture container 300 according to an embodiment of the present invention.
- FIG. 1 is a perspective view of a culture container 100 according to an embodiment of the present invention.
- FIG. 2 is a cross-sectional view taken along line A-A' in FIG.
- FIG. 3 is an enlarged view of the anchor portion in FIG. 1.
- FIG. 4 is a perspective view of a culture container 200 according to an embodiment
- FIG. 7 is (a) a plan view, (b) a front view, (c) a right side view, and (d) a bottom view of a culture container 300 according to an embodiment of the present invention.
- FIG. 8 is a perspective view of a culture container 400 according to an embodiment of the present invention.
- FIG. 9 is (a) a plan view, (b) a front view, (c) a right side view, and (d) a bottom view of a culture container 400 according to an embodiment of the present invention.
- FIG. 10 schematically shows a culture container 100 according to an embodiment of the present invention. In the culture container 100, the length (X) of the protrusion in the longitudinal direction is 4 cm, and the length (Y) of the protrusion in the transverse direction is 3 cm.
- Anchor part The member is a cylinder with a diameter of 0.5 mm and a height of 5 mm, and is installed with a spacing of 1 mm between center points.
- Convex portion promoting orientation the member has a height of 200 ⁇ m, a width of 50 ⁇ m, and a distance of 2 mm from the anchor.
- Figure 11-1 shows an SAA immunostaining image.
- FIG. 11-2 shows the color inverted image of FIG. 11-1.
- FIG. 12 is a graph showing changes over time in the amount of shrinkage of three-dimensional muscle tissue. Specifically, it shows the change in tissue width in the lateral direction. The vertical axis is the length (cm) of the tissue in the lateral direction.
- FIG. 13 is a graph showing changes over time in the amount of movement (amount of contraction) of three-dimensional muscle tissue.
- A The height of the convex portion is 100 ⁇ m
- B The height of the convex portion is 300 ⁇ m
- C The height of the convex portion is 500 ⁇ m.
- the method for producing three-dimensional muscle tissue of the present invention includes the following steps (A) and (B): (A) A step of supplying a hydrogel containing myoblasts to a culture container,
- the culture container is a culture container including a pair of anchor parts facing each other at the ends of the container, and a plurality of convex parts arranged between the pair of anchor parts, and (B) A step of inducing differentiation of myoblasts into myotubes in the culture vessel.
- three-dimensional muscle tissue mainly refers to muscles that are not derived from a living body but are artificially manufactured.
- the three-dimensional muscle tissue of the present invention is composed of muscle cells.
- the muscle cells are preferably striated muscle cells having contractile ability, specifically skeletal muscle cells or cardiac muscle cells.
- Myocytes are myoblast precursors in the form of multinucleated myotubes or myofibers.
- muscle fibers have myofibrils as constituent units, which are composed of actin filaments, which are proteins that make up muscles, and myosin filaments, which are proteins that make up muscles. Furthermore, myofibrils have a structure in which a plurality of sarcomere structures are connected in the longitudinal direction. It is known that muscle contraction and relaxation occur based on the interaction (sliding) of actin and myosin in sarcomeres.
- the three-dimensional muscle tissue of the present invention has a sarcomere structure. However, it does not matter whether or not sliding occurs in the sarcomere structure.
- Whether or not a three-dimensional muscle tissue has a sarcomere structure can be evaluated using a known method. For example, the presence of sarcomeric ⁇ -actinin (SAA), a protein that constitutes the Z membrane of the sarcomere structure, was evaluated by immunostaining of SAA, and the SAA immunostaining was positive and SAA was distributed in a regular stripe pattern. If there is a sarcomere structure, it can be determined that it has a sarcomere structure.
- SAA sarcomeric ⁇ -actinin
- the three-dimensional muscle tissue of the present invention is preferably an edible three-dimensional muscle tissue.
- the muscle cells constituting the muscle tissue are preferably skeletal muscle cells.
- Edible three-dimensional muscle tissue can be referred to as "cultured meat", "artificial meat”, etc.
- Step (A) is a step of supplying a hydrogel containing myoblasts to a culture container,
- the culture container has a plurality of substantially rectangular convex portions parallel to each other on the bottom, and anchor portions at both longitudinal ends of the convex portions.
- Myoblasts can be prepared by known techniques. For example, primary myoblasts obtained by treating muscle tissue derived from a living body with a degrading enzyme (eg, collagenase) can be used. For example, when the muscle cells constituting the three-dimensional tissue are skeletal muscle cells, the myoblasts are skeletal myoblasts. Note that filter processing can be performed to remove impurities such as connective tissue from primary myoblasts. On the other hand, it is not essential to completely remove cells other than myoblasts, and myoblasts can be used in a mixed state containing cells other than myoblasts.
- a degrading enzyme eg, collagenase
- myoblasts cells induced to differentiate from stem cells having pluripotency such as ES cells and iPS cells, or somatic stem cells having the ability to differentiate into myoblasts, can also be used.
- Myoblasts are derived from vertebrates such as mammals, birds, reptiles, amphibians, and fish.
- mammals include non-human mammals such as monkeys, cows, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, and mice.
- avian animals include ostriches, chickens, ducks, and sparrows.
- reptile animals include snakes, crocodiles, lizards, and turtles.
- Amphibians include frogs, newts, salamanders, and the like.
- fish animals include salmon, tuna, shark, sea bream, and carp.
- the myoblasts are preferably derived from mammals raised for livestock such as cows, pigs, sheep, goats, and horses, and more preferably from cows.
- myoblasts myoblasts that have been genetically modified by homologous recombination, CRISPR/Cas9, or other genome editing methods, or myoblasts that have not been genetically modified can be used.
- myoblasts that have not been genetically modified from the viewpoint of safety and consumer preference.
- hydrogel The hydrogel functions as a scaffolding material during the culture of three-dimensional muscle tissue.
- One of the preferred embodiments of the hydrogel includes fibrin, fibronectin, laminin, collagen (for example, type I, type II, type III, type V, type XI, etc.), agar, agarose, glycosaminoglycan, hyaluronic acid, A gel of components constituting the extracellular basement membrane matrix, such as proteoglycans, can be used.
- Commercial products can also be used as hydrogels. For example, components based on mouse EHS tumor extract (containing type IV collagen, laminin, heparan sulfate proteoglycans, etc.) sold under the trade name "Matrigel" can be used. can.
- collagen includes undenatured collagen and denatured collagen.
- denatured collagen is gelatin.
- the hydrogel includes a gel containing blood-derived plasma and a coagulant.
- the animal from which the blood is derived is derived from a mammalian animal raised for livestock production such as a cow, pig, sheep, goat, or horse, and is more preferably derived from a cow.
- the animal of origin may be a fetus before parturition or an adult after parturition.
- the age in days, months, and age of the source animal is not limited. In the case of an adult, it may be a young individual, a young individual, a mature individual, or an old individual after delivery from a female individual.
- the source animal is an adult cow, and more preferably an adult cow from the viewpoint of easy availability on the market and the amount to be slaughtered and slaughtered.
- Blood from adult cows can be obtained by collecting specimens from slaughtered cows at meat and wholesale markets, or by other methods.
- anticoagulants may be added to blood before delivery. Blood to which an anticoagulant has been added is separated into red blood cells and plasma containing fibrinogen by centrifugation. Here, if an anticoagulant is added to the blood, neither red blood cells nor plasma containing fibrinogen, which is a coagulation component, will coagulate or gel.
- the plasma gels.
- the hydrogel of this embodiment contains plasma, it is also expected to have a function of replenishing cell culture components, which are nutrients during cell culture.
- the adult bovine blood-derived plasma containing fibrinogen may be platelet-rich plasma (PRP plasma).
- PRP plasma is a platelet-rich plasma concentrate prepared by centrifuging plasma, and is enriched with platelets as well as soluble proteins and growth factors.
- Platelet-rich plasma can be collected by centrifugation, double centrifugation, selective filtration, etc. of blood to which an anticoagulant has been added. Specifically, plasma from which red blood cells have been removed may be further centrifuged and collected, or in the case of blood that also contains red blood cells, a layer of the plasma layer concentrated at the boundary with red blood cells may be collected.
- the anticoagulant is not limited as long as it prevents blood coagulation.
- anticoagulants include those that bind to calcium ions essential for blood coagulation, such as sodium citrate, EDTA (ethylenediaminetetraacetic acid), and sodium fluoride.
- sodium citrate it is more desirable to add it in an amount of 2% to 4% by volume, preferably around 3% by volume, based on the total amount of blood and anticoagulant.
- the coagulant is not limited as long as it can gel blood plasma containing an anticoagulant.
- examples of the coagulant include calcium chloride and DMEM (Dulbecco's Modified Eagle's Medium), with those containing calcium ions being more preferred.
- the coagulant is calcium chloride
- it can be added to the total amount of plasma and coagulant so that the calcium ion concentration is, for example, preferably 5mM to 70mM, more preferably 10mM to 65mM, even more preferably 10mM to 60mM.
- XX to YY means "more than or equal to XX and less than or equal to YY.”
- DMEM contains L-arginine, L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and calcium chloride.
- potassium chloride magnesium sulfate, sodium chloride, sodium dihydrogen phosphate, D-glucose, folic acid, nicotinamide, riboflavin, vitamin B12, choline, inositol, pantothenic acid, pyridoxal phosphate, thiamine, iron, and the like.
- the amount of blood-derived plasma is preferably 0.1 volume% or more and 35 volume% or less, more preferably 0.1 volume% or more and 30 volume% or less, and even more preferably 0.1 volume% or more and 30 volume% or less, relative to the amount of DMEM. can be added in an amount of 0.2% by volume or more and 30% by volume or less.
- the hydrogel may further include a medium component.
- Medium components include DMEM (for example, manufactured by GIBCO), EMEM (for example, manufactured by GIBCO), MEMALPHA (for example, manufactured by GIBCO), RPMI-1640 (Roswell Park Memorial Institute 1640 medium; manufactured by GIBCO), etc. can be mentioned.
- additive components commonly used in culture media can be appropriately blended into the culture medium components. Examples of additive components include antibiotics, vitamins, nucleic acids, amino acids, inorganic salts, sugars, polyamines, carbohydrates, proteins, fatty acids, lipids, pH adjusters, zinc, copper, selenium, and the like.
- the myoblasts in the hydrogel preferably have a cell density of about 1.0 x 10 6 cells/mL or more, more preferably about 1.0 x 10 7 cells/mL to about 1.0 x 10 8 cells/mL. , more preferably 5.0 ⁇ 10 7 cells/mL to about 1.0 ⁇ 10 8 cells/mL.
- the culture container used in the present invention is a culture container for producing three-dimensional muscle tissue, and includes a pair of anchor portions facing each other at the ends of the container, and a plurality of convex portions arranged between the pair of anchor portions. This is a culture container. It is thought that by providing the convex portion, the orientation of myotubes and myofiber bundles is efficiently formed during culture.
- FIG. 1 shows a perspective view of a configuration example of a culture container 100 according to the present embodiment.
- FIG. 2 shows a cross-sectional view of the culture container 100.
- the material constituting the culture container 100 is not limited.
- the material constituting the culture container 100 is a thermoplastic resin or a thermosetting resin.
- the thermoplastic resin include polyolefin resins such as polypropylene; polyester resins such as polyethylene terephthalate; acrylic resins; thermoplastic elastomers: silicone resins such as polydimethylsiloxane (PDMS);
- the material constituting the culture container 100 is preferably a material that is non-adhesive to cells or a material that has been surface-treated to become non-adhesive. Non-adhesive surface treatments include parylene coatings.
- the material constituting the culture container 100 may be transparent or opaque. From the viewpoint of ease of observation, it is preferably transparent.
- the culture container 100 can be manufactured using, for example, a 3D printer.
- the culture vessel is preferably rectangular.
- the length (X) of the protrusion in the longitudinal direction is preferably 1 cm or more, more preferably 1.5 cm or more
- the length (Y) of the protrusion in the lateral direction is preferably 1 cm or more, More preferably, it is 2 cm or more.
- the longitudinal direction of the convex portion is the direction in which a pair of anchor portions, which will be described later, face each other, and the lateral direction of the convex portion is the direction in which the convex portions are arranged.
- the ratio [(Y)/(X)] of the length (Y) of the convex portion in the transverse direction to the length (X) of the convex portion in the longitudinal direction is preferably 0.7 or more.
- [(Y)/(X)] can also be 1 or more, 1.5 or more, or 2 or more.
- the upper limit is not particularly limited, and is, for example, 4 or less.
- the culture container 100 has a plurality of convex portions arranged between a pair of anchor portions 20, which will be described later.
- the shape of the anchor part is not limited.
- the culture container has a substantially rectangular shape when viewed from above.
- the manner in which the plurality of convex portions are arranged is not limited.
- the plurality of convex portions are provided on the bottom so as to be parallel to each other.
- the height of the convex portion 10 is preferably 50 ⁇ m to 1 mm, more preferably 100 ⁇ m to 300 ⁇ m. Further, the distance between the convex portions is 10 ⁇ m to 5 mm, more preferably 50 ⁇ m to 2.5 mm, and even more preferably 100 ⁇ m to 1 mm.
- the culture container used in the present invention includes a pair of anchor portions facing each other at the ends of the container.
- the anchor part is not particularly limited as long as it is a means for fixing the hydrogel and the manufactured three-dimensional muscle tissue.
- a member that is fixed using a component having adhesive strength for example, hydrogel such as fibrin
- a plurality of cylindrical anchor portions 20 face each other at each end of the container.
- the cylindrical anchor portion 20 has a diameter of approximately 100 ⁇ m to 2 mm and a height of approximately 3 mm to 10 mm.
- FIGS. 4 to 9 show configuration examples of a culture container 200, a culture container 300, and a culture container 400 according to other embodiments using a perspective view, a top view, a front view, a right side view, and a bottom view.
- a hydrogel containing myoblasts is supplied to the culture vessel.
- a culture container is used as the bottom surface, a mold is provided on the side surface, and the hydrogel containing myoblasts is supplied.
- the amount of hydrogel containing myoblasts to be supplied can be set appropriately.
- the amount may be such that the thickness of the hydrogel is preferably 1 mm to 1 cm, more preferably 2 to 5 mm.
- the hydrogel supplied to the culture container can be heated and solidified.
- the temperature range for heating is preferably about 37°C.
- the heating time can be adjusted depending on the progress of gelation, and is exemplified to be about 5 minutes to 60 minutes, and more preferably about 10 minutes.
- the hydrogel containing myoblasts can be subjected to proliferation culture to allow the myoblasts contained therein to proliferate.
- the hydrogel contains a sufficient amount of myoblasts (for example, 1.0 ⁇ 10 8 cells/mL or more)
- the next step of inducing differentiation can be performed without performing proliferation culture.
- the next step of inducing differentiation can be performed after proliferation culture.
- the above-mentioned culture can be performed, for example, in the above-mentioned growth culture medium by a method known to those skilled in the art.
- a suitable culture method includes, but is not limited to, a method of culturing at about 37° C. and a carbon dioxide concentration of about 5 to 10% (v/v). Cultivation under the above conditions can be performed using, for example, a known CO 2 incubator.
- DMEM Dulbecco's Modified Eagle's Medium
- EMEM Eagle's minimal essential medium
- ⁇ MEM alpha Modified Add serum components
- Components such as horse serum (Horse serum, Fetal bovine serum (FBS), human serum, etc.
- growth factors It is possible to use a medium supplemented with antibiotics such as penicillin and streptomycin. can.
- fetal bovine serum When adding a serum component to the growth culture medium, fetal bovine serum can be used as the serum component.
- concentration of serum components can be about 10% (v/v).
- the culture period can be, for example, about 1 day to 2 weeks.
- the culture medium can be replaced if necessary.
- Culture conditions can be according to conventional methods.
- Step (B) is a step of inducing differentiation of myoblasts into myotubes in the culture vessel. Through this step, myoblasts become multinucleated by cell fusion with surrounding cells, and myotubes are formed. Myotubes further mature to form muscle fibers.
- the above culture can be performed, for example, in a medium for differentiation induction (multinucleation medium) by a method known to those skilled in the art.
- suitable culturing methods include, but are not limited to, culturing at a temperature of about 37° C. and a carbon dioxide concentration of about 5 to 10% (v/v). Cultivation under the above conditions can be performed using, for example, a known CO 2 incubator.
- myoblasts become depleted of nutrients, they engulf surrounding cells and begin to become multinucleated. Therefore, induction of differentiation into myotubes can be performed using a medium containing fewer nutrients than the aforementioned proliferation culture.
- the present invention also relates to a three-dimensional muscle tissue obtained by the above manufacturing method.
- Example 1 Preparation of large contractile muscle tissue using bovine myoblasts Bovine myoblasts were embedded in a hydrogel with the composition shown below, and both ends were fixed and cultured to produce a muscle tissue with a length of 7 mm. was created. We investigated the formation and maturation conditions of muscle tissue by changing the hydrogel composition and cell density.
- a culture container 100 shown in FIG. 10 was created using a 3D printer (Formlab 3B, manufactured by BLULE Inc.).
- the length (X) of the protrusion in the longitudinal direction was 4 cm
- the length (Y) of the protrusion in the transverse direction was 3 cm.
- the convex portion 10 for promoting the orientation of muscle cells had a height of 200 ⁇ m and a width of 5 ⁇ m, was separated from the anchor portion by 2 mm, and had a longitudinal length of 3 cm.
- the anchor part 20 had a cylindrical shape with a diameter of 0.5 mm and a height of 5 mm, and was provided so that the distance between the center points was 1 mm.
- the surface of the created culture container was coated with parylene using a parylene vapor deposition device (Lab coater PDS2010, manufactured by Specialty Coating Systems). Next, after sterilizing the culture container using an ozone sterilizer, the anchor portion 20 was coated with fibronectin to promote cell adhesion.
- control culture container was created in the same manner as culture container 100 except that no convex portion was provided.
- Fibrinogen 25mg/mL
- 480 ⁇ L final concentration 4mg/mL
- Matrigel 600 ⁇ L final concentration 20%
- Thrombin 200 Unit/mL
- DMEM 1690 ⁇ L (manufactured by Thermo Fisher Scientific, 11965118)
- a growth medium (10% FBS DMEM + 1% Penicillin/Streptomycin) was added and cultured for 2 days at 37° C. and 5% CO 2 . Thereafter, the medium was replaced with a differentiation medium (2% HS DMEM + 1% Penicillin/Streptomycin + 100 ⁇ M), and culture was continued for an additional 5 days to obtain three-dimensional muscle tissue. The medium was changed every other day. As the culture progressed over time, the three-dimensional muscle tissue contracted (shrinked) in the short direction of the convex part.
- a differentiation medium 2% HS DMEM + 1% Penicillin/Streptomycin + 100 ⁇ M
- Electrical stimulation was applied to the three-dimensional muscle tissue obtained on the 5th and 7th day of culture using the electrical stimulation culture system C-Pace (manufactured by IonOptic) under the following conditions: frequency: 1 Hz, strong Power: 0.3V/mm, Duration: 40ms. The presence or absence of contraction movement in response to electrical stimulation was observed by microscopic observation. Clear contraction movement was observed in the three-dimensional muscle tissue obtained using the culture vessel 100. On the other hand, no contraction movement could be observed in the three-dimensional muscle tissue obtained using the control culture vessel.
- Example 2 Examination of the size of cultured muscle tissue (fabrication of culture vessels 200, 300, and 400)
- the culture container 200 has a length (X) of the convex part in the longitudinal direction: a length (Y) of the convex part in the short direction of 4 cm: 3 cm; 4 cm: 8 cm.
- a culture vessel 300 with a diameter of 10 cm and a culture vessel 400 with a diameter of 10 cm were produced.
- a hydrogel was produced in the same manner as in Example 1. 3 mL of the resulting cell-embedded hydrogel was poured into the culture container 200, 8 mL into the culture container 300, and 24 mL into the culture container 400, and left standing in a CO 2 incubator at 37° C. for 30 minutes. The gel was solidified. The cells were cultured for 7 days in the same manner as in Example 1 to obtain three-dimensional muscle tissue. As the culture progressed over time, the three-dimensional muscle tissue contracted (shrinked) in the short direction of the convex part.
- the shrinkage amount of the three-dimensional muscle tissue using culture container 200 and culture container 300 was about 2 cm
- the shrinkage amount of the three-dimensional muscle tissue using culture container 400 was about 5 cm. This suggests that the amount of shrinkage of three-dimensional muscle tissue is approximately half the length of the convex part of the culture container in the transverse direction, and a culture container of the size estimated for the amount of shrinkage was used. By doing this, it became clear that it was possible to create three-dimensional muscle tissue of any size.
- Example 3 Production of hydrogel using edible pig blood (production of culture container 500)
- a culture container 500 was produced in which the length of the protrusion in the longitudinal direction (X): the length of the protrusion in the transverse direction (Y) was 8 cm:12 cm.
- Example 1 (Preparation and culture of hydrogel)
- a hydrogel was produced using the above composition using edible pig blood instead of Matrigel.
- 2.5 ⁇ 10 8 myoblast cells were mixed with the hydrogel at a concentration of 1.0 ⁇ 10 7 cells/mL to produce a hydrogel in which the cells were embedded.
- the culture container 500 or the control culture container was used as the bottom, a mold was provided on the side, and the obtained hydrogel in which cells were embedded was poured into the mold.
- the hydrogel was left standing in a CO 2 incubator at 37° C. for 30 minutes to solidify the hydrogel.
- the cells were cultured for 14 days in the same manner as in Example 1 except that the culture period was changed to 14 days to obtain a three-dimensional muscle tissue.
- Example 4 Examination of height of convex portion (fabrication of culture container)
- a culture container 600 in which the height of the projection 10 is 100 ⁇ m, a culture container 700 in which the height of the projection 10 is 300 ⁇ m, and a culture container 700 in which the height of the projection 10 is 300 ⁇ m are prepared in the same manner as the culture container 100 in Example 1 except for the height of the projection 10.
- Preparation and culture of hydrogel A hydrogel was prepared and cultured in the same manner as in Example 1.
- the amount of movement was particularly large when the height of the convex portion 10 was 100 ⁇ m.
Landscapes
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Sustainable Development (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a method for producing three-dimensional muscle tissue, the method including the following steps (A) and (B): (A) a step for supplying a myoblast-containing hydrogel to a culture container, the culture container comprising a pair of anchor sections on opposite edges of the container and a plurality of convexities arranged spanning between the pair of anchor sections; and (B) a step for inducing the myoblasts to differentiate into myotubes in the culture container.
Description
本発明は、三次元筋組織の製造方法、及び三次元筋組織を製造するための培養容器に関する。
The present invention relates to a method for producing three-dimensional muscle tissue and a culture vessel for producing three-dimensional muscle tissue.
世界的な人口増加、並びに、新興国での経済発展及び食生活の多様化に伴って、世界的に食肉需要が加速している。しかしながら、家畜の生産には飼育コストもかかり、家畜生産量は有限であり、やがて食肉の供給が追いつかなくなることが予想されている。
そこで、食肉の安定的供給の解決策の１つとして、代替肉の開発研究がなされてきている。 Demand for meat is accelerating worldwide due to global population growth, economic development in emerging countries, and diversification of dietary habits. However, livestock production requires breeding costs, and livestock production is limited, and it is predicted that the supply of meat will eventually not be able to keep up.
Therefore, as one solution to ensuring a stable supply of meat, research is being conducted to develop alternative meats.
そこで、食肉の安定的供給の解決策の１つとして、代替肉の開発研究がなされてきている。 Demand for meat is accelerating worldwide due to global population growth, economic development in emerging countries, and diversification of dietary habits. However, livestock production requires breeding costs, and livestock production is limited, and it is predicted that the supply of meat will eventually not be able to keep up.
Therefore, as one solution to ensuring a stable supply of meat, research is being conducted to develop alternative meats.
家畜から生産される食肉の代替肉には複数種類があり、肉もどき、植物肉、培養ミンチ肉、培養ステーキ肉等に分類される。このうち、培養ミンチ肉と培養ステーキ肉は培養肉と呼ばれる。培養ミンチ肉がバラバラの筋細胞の集合体であり食肉の食感が劣るのに対し、培養ステーキ肉は筋組織の立体構造を再現したものであり食肉の食感を感じることが可能な代替肉である。
There are multiple types of meat substitutes produced from livestock, which are classified into meat imitations, plant meats, cultured minced meat, cultured steak meat, etc. Among these, cultured minced meat and cultured steak meat are called cultured meat. Cultured minced meat is a collection of disparate muscle cells and has an inferior texture, whereas cultured steak meat reproduces the three-dimensional structure of muscle tissue, making it an alternative meat that allows you to experience the texture of meat. It is.
ここで、筋組織の立体構造を再現するには、すなわち、三次元筋組織を生体外で構築するためには、三次元的に細胞を培養することが必要である。しかし、三次元的に細胞を培養するのに乗り越えるべき技術的課題も多い。
Here, in order to reproduce the three-dimensional structure of muscle tissue, that is, to construct three-dimensional muscle tissue in vitro, it is necessary to culture cells three-dimensionally. However, there are many technical challenges that must be overcome in order to culture cells three-dimensionally.
引用文献１には、特に食用に適した三次元筋組織の製造方法として、ハイドロゲルに骨格筋芽細胞を含んでおり、互いに平行な略長方形の穴を複数有する略長方形の第１の細胞モジュール、及び、ハイドロゲルに骨格筋芽細胞を含んでおり、垂直方向において前記第１の細胞モジュールとは異なる位置に互いに平行な略長方形の穴を複数有する略長方形の第２の細胞モジュールを作成する工程、得られた前記第１の細胞モジュールと前記第２の細胞モジュールとを交互に積層し積層体を得る工程、得られた積層体に含まれる骨格筋芽細胞を増殖培養する工程、及び増殖した骨格筋芽細胞を筋管へと分化誘導する工程を含む、三次元筋組織の製造方法が開示されている。
Cited Document 1 describes a method for producing a three-dimensional muscle tissue particularly suitable for edible food, in which a hydrogel contains skeletal myoblasts and a substantially rectangular first cell module having a plurality of mutually parallel substantially rectangular holes is disclosed. , and creating a substantially rectangular second cell module containing skeletal myoblasts in the hydrogel and having a plurality of substantially rectangular holes parallel to each other at positions different from the first cell module in the vertical direction. step, a step of alternately stacking the obtained first cell module and the second cell module to obtain a laminate, a step of proliferating and culturing skeletal myoblasts contained in the obtained laminate, and proliferation. A method for producing three-dimensional muscle tissue is disclosed, which includes a step of inducing differentiation of skeletal myoblasts into myotubes.
引用文献１に記載の技術により、特に食用に適した三次元筋組織、より具体的には、生体由来の筋肉と同様にサルコメア構造を有し、食用に供した際に畜産された肉に近い食感が期待できる三次元筋組織の製造方法が提供される。
一方で、食用に適した十分な大きさを有する三次元筋組織を簡便に製造することに関し、更なる改善が必要であった。
本発明は、食用に適した十分な大きさを有する三次元筋組織を簡便に製造することができる三次元筋組織の製造方法及び三次元筋組織を製造するための培養容器を提供することを課題とする。 The technology described in CitedDocument 1 produces three-dimensional muscle tissue that is especially suitable for human consumption, and more specifically, has a sarcomere structure similar to that of living body-derived muscle, and is similar to livestock meat when used for human consumption. A method for producing three-dimensional muscle tissue with a promising texture is provided.
On the other hand, further improvements are needed regarding the simple production of three-dimensional muscle tissue that is large enough to be eaten.
The present invention aims to provide a method for producing three-dimensional muscle tissue that can easily produce three-dimensional muscle tissue having a sufficient size suitable for human consumption, and a culture container for producing three-dimensional muscle tissue. Take it as a challenge.
一方で、食用に適した十分な大きさを有する三次元筋組織を簡便に製造することに関し、更なる改善が必要であった。
本発明は、食用に適した十分な大きさを有する三次元筋組織を簡便に製造することができる三次元筋組織の製造方法及び三次元筋組織を製造するための培養容器を提供することを課題とする。 The technology described in Cited
On the other hand, further improvements are needed regarding the simple production of three-dimensional muscle tissue that is large enough to be eaten.
The present invention aims to provide a method for producing three-dimensional muscle tissue that can easily produce three-dimensional muscle tissue having a sufficient size suitable for human consumption, and a culture container for producing three-dimensional muscle tissue. Take it as a challenge.
本発明者は、上記課題を鑑みて鋭意検討を重ねた結果、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器を使用することにより上記の課題を解決できることを見いだした。本発明は、このような発見にさらに検討を重ねて完成したものである。
As a result of intensive studies in view of the above-mentioned problems, the present inventors have discovered the use of a culture container comprising a pair of anchor parts facing each other at the ends of the container, and a plurality of convex parts arranged between the pair of anchor parts. We have found that the above problems can be solved by The present invention has been completed through further study of these discoveries.
本発明は、以下の態様を包含する。
〔１〕 下記の工程（Ａ）及び（Ｂ）を含む三次元筋組織の製造方法：
（Ａ）筋芽細胞を含むハイドロゲルを培養容器へ供給する工程であって、
前記培養容器は、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器である工程、並びに、
（Ｂ）前記培養容器中で、筋芽細胞を筋管へと分化誘導する工程。
〔２〕 前記培養容器において、前記凸部の長手方向の長さ（Ｘ）が１ｃｍ以上、かつ、前記凸部の短手方向の長さ（Ｙ）が１ｃｍ以上である、上記〔１〕に記載の三次元筋組織の製造方法。
〔３〕 前記培養容器において、前記凸部の長手方向の長さ（Ｘ）に対する前記凸部の短手方向の長さ（Ｙ）の比〔（Ｙ）／（Ｘ）〕が０．７以上である、上記〔１〕又は〔２〕に記載の三次元筋組織の製造方法。
〔４〕 前記凸部が、高さ５０μｍ～１ｍｍである、上記〔１〕～〔３〕のいずれか１項に記載の三次元筋組織の製造方法。
〔５〕 前記凸部の互いの間隔が、１０μｍ～１ｍｍである、上記〔１〕～〔４〕のいずれか１項に記載の三次元筋組織の製造方法。
〔６〕 上記〔１〕～〔５〕のいずれか１項に記載の製造方法により得られる三次元筋組織。
〔７〕 三次元筋組織を製造するための培養容器であって、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる、培養容器。 The present invention includes the following aspects.
[1] Method for producing three-dimensional muscle tissue including the following steps (A) and (B):
(A) A step of supplying a hydrogel containing myoblasts to a culture container,
a step in which the culture container is a culture container including a pair of anchor portions facing each other at ends of the container, and a plurality of convex portions arranged between the pair of anchor portions, and
(B) A step of inducing differentiation of myoblasts into myotubes in the culture vessel.
[2] In the above [1], in the culture container, the length (X) of the protrusion in the longitudinal direction is 1 cm or more, and the length (Y) of the protrusion in the transverse direction is 1 cm or more. The described method for producing three-dimensional muscle tissue.
[3] In the culture container, the ratio [(Y)/(X)] of the length (Y) of the convex part in the transverse direction to the length (X) of the convex part in the longitudinal direction is 0.7 or more. The method for producing three-dimensional muscle tissue according to [1] or [2] above.
[4] The method for producing three-dimensional muscle tissue according to any one of [1] to [3] above, wherein the convex portion has a height of 50 μm to 1 mm.
[5] The method for producing three-dimensional muscle tissue according to any one of [1] to [4] above, wherein the distance between the convex portions is 10 μm to 1 mm.
[6] Three-dimensional muscle tissue obtained by the production method according to any one of [1] to [5] above.
[7] A culture container for producing three-dimensional muscle tissue, comprising a pair of anchor parts facing each other at the ends of the container, and a plurality of convex parts arranged between the pair of anchor parts.
〔１〕 下記の工程（Ａ）及び（Ｂ）を含む三次元筋組織の製造方法：
（Ａ）筋芽細胞を含むハイドロゲルを培養容器へ供給する工程であって、
前記培養容器は、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器である工程、並びに、
（Ｂ）前記培養容器中で、筋芽細胞を筋管へと分化誘導する工程。
〔２〕 前記培養容器において、前記凸部の長手方向の長さ（Ｘ）が１ｃｍ以上、かつ、前記凸部の短手方向の長さ（Ｙ）が１ｃｍ以上である、上記〔１〕に記載の三次元筋組織の製造方法。
〔３〕 前記培養容器において、前記凸部の長手方向の長さ（Ｘ）に対する前記凸部の短手方向の長さ（Ｙ）の比〔（Ｙ）／（Ｘ）〕が０．７以上である、上記〔１〕又は〔２〕に記載の三次元筋組織の製造方法。
〔４〕 前記凸部が、高さ５０μｍ～１ｍｍである、上記〔１〕～〔３〕のいずれか１項に記載の三次元筋組織の製造方法。
〔５〕 前記凸部の互いの間隔が、１０μｍ～１ｍｍである、上記〔１〕～〔４〕のいずれか１項に記載の三次元筋組織の製造方法。
〔６〕 上記〔１〕～〔５〕のいずれか１項に記載の製造方法により得られる三次元筋組織。
〔７〕 三次元筋組織を製造するための培養容器であって、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる、培養容器。 The present invention includes the following aspects.
[1] Method for producing three-dimensional muscle tissue including the following steps (A) and (B):
(A) A step of supplying a hydrogel containing myoblasts to a culture container,
a step in which the culture container is a culture container including a pair of anchor portions facing each other at ends of the container, and a plurality of convex portions arranged between the pair of anchor portions, and
(B) A step of inducing differentiation of myoblasts into myotubes in the culture vessel.
[2] In the above [1], in the culture container, the length (X) of the protrusion in the longitudinal direction is 1 cm or more, and the length (Y) of the protrusion in the transverse direction is 1 cm or more. The described method for producing three-dimensional muscle tissue.
[3] In the culture container, the ratio [(Y)/(X)] of the length (Y) of the convex part in the transverse direction to the length (X) of the convex part in the longitudinal direction is 0.7 or more. The method for producing three-dimensional muscle tissue according to [1] or [2] above.
[4] The method for producing three-dimensional muscle tissue according to any one of [1] to [3] above, wherein the convex portion has a height of 50 μm to 1 mm.
[5] The method for producing three-dimensional muscle tissue according to any one of [1] to [4] above, wherein the distance between the convex portions is 10 μm to 1 mm.
[6] Three-dimensional muscle tissue obtained by the production method according to any one of [1] to [5] above.
[7] A culture container for producing three-dimensional muscle tissue, comprising a pair of anchor parts facing each other at the ends of the container, and a plurality of convex parts arranged between the pair of anchor parts.
本発明により、特に食用に適した三次元筋組織の製造方法が提供される。本発明の製造方法により製造される三次元筋組織は、生体由来の筋肉と同様に、サルコメア構造を有する。また、食用に適した十分な大きさを有する三次元筋組織を製造することができる。そのため、本発明の製造方法により製造された三次元筋組織は、食用に供した際に畜産された肉に近い食感が期待できる。
The present invention provides a method for producing three-dimensional muscle tissue that is particularly suitable for human consumption. The three-dimensional muscle tissue produced by the production method of the present invention has a sarcomere structure, similar to muscles of biological origin. Moreover, three-dimensional muscle tissue having a sufficient size suitable for consumption can be produced. Therefore, the three-dimensional muscle tissue produced by the production method of the present invention can be expected to have a texture similar to that of livestock meat when used for human consumption.
［三次元筋組織の製造方法］
本発明の三次元筋組織の製造方法は、下記の工程（Ａ）及び（Ｂ）を含む：
（Ａ）筋芽細胞を含むハイドロゲルを培養容器へ供給する工程であって、
前記培養容器は、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器である工程、並びに、
（Ｂ）前記培養容器中で、筋芽細胞を筋管へと分化誘導する工程。 [Method for manufacturing three-dimensional muscle tissue]
The method for producing three-dimensional muscle tissue of the present invention includes the following steps (A) and (B):
(A) A step of supplying a hydrogel containing myoblasts to a culture container,
The culture container is a culture container including a pair of anchor parts facing each other at the ends of the container, and a plurality of convex parts arranged between the pair of anchor parts, and
(B) A step of inducing differentiation of myoblasts into myotubes in the culture vessel.
本発明の三次元筋組織の製造方法は、下記の工程（Ａ）及び（Ｂ）を含む：
（Ａ）筋芽細胞を含むハイドロゲルを培養容器へ供給する工程であって、
前記培養容器は、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器である工程、並びに、
（Ｂ）前記培養容器中で、筋芽細胞を筋管へと分化誘導する工程。 [Method for manufacturing three-dimensional muscle tissue]
The method for producing three-dimensional muscle tissue of the present invention includes the following steps (A) and (B):
(A) A step of supplying a hydrogel containing myoblasts to a culture container,
The culture container is a culture container including a pair of anchor parts facing each other at the ends of the container, and a plurality of convex parts arranged between the pair of anchor parts, and
(B) A step of inducing differentiation of myoblasts into myotubes in the culture vessel.
＜三次元筋組織＞
本発明において、三次元筋組織とは、生体に由来せず、人工的に製造された筋肉を主に意味する。本発明の三次元筋組織は、筋細胞から構成される。筋細胞は、好ましくは収縮能を有する横紋筋細胞であり、具体的には骨格筋細胞又は心筋細胞である。筋細胞は、その前駆体である筋芽細胞が多核化した筋管又は筋線維の形態である。 <Three-dimensional muscle tissue>
In the present invention, three-dimensional muscle tissue mainly refers to muscles that are not derived from a living body but are artificially manufactured. The three-dimensional muscle tissue of the present invention is composed of muscle cells. The muscle cells are preferably striated muscle cells having contractile ability, specifically skeletal muscle cells or cardiac muscle cells. Myocytes are myoblast precursors in the form of multinucleated myotubes or myofibers.
本発明において、三次元筋組織とは、生体に由来せず、人工的に製造された筋肉を主に意味する。本発明の三次元筋組織は、筋細胞から構成される。筋細胞は、好ましくは収縮能を有する横紋筋細胞であり、具体的には骨格筋細胞又は心筋細胞である。筋細胞は、その前駆体である筋芽細胞が多核化した筋管又は筋線維の形態である。 <Three-dimensional muscle tissue>
In the present invention, three-dimensional muscle tissue mainly refers to muscles that are not derived from a living body but are artificially manufactured. The three-dimensional muscle tissue of the present invention is composed of muscle cells. The muscle cells are preferably striated muscle cells having contractile ability, specifically skeletal muscle cells or cardiac muscle cells. Myocytes are myoblast precursors in the form of multinucleated myotubes or myofibers.
一般に、筋線維は、筋肉を構成するタンパク質であるアクチンの線維（アクチンフィラメント）及び筋肉を構成するタンパク質であるミオシンの線維（ミオシンフィラメント）から構成される筋原線維を構成単位とする。さらに、筋原線維は複数のサルコメア構造が長軸方向に連なった構造を有している。サルコメアにおけるアクチン及びミオシンの相互作用（滑り込み）に基づき、筋肉の収縮及び弛緩が発生することが知られている。
In general, muscle fibers have myofibrils as constituent units, which are composed of actin filaments, which are proteins that make up muscles, and myosin filaments, which are proteins that make up muscles. Furthermore, myofibrils have a structure in which a plurality of sarcomere structures are connected in the longitudinal direction. It is known that muscle contraction and relaxation occur based on the interaction (sliding) of actin and myosin in sarcomeres.
本発明の三次元筋組織は、サルコメア構造を有する。ただし、サルコメア構造における滑り込みが生じるか否かは問わない。
The three-dimensional muscle tissue of the present invention has a sarcomere structure. However, it does not matter whether or not sliding occurs in the sarcomere structure.
三次元筋組織は、サルコメア構造を有するか否かは、公知の手法により評価することができる。例えば、サルコメア構造のＺ膜を構成するタンパク質であるｓａｒｃｏｍｅｒｉｃ α－ａｃｔｉｎｉｎ（ＳＡＡ）が存在することをＳＡＡの免疫染色により評価し、ＳＡＡ免疫染色が陽性かつＳＡＡが規則的な縞状に分布している場合にサルコメア構造を有すると判定することができる。
Whether or not a three-dimensional muscle tissue has a sarcomere structure can be evaluated using a known method. For example, the presence of sarcomeric α-actinin (SAA), a protein that constitutes the Z membrane of the sarcomere structure, was evaluated by immunostaining of SAA, and the SAA immunostaining was positive and SAA was distributed in a regular stripe pattern. If there is a sarcomere structure, it can be determined that it has a sarcomere structure.
本発明の三次元筋組織は、好ましくは食用の三次元筋組織である。例えば食用の三次元筋組織である場合、筋組織を構成する筋細胞は、好ましくは骨格筋細胞である。食用の三次元筋組織は、「培養肉」、「人工食肉」等と換言することができる。
The three-dimensional muscle tissue of the present invention is preferably an edible three-dimensional muscle tissue. For example, in the case of edible three-dimensional muscle tissue, the muscle cells constituting the muscle tissue are preferably skeletal muscle cells. Edible three-dimensional muscle tissue can be referred to as "cultured meat", "artificial meat", etc.
＜工程（Ａ）＞
工程（Ａ）は、筋芽細胞を含むハイドロゲルを培養容器へ供給する工程であって、
前記培養容器は、底部に複数の互いに平行な略長方形の凸部、及び前記凸部の長手方向の両端にアンカー部を備えた培養容器である。 <Process (A)>
Step (A) is a step of supplying a hydrogel containing myoblasts to a culture container,
The culture container has a plurality of substantially rectangular convex portions parallel to each other on the bottom, and anchor portions at both longitudinal ends of the convex portions.
工程（Ａ）は、筋芽細胞を含むハイドロゲルを培養容器へ供給する工程であって、
前記培養容器は、底部に複数の互いに平行な略長方形の凸部、及び前記凸部の長手方向の両端にアンカー部を備えた培養容器である。 <Process (A)>
Step (A) is a step of supplying a hydrogel containing myoblasts to a culture container,
The culture container has a plurality of substantially rectangular convex portions parallel to each other on the bottom, and anchor portions at both longitudinal ends of the convex portions.
〔筋芽細胞を含むハイドロゲル〕
（筋芽細胞）
筋芽細胞は、公知の手法により調製することができる。例えば、生体由来の筋組織を分解酵素（例えば、コラゲナーゼ）の処理を施して得られる初代筋芽細胞を使用することができる。
例えば、三次元組織を構成する筋細胞が骨格筋細胞であるとき、筋芽細胞は骨格筋芽細胞である。
なお、初代筋芽細胞から結合組織などの不純物を除去するためにフィルター処理を施すことができる。一方で、筋芽細胞以外の細胞を完全に除去することは必須ではなく、筋芽細胞は、筋芽細胞以外の細胞を含む混合状態で使用することができる。 [Hydrogel containing myoblasts]
(myoblast)
Myoblasts can be prepared by known techniques. For example, primary myoblasts obtained by treating muscle tissue derived from a living body with a degrading enzyme (eg, collagenase) can be used.
For example, when the muscle cells constituting the three-dimensional tissue are skeletal muscle cells, the myoblasts are skeletal myoblasts.
Note that filter processing can be performed to remove impurities such as connective tissue from primary myoblasts. On the other hand, it is not essential to completely remove cells other than myoblasts, and myoblasts can be used in a mixed state containing cells other than myoblasts.
（筋芽細胞）
筋芽細胞は、公知の手法により調製することができる。例えば、生体由来の筋組織を分解酵素（例えば、コラゲナーゼ）の処理を施して得られる初代筋芽細胞を使用することができる。
例えば、三次元組織を構成する筋細胞が骨格筋細胞であるとき、筋芽細胞は骨格筋芽細胞である。
なお、初代筋芽細胞から結合組織などの不純物を除去するためにフィルター処理を施すことができる。一方で、筋芽細胞以外の細胞を完全に除去することは必須ではなく、筋芽細胞は、筋芽細胞以外の細胞を含む混合状態で使用することができる。 [Hydrogel containing myoblasts]
(myoblast)
Myoblasts can be prepared by known techniques. For example, primary myoblasts obtained by treating muscle tissue derived from a living body with a degrading enzyme (eg, collagenase) can be used.
For example, when the muscle cells constituting the three-dimensional tissue are skeletal muscle cells, the myoblasts are skeletal myoblasts.
Note that filter processing can be performed to remove impurities such as connective tissue from primary myoblasts. On the other hand, it is not essential to completely remove cells other than myoblasts, and myoblasts can be used in a mixed state containing cells other than myoblasts.
また、筋芽細胞は、ＥＳ細胞、ｉＰＳ細胞のような万能性を有する幹細胞や筋芽細胞へ分化する能力を有する体性幹細胞から分化誘導した細胞を用いることもできる。
Furthermore, as the myoblasts, cells induced to differentiate from stem cells having pluripotency such as ES cells and iPS cells, or somatic stem cells having the ability to differentiate into myoblasts, can also be used.
筋芽細胞は、ほ乳類動物、鳥類動物、は虫類動物、両生類動物、魚類動物等の脊椎動物に由来する。ほ乳類動物としては、サル、ウシ、ウマ、ブタ、ヒツジ、ヤギ、イヌ、ネコ、モルモット、ラット、マウス等の非ヒトほ乳類動物が挙げられる。鳥類動物としては、ダチョウ、ニワトリ、カモ、スズメ等が挙げられる。は虫類動物としては、ヘビ、ワニ、トカゲ、カメ等が挙げられる。両生類動物としては、カエル、イモリ、サンショウウオ等が挙げられる。魚類動物としては、サケ、マグロ、サメ、タイ、コイ等が挙げられる。三次元筋組織を食用とする場合、筋芽細胞はウシ、ブタ、ヒツジ、ヤギ、ウマ等の畜産のために飼育されるほ乳類動物に由来することが好ましく、ウシ由来であることがより好ましい。
Myoblasts are derived from vertebrates such as mammals, birds, reptiles, amphibians, and fish. Examples of mammals include non-human mammals such as monkeys, cows, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, and mice. Examples of avian animals include ostriches, chickens, ducks, and sparrows. Examples of reptile animals include snakes, crocodiles, lizards, and turtles. Amphibians include frogs, newts, salamanders, and the like. Examples of fish animals include salmon, tuna, shark, sea bream, and carp. When the three-dimensional muscle tissue is used as food, the myoblasts are preferably derived from mammals raised for livestock such as cows, pigs, sheep, goats, and horses, and more preferably from cows.
筋芽細胞は、相同組み替え法、ＣＲＩＳＰＲ／Ｃａｓ９法等のゲノム編集の手法等により遺伝子改変をされた筋芽細胞または遺伝子改変されていない筋芽細胞を用いることができる。三次元筋組織を食用とする場合の一つの態様においては、安全性及び消費者の嗜好の観点から、筋芽細胞は遺伝子改変されていない筋芽細胞を用いることが好ましい。
As myoblasts, myoblasts that have been genetically modified by homologous recombination, CRISPR/Cas9, or other genome editing methods, or myoblasts that have not been genetically modified can be used. In one embodiment when the three-dimensional muscle tissue is used as food, it is preferable to use myoblasts that have not been genetically modified from the viewpoint of safety and consumer preference.
（ハイドロゲル）
ハイドロゲルは、三次元筋組織の培養時の足場材として機能する。
ハイドロゲルの好ましい態様の１つとしては、フィブリン、フィブロネクチン、ラミニン、コラーゲン（例えば、Ｉ型、ＩＩ型、ＩＩＩ型、Ｖ型、ＸＩ型など）、寒天、アガロース、グリコサミノグリカン、ヒアルロン酸、プロテオグリカンなどを等の細胞外基底膜マトリックスを構成する成分のゲルを使用することができる。ハイドロゲルとして市販品を使用することもできる例えば、「マトリゲル」の商品名で販売されるマウスＥＨＳ腫瘍抽出物（ＩＶ型コラーゲン、ラミニン、ヘパラン硫酸プロテオグリカンなどを含む）に基づく成分を使用することができる。 (hydrogel)
The hydrogel functions as a scaffolding material during the culture of three-dimensional muscle tissue.
One of the preferred embodiments of the hydrogel includes fibrin, fibronectin, laminin, collagen (for example, type I, type II, type III, type V, type XI, etc.), agar, agarose, glycosaminoglycan, hyaluronic acid, A gel of components constituting the extracellular basement membrane matrix, such as proteoglycans, can be used. Commercial products can also be used as hydrogels. For example, components based on mouse EHS tumor extract (containing type IV collagen, laminin, heparan sulfate proteoglycans, etc.) sold under the trade name "Matrigel" can be used. can.
ハイドロゲルは、三次元筋組織の培養時の足場材として機能する。
ハイドロゲルの好ましい態様の１つとしては、フィブリン、フィブロネクチン、ラミニン、コラーゲン（例えば、Ｉ型、ＩＩ型、ＩＩＩ型、Ｖ型、ＸＩ型など）、寒天、アガロース、グリコサミノグリカン、ヒアルロン酸、プロテオグリカンなどを等の細胞外基底膜マトリックスを構成する成分のゲルを使用することができる。ハイドロゲルとして市販品を使用することもできる例えば、「マトリゲル」の商品名で販売されるマウスＥＨＳ腫瘍抽出物（ＩＶ型コラーゲン、ラミニン、ヘパラン硫酸プロテオグリカンなどを含む）に基づく成分を使用することができる。 (hydrogel)
The hydrogel functions as a scaffolding material during the culture of three-dimensional muscle tissue.
One of the preferred embodiments of the hydrogel includes fibrin, fibronectin, laminin, collagen (for example, type I, type II, type III, type V, type XI, etc.), agar, agarose, glycosaminoglycan, hyaluronic acid, A gel of components constituting the extracellular basement membrane matrix, such as proteoglycans, can be used. Commercial products can also be used as hydrogels. For example, components based on mouse EHS tumor extract (containing type IV collagen, laminin, heparan sulfate proteoglycans, etc.) sold under the trade name "Matrigel" can be used. can.
なお、本明細書において「コラーゲン」は、未変性のコラーゲン及び変性したコラーゲンを包含する。変性したコラーゲンとしては、ゼラチンが例示される。
Note that in this specification, "collagen" includes undenatured collagen and denatured collagen. An example of denatured collagen is gelatin.
ハイドロゲルの別の好ましい態様の１つとして、血液由来血漿及び凝固剤を含むゲルが挙げられる。
血液の由来動物は、入手容易性の観点から、ウシ、ブタ、ヒツジ、ヤギ、ウマ等の畜産のために飼育されるほ乳類動物に由来であり、より好ましくはウシ由来である。
由来動物は分娩前の胎児であっても、又は、分娩後の成体のいずれであってもよい。由来動物の日齢、月齢、年齢は限定されない。成体である場合、雌性個体から分娩された後の、幼い個体、若い個体、成熟した個体、老いた個体のいずれであってもよい。
好ましい態様において由来動物は成体のウシであり、中でも市場からの入手のしやすさ及び屠畜解体される量から、より好ましくは成熟したウシである。
成牛の血液は、食肉市場や卸売市場において屠畜解体される牛から検体として採取する方法や、その他の方法により得られる。 Another preferred embodiment of the hydrogel includes a gel containing blood-derived plasma and a coagulant.
From the viewpoint of availability, the animal from which the blood is derived is derived from a mammalian animal raised for livestock production such as a cow, pig, sheep, goat, or horse, and is more preferably derived from a cow.
The animal of origin may be a fetus before parturition or an adult after parturition. The age in days, months, and age of the source animal is not limited. In the case of an adult, it may be a young individual, a young individual, a mature individual, or an old individual after delivery from a female individual.
In a preferred embodiment, the source animal is an adult cow, and more preferably an adult cow from the viewpoint of easy availability on the market and the amount to be slaughtered and slaughtered.
Blood from adult cows can be obtained by collecting specimens from slaughtered cows at meat and wholesale markets, or by other methods.
血液の由来動物は、入手容易性の観点から、ウシ、ブタ、ヒツジ、ヤギ、ウマ等の畜産のために飼育されるほ乳類動物に由来であり、より好ましくはウシ由来である。
由来動物は分娩前の胎児であっても、又は、分娩後の成体のいずれであってもよい。由来動物の日齢、月齢、年齢は限定されない。成体である場合、雌性個体から分娩された後の、幼い個体、若い個体、成熟した個体、老いた個体のいずれであってもよい。
好ましい態様において由来動物は成体のウシであり、中でも市場からの入手のしやすさ及び屠畜解体される量から、より好ましくは成熟したウシである。
成牛の血液は、食肉市場や卸売市場において屠畜解体される牛から検体として採取する方法や、その他の方法により得られる。 Another preferred embodiment of the hydrogel includes a gel containing blood-derived plasma and a coagulant.
From the viewpoint of availability, the animal from which the blood is derived is derived from a mammalian animal raised for livestock production such as a cow, pig, sheep, goat, or horse, and is more preferably derived from a cow.
The animal of origin may be a fetus before parturition or an adult after parturition. The age in days, months, and age of the source animal is not limited. In the case of an adult, it may be a young individual, a young individual, a mature individual, or an old individual after delivery from a female individual.
In a preferred embodiment, the source animal is an adult cow, and more preferably an adult cow from the viewpoint of easy availability on the market and the amount to be slaughtered and slaughtered.
Blood from adult cows can be obtained by collecting specimens from slaughtered cows at meat and wholesale markets, or by other methods.
血液は、流通や保管時の凝固を防ぐため、抗凝固剤を添加してから配送することがある。抗凝固剤が添加された血液は、遠心分離により、赤血球と、フィブリノーゲンを含む血漿に分離される。ここで、血液に抗凝固剤が添加されている場合は、赤血球も、凝固成分となるフィブリノーゲンを含む血漿も、凝固及びゲル化しない。
To prevent blood from coagulating during distribution and storage, anticoagulants may be added to blood before delivery. Blood to which an anticoagulant has been added is separated into red blood cells and plasma containing fibrinogen by centrifugation. Here, if an anticoagulant is added to the blood, neither red blood cells nor plasma containing fibrinogen, which is a coagulation component, will coagulate or gel.
遠心分離により得られたフィブリノーゲンを含む血液由来血漿に、凝固剤を添加すると、血漿はゲル化する。
なお、本態様のハイドロゲルは、血漿を含むため、細胞培養時の栄養分たる細胞培養成分の補給機能も期待される。 When a coagulant is added to blood-derived plasma containing fibrinogen obtained by centrifugation, the plasma gels.
In addition, since the hydrogel of this embodiment contains plasma, it is also expected to have a function of replenishing cell culture components, which are nutrients during cell culture.
なお、本態様のハイドロゲルは、血漿を含むため、細胞培養時の栄養分たる細胞培養成分の補給機能も期待される。 When a coagulant is added to blood-derived plasma containing fibrinogen obtained by centrifugation, the plasma gels.
In addition, since the hydrogel of this embodiment contains plasma, it is also expected to have a function of replenishing cell culture components, which are nutrients during cell culture.
また、上記フィブリノーゲンを含む成牛血液由来血漿は、多血小板血漿（ＰＲＰ血漿；Ｐｌａｔｅｌｅｔ－ｒｉｃｈ Ｐｌａｓｍａ）であってもよい。多血小板血漿は、血漿を遠心分離して調製した、血小板に富む血漿濃縮物であり、血小板の他、可溶性タンパク質や成長因子が濃縮されている。
Further, the adult bovine blood-derived plasma containing fibrinogen may be platelet-rich plasma (PRP plasma). Platelet-rich plasma is a platelet-rich plasma concentrate prepared by centrifuging plasma, and is enriched with platelets as well as soluble proteins and growth factors.
多血小板血漿は、抗凝固剤が添加された血液の遠心分離、二重遠心分離、選択的濾過等で回収することができる。具体的には赤血球を除いた血漿をさらに遠心分離して回収してもよいし、赤血球も含まれる血液では血漿層のうち赤血球との境界に濃縮した層を回収してもよい。
Platelet-rich plasma can be collected by centrifugation, double centrifugation, selective filtration, etc. of blood to which an anticoagulant has been added. Specifically, plasma from which red blood cells have been removed may be further centrifuged and collected, or in the case of blood that also contains red blood cells, a layer of the plasma layer concentrated at the boundary with red blood cells may be collected.
多血小板血漿を用いた場合は、血漿全体を用いた場合に比べ、ゲル化が速やかに進む傾向にある。このため、多血小板血漿を用いた細胞培養用ゲルでは、細胞の沈殿が起こりにくく、細胞をより均一に分散させることができる。
When platelet-rich plasma is used, gelation tends to proceed more quickly than when the whole plasma is used. Therefore, in cell culture gels using platelet-rich plasma, cell precipitation is less likely to occur, and cells can be more uniformly dispersed.
抗凝固剤は、血液の凝固を防ぐものであれば限定されない。抗凝固剤としては、クエン酸ナトリウム、ＥＤＴＡ（エチレンジアミン四酢酸）、フッ化ナトリウム等の、血液の凝固に不可欠なカルシウムイオンと結合するものが例示される。
クエン酸ナトリウムの場合、血液及び抗凝固剤全量に対し、２体積％～４体積％、好ましくは、３体積％前後となるように添加することがより望ましい。 The anticoagulant is not limited as long as it prevents blood coagulation. Examples of anticoagulants include those that bind to calcium ions essential for blood coagulation, such as sodium citrate, EDTA (ethylenediaminetetraacetic acid), and sodium fluoride.
In the case of sodium citrate, it is more desirable to add it in an amount of 2% to 4% by volume, preferably around 3% by volume, based on the total amount of blood and anticoagulant.
クエン酸ナトリウムの場合、血液及び抗凝固剤全量に対し、２体積％～４体積％、好ましくは、３体積％前後となるように添加することがより望ましい。 The anticoagulant is not limited as long as it prevents blood coagulation. Examples of anticoagulants include those that bind to calcium ions essential for blood coagulation, such as sodium citrate, EDTA (ethylenediaminetetraacetic acid), and sodium fluoride.
In the case of sodium citrate, it is more desirable to add it in an amount of 2% to 4% by volume, preferably around 3% by volume, based on the total amount of blood and anticoagulant.
凝固剤は、抗凝固剤を含む血液の血漿をゲル化できるものであれば限定されない。凝固剤としては、塩化カルシウム、ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ Ｍｅｄｉｕｍ）等が例示され、カルシウムイオンを含有するものがより好ましい。
The coagulant is not limited as long as it can gel blood plasma containing an anticoagulant. Examples of the coagulant include calcium chloride and DMEM (Dulbecco's Modified Eagle's Medium), with those containing calcium ions being more preferred.
凝固剤が塩化カルシウムの場合、血漿及び凝固剤全量に対し、カルシウムイオン濃度として例えば、好ましくは５ｍＭ～７０ｍＭ、より好ましくは１０ｍＭ～６５ｍＭ、さらに好ましくは１０ｍＭ～６０ｍＭとなるように添加することができる。
なお、本明細書において。「ＸＸ～ＹＹ」は、「ＸＸ以上ＹＹ以下」を意味する。 When the coagulant is calcium chloride, it can be added to the total amount of plasma and coagulant so that the calcium ion concentration is, for example, preferably 5mM to 70mM, more preferably 10mM to 65mM, even more preferably 10mM to 60mM. .
Note that in this specification. "XX to YY" means "more than or equal to XX and less than or equal to YY."
なお、本明細書において。「ＸＸ～ＹＹ」は、「ＸＸ以上ＹＹ以下」を意味する。 When the coagulant is calcium chloride, it can be added to the total amount of plasma and coagulant so that the calcium ion concentration is, for example, preferably 5mM to 70mM, more preferably 10mM to 65mM, even more preferably 10mM to 60mM. .
Note that in this specification. "XX to YY" means "more than or equal to XX and less than or equal to YY."
ＤＭＥＭには、Ｌ－アルギニン、Ｌ－シスチン、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－ロイシン、Ｌ－リシン、Ｌ－フェニルアラニン、Ｌ－トレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、塩化カルシウム、塩化カリウム、硫酸マグネシウム、ナトリウム塩化物、リン酸二水素ナトリウム、Ｄ－グルコース、葉酸、ニコチンアミド、リボフラビン、ビタミンＢ１２、コリン、イノシトール、パントテン酸、ピリドキサールリン酸、チアミン、鉄等が含まれる。
凝固剤がＤＭＥＭの場合、ＤＭＥＭの量に対し、血液由来血漿の量を、例えば好ましくは０．１体積％以上３５体積％以下、より好ましくは０．１体積％以上３０体積％以下、さらに好ましくは０．２体積％以上３０体積％以下となるように添加することができる。 DMEM contains L-arginine, L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and calcium chloride. , potassium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate, D-glucose, folic acid, nicotinamide, riboflavin, vitamin B12, choline, inositol, pantothenic acid, pyridoxal phosphate, thiamine, iron, and the like.
When the coagulant is DMEM, the amount of blood-derived plasma is preferably 0.1 volume% or more and 35 volume% or less, more preferably 0.1 volume% or more and 30 volume% or less, and even more preferably 0.1 volume% or more and 30 volume% or less, relative to the amount of DMEM. can be added in an amount of 0.2% by volume or more and 30% by volume or less.
凝固剤がＤＭＥＭの場合、ＤＭＥＭの量に対し、血液由来血漿の量を、例えば好ましくは０．１体積％以上３５体積％以下、より好ましくは０．１体積％以上３０体積％以下、さらに好ましくは０．２体積％以上３０体積％以下となるように添加することができる。 DMEM contains L-arginine, L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and calcium chloride. , potassium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate, D-glucose, folic acid, nicotinamide, riboflavin, vitamin B12, choline, inositol, pantothenic acid, pyridoxal phosphate, thiamine, iron, and the like.
When the coagulant is DMEM, the amount of blood-derived plasma is preferably 0.1 volume% or more and 35 volume% or less, more preferably 0.1 volume% or more and 30 volume% or less, and even more preferably 0.1 volume% or more and 30 volume% or less, relative to the amount of DMEM. can be added in an amount of 0.2% by volume or more and 30% by volume or less.
ハイドロゲルは、さらに培地成分を含んでもよい。
培地成分としては、ＤＭＥＭ（例えば、ＧＩＢＣＯ社製等）や、ＥＭＥＭ（例えば、ＧＩＢＣＯ社製）、ＭＥＭＡＬＰＨＡ（例えば、ＧＩＢＣＯ社製）、ＲＰＭＩ－１６４０（Ｒｏｓｗｅｌｌ Ｐａｒｋ Ｍｅｍｏｒｉａｌ Ｉｎｓｔｉｔｕｔｅ １６４０培地；ＧＩＢＣＯ社製）等が挙げられる。
さらに培地成分には、通常培地で使用する添加剤成分を適宜配合することができる。添加剤成分としては、抗生物質の他、ビタミン類、核酸、アミノ酸、無機塩、糖、ポリアミン、炭水化物、タンパク質、脂肪酸、脂質、ｐＨ調整剤、亜鉛、銅、セレン等が例示される。 The hydrogel may further include a medium component.
Medium components include DMEM (for example, manufactured by GIBCO), EMEM (for example, manufactured by GIBCO), MEMALPHA (for example, manufactured by GIBCO), RPMI-1640 (Roswell Park Memorial Institute 1640 medium; manufactured by GIBCO), etc. can be mentioned.
Furthermore, additive components commonly used in culture media can be appropriately blended into the culture medium components. Examples of additive components include antibiotics, vitamins, nucleic acids, amino acids, inorganic salts, sugars, polyamines, carbohydrates, proteins, fatty acids, lipids, pH adjusters, zinc, copper, selenium, and the like.
培地成分としては、ＤＭＥＭ（例えば、ＧＩＢＣＯ社製等）や、ＥＭＥＭ（例えば、ＧＩＢＣＯ社製）、ＭＥＭＡＬＰＨＡ（例えば、ＧＩＢＣＯ社製）、ＲＰＭＩ－１６４０（Ｒｏｓｗｅｌｌ Ｐａｒｋ Ｍｅｍｏｒｉａｌ Ｉｎｓｔｉｔｕｔｅ １６４０培地；ＧＩＢＣＯ社製）等が挙げられる。
さらに培地成分には、通常培地で使用する添加剤成分を適宜配合することができる。添加剤成分としては、抗生物質の他、ビタミン類、核酸、アミノ酸、無機塩、糖、ポリアミン、炭水化物、タンパク質、脂肪酸、脂質、ｐＨ調整剤、亜鉛、銅、セレン等が例示される。 The hydrogel may further include a medium component.
Medium components include DMEM (for example, manufactured by GIBCO), EMEM (for example, manufactured by GIBCO), MEMALPHA (for example, manufactured by GIBCO), RPMI-1640 (Roswell Park Memorial Institute 1640 medium; manufactured by GIBCO), etc. can be mentioned.
Furthermore, additive components commonly used in culture media can be appropriately blended into the culture medium components. Examples of additive components include antibiotics, vitamins, nucleic acids, amino acids, inorganic salts, sugars, polyamines, carbohydrates, proteins, fatty acids, lipids, pH adjusters, zinc, copper, selenium, and the like.
ハイドロゲルにおける筋芽細胞は、例えば細胞密度が好ましくは約１．０×１０６個／ｍＬ以上、より好ましくは約１．０×１０７個／ｍＬ～約１．０×１０８個／ｍＬ、さらに好ましくは５．０×１０７個／ｍＬ～約１．０×１０８個／ｍＬである。
For example, the myoblasts in the hydrogel preferably have a cell density of about 1.0 x 10 6 cells/mL or more, more preferably about 1.0 x 10 7 cells/mL to about 1.0 x 10 8 cells/mL. , more preferably 5.0×10 7 cells/mL to about 1.0×10 8 cells/mL.
（培養容器）
本発明で使用する培養容器は、三次元筋組織を製造するための培養容器であって、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器である。
該凸部を備えることで、培養時に筋管及び筋線維の束の配向が効率的に形成されると考えられる。 (Culture container)
The culture container used in the present invention is a culture container for producing three-dimensional muscle tissue, and includes a pair of anchor portions facing each other at the ends of the container, and a plurality of convex portions arranged between the pair of anchor portions. This is a culture container.
It is thought that by providing the convex portion, the orientation of myotubes and myofiber bundles is efficiently formed during culture.
本発明で使用する培養容器は、三次元筋組織を製造するための培養容器であって、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器である。
該凸部を備えることで、培養時に筋管及び筋線維の束の配向が効率的に形成されると考えられる。 (Culture container)
The culture container used in the present invention is a culture container for producing three-dimensional muscle tissue, and includes a pair of anchor portions facing each other at the ends of the container, and a plurality of convex portions arranged between the pair of anchor portions. This is a culture container.
It is thought that by providing the convex portion, the orientation of myotubes and myofiber bundles is efficiently formed during culture.
本発明を実施するための好適な実施形態について、図面を用いて説明する。
図１は、本実施形態に係る培養容器１００の構成例の斜視図を示す。図２は、培養容器１００の断面図を示す。 Preferred embodiments for carrying out the present invention will be described with reference to the drawings.
FIG. 1 shows a perspective view of a configuration example of aculture container 100 according to the present embodiment. FIG. 2 shows a cross-sectional view of the culture container 100.
図１は、本実施形態に係る培養容器１００の構成例の斜視図を示す。図２は、培養容器１００の断面図を示す。 Preferred embodiments for carrying out the present invention will be described with reference to the drawings.
FIG. 1 shows a perspective view of a configuration example of a
培養容器１００を構成する素材は限定されない。例えば、培養容器１００を構成する素材は熱可塑性樹脂又は熱硬化性樹脂である。熱可塑性樹脂としては、ポリプロピレン等のポリオレフィン系樹脂；ポリエチレンテレフタレート等のポリエステル系樹脂；アクリル系樹脂；熱可塑性エラストマー：ポリジメチルシロキサン（ＰＤＭＳ）等のシリコーン系樹脂等が挙げられる。
また、培養容器１００を構成する素材は、好ましくは細胞に対して非接着性の材質又は非接着性となる表面処理が施されている素材である。非接着性となる表面処理としては、パリレンコーティングが挙げられる。
培養容器１００を構成する素材は、透明又は不透明のいずれであってもよい。観察の容易性の観点から、好ましくは透明である。
培養容器１００は、例えば３Ｄプリンターを用いて製造することができる。 The material constituting theculture container 100 is not limited. For example, the material constituting the culture container 100 is a thermoplastic resin or a thermosetting resin. Examples of the thermoplastic resin include polyolefin resins such as polypropylene; polyester resins such as polyethylene terephthalate; acrylic resins; thermoplastic elastomers: silicone resins such as polydimethylsiloxane (PDMS);
Further, the material constituting theculture container 100 is preferably a material that is non-adhesive to cells or a material that has been surface-treated to become non-adhesive. Non-adhesive surface treatments include parylene coatings.
The material constituting theculture container 100 may be transparent or opaque. From the viewpoint of ease of observation, it is preferably transparent.
Theculture container 100 can be manufactured using, for example, a 3D printer.
また、培養容器１００を構成する素材は、好ましくは細胞に対して非接着性の材質又は非接着性となる表面処理が施されている素材である。非接着性となる表面処理としては、パリレンコーティングが挙げられる。
培養容器１００を構成する素材は、透明又は不透明のいずれであってもよい。観察の容易性の観点から、好ましくは透明である。
培養容器１００は、例えば３Ｄプリンターを用いて製造することができる。 The material constituting the
Further, the material constituting the
The material constituting the
The
培養容器は好ましくは四角形である。この場合、前記凸部の長手方向の長さ（Ｘ）が好ましくは１ｃｍ以上、より好ましくは１．５ｃｍ以上、かつ、前記凸部の短手方向の長さ（Ｙ）が好ましくは１ｃｍ以上、より好ましくは２ｃｍ以上である。凸部の長手方向とは、後述する一対のアンカー部が向かい合う方向であり、凸部の短手方向とは凸部が配列される方向である。
また、前記凸部の長手方向の長さ（Ｘ）に対する前記凸部の短手方向の長さ（Ｙ）の比〔（Ｙ）／（Ｘ）〕は、好ましくは０．７以上である。〔（Ｙ）／（Ｘ）〕は、１以上、１．５以上、２以上とすることもできる。上限は特に限定されず、例えば４以下である。 The culture vessel is preferably rectangular. In this case, the length (X) of the protrusion in the longitudinal direction is preferably 1 cm or more, more preferably 1.5 cm or more, and the length (Y) of the protrusion in the lateral direction is preferably 1 cm or more, More preferably, it is 2 cm or more. The longitudinal direction of the convex portion is the direction in which a pair of anchor portions, which will be described later, face each other, and the lateral direction of the convex portion is the direction in which the convex portions are arranged.
Further, the ratio [(Y)/(X)] of the length (Y) of the convex portion in the transverse direction to the length (X) of the convex portion in the longitudinal direction is preferably 0.7 or more. [(Y)/(X)] can also be 1 or more, 1.5 or more, or 2 or more. The upper limit is not particularly limited, and is, for example, 4 or less.
また、前記凸部の長手方向の長さ（Ｘ）に対する前記凸部の短手方向の長さ（Ｙ）の比〔（Ｙ）／（Ｘ）〕は、好ましくは０．７以上である。〔（Ｙ）／（Ｘ）〕は、１以上、１．５以上、２以上とすることもできる。上限は特に限定されず、例えば４以下である。 The culture vessel is preferably rectangular. In this case, the length (X) of the protrusion in the longitudinal direction is preferably 1 cm or more, more preferably 1.5 cm or more, and the length (Y) of the protrusion in the lateral direction is preferably 1 cm or more, More preferably, it is 2 cm or more. The longitudinal direction of the convex portion is the direction in which a pair of anchor portions, which will be described later, face each other, and the lateral direction of the convex portion is the direction in which the convex portions are arranged.
Further, the ratio [(Y)/(X)] of the length (Y) of the convex portion in the transverse direction to the length (X) of the convex portion in the longitudinal direction is preferably 0.7 or more. [(Y)/(X)] can also be 1 or more, 1.5 or more, or 2 or more. The upper limit is not particularly limited, and is, for example, 4 or less.
培養容器１００は、後述する一対のアンカー部２０間にわたって複数の凸部が配列してなる。
アンカー部の形状は限定されない。例えば、培養容器の上部からの平面視の形状が略長方形である。 Theculture container 100 has a plurality of convex portions arranged between a pair of anchor portions 20, which will be described later.
The shape of the anchor part is not limited. For example, the culture container has a substantially rectangular shape when viewed from above.
アンカー部の形状は限定されない。例えば、培養容器の上部からの平面視の形状が略長方形である。 The
The shape of the anchor part is not limited. For example, the culture container has a substantially rectangular shape when viewed from above.
複数の凸部が配列する態様は限定されない。好ましくは、複数の凸部は底部に互いに平行となるように設けられている。
凸部１０は、好ましくは高さ５０μｍ～１ｍｍ、より好ましくは１００μｍ～３００μｍである。
また、前記凸部の互いの間隔が、１０μｍ～５ｍｍ、より好ましくは５０μｍ～２．５ｍｍ、更に好ましくは１００μｍ～１ｍｍである。 The manner in which the plurality of convex portions are arranged is not limited. Preferably, the plurality of convex portions are provided on the bottom so as to be parallel to each other.
The height of theconvex portion 10 is preferably 50 μm to 1 mm, more preferably 100 μm to 300 μm.
Further, the distance between the convex portions is 10 μm to 5 mm, more preferably 50 μm to 2.5 mm, and even more preferably 100 μm to 1 mm.
凸部１０は、好ましくは高さ５０μｍ～１ｍｍ、より好ましくは１００μｍ～３００μｍである。
また、前記凸部の互いの間隔が、１０μｍ～５ｍｍ、より好ましくは５０μｍ～２．５ｍｍ、更に好ましくは１００μｍ～１ｍｍである。 The manner in which the plurality of convex portions are arranged is not limited. Preferably, the plurality of convex portions are provided on the bottom so as to be parallel to each other.
The height of the
Further, the distance between the convex portions is 10 μm to 5 mm, more preferably 50 μm to 2.5 mm, and even more preferably 100 μm to 1 mm.
本発明で使用する培養容器は、容器端部に向かい合う一対のアンカー部を備える。アンカー部は、ハイドロゲル及び製造される三次元筋組織を固定する手段であれば、特に限定されない。例えば、接着力を有する成分（例えば、フィブリン等のハイドロゲル）を用いて固定する部材が挙げられる。
図１に示す好ましい態様である培養容器１００は、容器端部のそれぞれに複数の円柱状からなるのアンカー部２０が向かい合っている。円柱状のアンカー部２０は、直径が１００μｍ～２ｍｍ程度、高さが３ｍｍ～１０ｍｍ程度である。 The culture container used in the present invention includes a pair of anchor portions facing each other at the ends of the container. The anchor part is not particularly limited as long as it is a means for fixing the hydrogel and the manufactured three-dimensional muscle tissue. For example, a member that is fixed using a component having adhesive strength (for example, hydrogel such as fibrin) can be mentioned.
In the preferred embodiment of theculture container 100 shown in FIG. 1, a plurality of cylindrical anchor portions 20 face each other at each end of the container. The cylindrical anchor portion 20 has a diameter of approximately 100 μm to 2 mm and a height of approximately 3 mm to 10 mm.
図１に示す好ましい態様である培養容器１００は、容器端部のそれぞれに複数の円柱状からなるのアンカー部２０が向かい合っている。円柱状のアンカー部２０は、直径が１００μｍ～２ｍｍ程度、高さが３ｍｍ～１０ｍｍ程度である。 The culture container used in the present invention includes a pair of anchor portions facing each other at the ends of the container. The anchor part is not particularly limited as long as it is a means for fixing the hydrogel and the manufactured three-dimensional muscle tissue. For example, a member that is fixed using a component having adhesive strength (for example, hydrogel such as fibrin) can be mentioned.
In the preferred embodiment of the
図４～９は、他の実施形態に係る培養容器２００、培養容器３００、及び培養容器４００の構成例を斜視図、並びに、平面図、正面図、右側面図及び底面図により示す。
FIGS. 4 to 9 show configuration examples of a culture container 200, a culture container 300, and a culture container 400 according to other embodiments using a perspective view, a top view, a front view, a right side view, and a bottom view.
（ハイドロゲルの供給）
筋芽細胞を含むハイドロゲルは、上記培養容器に供給される。好ましくは、培養容器を底面とし、側面に型枠を設けて、筋芽細胞を含むハイドロゲルを供給する。
供給する筋芽細胞を含むハイドロゲルの量は適宜設定することができる。例えば、ハイドロゲルの厚さが好ましくは１ｍｍ～１ｃｍ、より好ましくは２～５ｍｍとなる量とすることができる。 (Hydrogel supply)
A hydrogel containing myoblasts is supplied to the culture vessel. Preferably, a culture container is used as the bottom surface, a mold is provided on the side surface, and the hydrogel containing myoblasts is supplied.
The amount of hydrogel containing myoblasts to be supplied can be set appropriately. For example, the amount may be such that the thickness of the hydrogel is preferably 1 mm to 1 cm, more preferably 2 to 5 mm.
筋芽細胞を含むハイドロゲルは、上記培養容器に供給される。好ましくは、培養容器を底面とし、側面に型枠を設けて、筋芽細胞を含むハイドロゲルを供給する。
供給する筋芽細胞を含むハイドロゲルの量は適宜設定することができる。例えば、ハイドロゲルの厚さが好ましくは１ｍｍ～１ｃｍ、より好ましくは２～５ｍｍとなる量とすることができる。 (Hydrogel supply)
A hydrogel containing myoblasts is supplied to the culture vessel. Preferably, a culture container is used as the bottom surface, a mold is provided on the side surface, and the hydrogel containing myoblasts is supplied.
The amount of hydrogel containing myoblasts to be supplied can be set appropriately. For example, the amount may be such that the thickness of the hydrogel is preferably 1 mm to 1 cm, more preferably 2 to 5 mm.
培養容器に供給されたハイドロゲルは、加温し固化することができる。加温の温度範囲は、好ましくは３７℃程度である。また、加温時間は、ゲル化の進み具合で調整することができ、５分間～６０分間程度が例示され、より好ましくは約１０分間程度である。
The hydrogel supplied to the culture container can be heated and solidified. The temperature range for heating is preferably about 37°C. Further, the heating time can be adjusted depending on the progress of gelation, and is exemplified to be about 5 minutes to 60 minutes, and more preferably about 10 minutes.
また、筋芽細胞を含むハイドロゲルは、増殖培養に供し、含まれる筋芽細胞を細胞増殖させることができる。
例えば、筋芽細胞がハイドロゲルに十分量（例えば、１．０×１０８個／ｍＬ以上）含まれる場合、増殖培養を行うことなく次の分化誘導の工程を行うことができる。例えば、筋芽細胞を増殖させる必要がある場合は、増殖培養を行った後に次の分化誘導の工程を行うことができる。 Furthermore, the hydrogel containing myoblasts can be subjected to proliferation culture to allow the myoblasts contained therein to proliferate.
For example, if the hydrogel contains a sufficient amount of myoblasts (for example, 1.0×10 8 cells/mL or more), the next step of inducing differentiation can be performed without performing proliferation culture. For example, if it is necessary to proliferate myoblasts, the next step of inducing differentiation can be performed after proliferation culture.
例えば、筋芽細胞がハイドロゲルに十分量（例えば、１．０×１０８個／ｍＬ以上）含まれる場合、増殖培養を行うことなく次の分化誘導の工程を行うことができる。例えば、筋芽細胞を増殖させる必要がある場合は、増殖培養を行った後に次の分化誘導の工程を行うことができる。 Furthermore, the hydrogel containing myoblasts can be subjected to proliferation culture to allow the myoblasts contained therein to proliferate.
For example, if the hydrogel contains a sufficient amount of myoblasts (for example, 1.0×10 8 cells/mL or more), the next step of inducing differentiation can be performed without performing proliferation culture. For example, if it is necessary to proliferate myoblasts, the next step of inducing differentiation can be performed after proliferation culture.
上記培養は、例えば上記の増殖培養用の培地中で、当業者に公知の手法で行うことができる。好適な培養を行う手法として、約３７℃程度および二酸化炭素濃度約５～１０％（ｖ／ｖ）程度の条件下で培養する手法が例示されるが、これに限定されるものではない。上記条件での培養は、例えば公知のＣＯ２インキュベータを用いて行うことができる。
The above-mentioned culture can be performed, for example, in the above-mentioned growth culture medium by a method known to those skilled in the art. A suitable culture method includes, but is not limited to, a method of culturing at about 37° C. and a carbon dioxide concentration of about 5 to 10% (v/v). Cultivation under the above conditions can be performed using, for example, a known CO 2 incubator.
増殖培養用の培地としては、ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ Ｍｅｄｉｕｍ）、ＥＭＥＭ（Ｅａｇｌｅ’ｓ ｍｉｎｉｍａｌ ｅｓｓｅｎｔｉａｌ ｍｅｄｉｕｍ）、αＭＥＭ（ａｌｐｈａ Ｍｏｄｉｆｉｅｄ Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ）などの通常の液体培地に、血清成分（例えば、ウマ血清（Ｈｏｒｓｅ Ｓｅｒｕｍ）ウシ胎児血清（Ｆｅｔａｌ Ｂｏｖｉｎｅ Ｓｅｒｕｍ（ＦＢＳ））、ヒト血清（Ｈｕｍａｎ Ｓｅｒｕｍ）など）、成長因子等の成分；ペニシリン、ストレプトマイシン等の抗生物質を添加した培地を使用することができる。
As a culture medium for proliferation culture, DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle's minimal essential medium), αMEM (alpha Modified Add serum components (e.g. Components such as horse serum (Horse serum, Fetal bovine serum (FBS), human serum, etc.), growth factors; It is possible to use a medium supplemented with antibiotics such as penicillin and streptomycin. can.
増殖培養用の培地に血清成分を添加する場合、血清成分としてはウシ胎児血清を用いることができる。血清成分の濃度は１０％（ｖ／ｖ）程度とすることができる。
When adding a serum component to the growth culture medium, fetal bovine serum can be used as the serum component. The concentration of serum components can be about 10% (v/v).
培養期間は、例えば、１日間～２週間程度とすることができる。
The culture period can be, for example, about 1 day to 2 weeks.
必要に応じて、培地交換を行うことができる。培養条件は、常法に準じることができる。
The culture medium can be replaced if necessary. Culture conditions can be according to conventional methods.
＜工程（Ｂ）＞
工程（Ｂ）は、前記培養容器中で、筋芽細胞を筋管へと分化誘導する工程である。
当該工程により、筋芽細胞は周囲の細胞と細胞融合により多核化し、筋管が形成される。筋管はさらに成熟することで筋線維を形成する。 <Process (B)>
Step (B) is a step of inducing differentiation of myoblasts into myotubes in the culture vessel.
Through this step, myoblasts become multinucleated by cell fusion with surrounding cells, and myotubes are formed. Myotubes further mature to form muscle fibers.
工程（Ｂ）は、前記培養容器中で、筋芽細胞を筋管へと分化誘導する工程である。
当該工程により、筋芽細胞は周囲の細胞と細胞融合により多核化し、筋管が形成される。筋管はさらに成熟することで筋線維を形成する。 <Process (B)>
Step (B) is a step of inducing differentiation of myoblasts into myotubes in the culture vessel.
Through this step, myoblasts become multinucleated by cell fusion with surrounding cells, and myotubes are formed. Myotubes further mature to form muscle fibers.
上記培養は、例えば分化誘導用（多核化用培地）の培地中で、当業者に公知の手法で行うことができる。好適な培養を行う手法として、約３７℃程度および二酸化炭素濃度約５～１０％（ｖ／ｖ）程度の条件下で培養する手法が例示されるが、これに限定されるものではない。上記条件での培養は、例えば公知のＣＯ２インキュベータを用いて行うことができる。
The above culture can be performed, for example, in a medium for differentiation induction (multinucleation medium) by a method known to those skilled in the art. Examples of suitable culturing methods include, but are not limited to, culturing at a temperature of about 37° C. and a carbon dioxide concentration of about 5 to 10% (v/v). Cultivation under the above conditions can be performed using, for example, a known CO 2 incubator.
筋芽細胞は栄養分が少なくなると，周囲の細胞を巻きこみ多核化を開始することが知られている。そのため、筋管への分化誘導は、前記の増殖培養よりも栄養分が少ない培地を用いて行うことができる。
It is known that when myoblasts become depleted of nutrients, they engulf surrounding cells and begin to become multinucleated. Therefore, induction of differentiation into myotubes can be performed using a medium containing fewer nutrients than the aforementioned proliferation culture.
かくして三次元筋組織が製造される。
本発明は、上記の製造方法により得られる三次元筋組織にも関する。 A three-dimensional muscle tissue is thus produced.
The present invention also relates to a three-dimensional muscle tissue obtained by the above manufacturing method.
本発明は、上記の製造方法により得られる三次元筋組織にも関する。 A three-dimensional muscle tissue is thus produced.
The present invention also relates to a three-dimensional muscle tissue obtained by the above manufacturing method.
次に実施例により本発明を更に具体的に説明する。しかし下記の実施例は本発明の範囲を限定するものではない。
Next, the present invention will be explained in more detail with reference to Examples. However, the following examples are not intended to limit the scope of the invention.
実施例１：ウシ筋芽細胞を用いた収縮可能な大型筋組織の作製
ウシ筋芽細胞を下記に示す組成のハイドロゲルに包埋し、両端を固定し培養することにより長さ７ｍｍの筋組織を作製した。ハイドロゲル組成および細胞密度を変化させ、筋組織の形成条件並びに成熟条件を検討した。 Example 1: Preparation of large contractile muscle tissue using bovine myoblasts Bovine myoblasts were embedded in a hydrogel with the composition shown below, and both ends were fixed and cultured to produce a muscle tissue with a length of 7 mm. was created. We investigated the formation and maturation conditions of muscle tissue by changing the hydrogel composition and cell density.
ウシ筋芽細胞を下記に示す組成のハイドロゲルに包埋し、両端を固定し培養することにより長さ７ｍｍの筋組織を作製した。ハイドロゲル組成および細胞密度を変化させ、筋組織の形成条件並びに成熟条件を検討した。 Example 1: Preparation of large contractile muscle tissue using bovine myoblasts Bovine myoblasts were embedded in a hydrogel with the composition shown below, and both ends were fixed and cultured to produce a muscle tissue with a length of 7 mm. was created. We investigated the formation and maturation conditions of muscle tissue by changing the hydrogel composition and cell density.
（培養容器１００の作製）
３Ｄプリンター（ＢＬＵＬＥ Ｉｎｃ社製、Ｆｏｒｍｌａｂ３Ｂ）を用いて図１０に示す培養容器１００を作成した。培養容器１００は凸部の長手方向の長さ（Ｘ）が４ｃｍ、凸部の短手方向の長さ（Ｙ）が３ｃｍであった。筋細胞の配向を促すための凸部１０は高さが２００μｍ及び幅５μｍであり、アンカー部と２ｍｍ離し、長手方向の長さが３ｃｍであった。
アンカー部２０は、直径０．５ｍｍ、高さ５ｍｍの円柱状であり、中心点の間隔が１ｍｍとなるように設けた。
作成した培養容器の表面を、パリレン蒸着装置（Ｓｐｅｃｉａｌｉｔｙ Ｃｏａｔｉｎｇ Ｓｙｓｔｅｍ社製、ラボコーターＰＤＳ２０１０）を用いて表面をパリレンコーティングした。次いで、オゾン滅菌装置を用いて培養容器を滅菌後、細胞接着を促すためアンカー部２０にフィブロネクチンコーティングを施した。 (Preparation of culture container 100)
Aculture container 100 shown in FIG. 10 was created using a 3D printer (Formlab 3B, manufactured by BLULE Inc.). In the culture container 100, the length (X) of the protrusion in the longitudinal direction was 4 cm, and the length (Y) of the protrusion in the transverse direction was 3 cm. The convex portion 10 for promoting the orientation of muscle cells had a height of 200 μm and a width of 5 μm, was separated from the anchor portion by 2 mm, and had a longitudinal length of 3 cm.
Theanchor part 20 had a cylindrical shape with a diameter of 0.5 mm and a height of 5 mm, and was provided so that the distance between the center points was 1 mm.
The surface of the created culture container was coated with parylene using a parylene vapor deposition device (Lab coater PDS2010, manufactured by Specialty Coating Systems). Next, after sterilizing the culture container using an ozone sterilizer, theanchor portion 20 was coated with fibronectin to promote cell adhesion.
３Ｄプリンター（ＢＬＵＬＥ Ｉｎｃ社製、Ｆｏｒｍｌａｂ３Ｂ）を用いて図１０に示す培養容器１００を作成した。培養容器１００は凸部の長手方向の長さ（Ｘ）が４ｃｍ、凸部の短手方向の長さ（Ｙ）が３ｃｍであった。筋細胞の配向を促すための凸部１０は高さが２００μｍ及び幅５μｍであり、アンカー部と２ｍｍ離し、長手方向の長さが３ｃｍであった。
アンカー部２０は、直径０．５ｍｍ、高さ５ｍｍの円柱状であり、中心点の間隔が１ｍｍとなるように設けた。
作成した培養容器の表面を、パリレン蒸着装置（Ｓｐｅｃｉａｌｉｔｙ Ｃｏａｔｉｎｇ Ｓｙｓｔｅｍ社製、ラボコーターＰＤＳ２０１０）を用いて表面をパリレンコーティングした。次いで、オゾン滅菌装置を用いて培養容器を滅菌後、細胞接着を促すためアンカー部２０にフィブロネクチンコーティングを施した。 (Preparation of culture container 100)
A
The
The surface of the created culture container was coated with parylene using a parylene vapor deposition device (Lab coater PDS2010, manufactured by Specialty Coating Systems). Next, after sterilizing the culture container using an ozone sterilizer, the
（対照培養容器の作製）
凸部を設けない以外は培養容器１００と同様にして、対照培養容器を作成した。 (Preparation of control culture container)
A control culture container was created in the same manner asculture container 100 except that no convex portion was provided.
凸部を設けない以外は培養容器１００と同様にして、対照培養容器を作成した。 (Preparation of control culture container)
A control culture container was created in the same manner as
（ハイドロゲル組成（１））
フィブリノーゲン（２５ｍｇ／ｍＬ） ４８０μＬ（終濃度４ｍｇ／ｍＬ）（Ｓｉｇｍａ社製、Ｆ８６３０）
マトリゲル ６００μＬ（終濃度２０％）（Ｃｏｒｎｉｎｇ社製、３５６２３１）
Ｔｈｒｏｍｂｉｎ（２００Ｕｎｉｔ／ｍＬ） ３０μＬ（終濃度２Ｕｎｉｔ／ｍＬ）（Ｓｉｇｍａ社製、Ｔ４６４８－１０ＫＵ）
ＤＭＥＭ １８９０μＬ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製、１１９６５１１８） (Hydrogel composition (1))
Fibrinogen (25mg/mL) 480μL (final concentration 4mg/mL) (Sigma, F8630)
Matrigel 600 μL (final concentration 20%) (Corning, 356231)
Thrombin (200 Unit/mL) 30 μL (final concentration 2 Unit/mL) (manufactured by Sigma, T4648-10KU)
DMEM 1890 μL (manufactured by Thermo Fisher Scientific, 11965118)
フィブリノーゲン（２５ｍｇ／ｍＬ） ４８０μＬ（終濃度４ｍｇ／ｍＬ）（Ｓｉｇｍａ社製、Ｆ８６３０）
マトリゲル ６００μＬ（終濃度２０％）（Ｃｏｒｎｉｎｇ社製、３５６２３１）
Ｔｈｒｏｍｂｉｎ（２００Ｕｎｉｔ／ｍＬ） ３０μＬ（終濃度２Ｕｎｉｔ／ｍＬ）（Ｓｉｇｍａ社製、Ｔ４６４８－１０ＫＵ）
ＤＭＥＭ １８９０μＬ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製、１１９６５１１８） (Hydrogel composition (1))
Fibrinogen (25mg/mL) 480μL (final concentration 4mg/mL) (Sigma, F8630)
Matrigel 600 μL (
Thrombin (200 Unit/mL) 30 μL (
DMEM 1890 μL (manufactured by Thermo Fisher Scientific, 11965118)
（ハイドロゲルの作製）
筋芽細胞３．０×１０７ｃｅｌｌｓを、１．０×１０７ｃｅｌｌｓ／ｍＬとなるようにハイドロゲルと混合し、細胞を包埋したハイドロゲルを作製した。培養容器１００又は対照培養容器を底面とし、側面に型枠を設けて、そこに得られた細胞を包埋したハイドロゲルを流し込んだ。３７℃、３０分間、ＣＯ２インキュベータ内で静置し、ハイドロゲルを固化した。 (Preparation of hydrogel)
3.0×10 7 cells of myoblasts were mixed with the hydrogel at 1.0×10 7 cells/mL to prepare a hydrogel in which the cells were embedded. Theculture container 100 or the control culture container was used as the bottom, a mold was provided on the side, and the obtained hydrogel in which cells were embedded was poured into the mold. The hydrogel was left standing in a CO 2 incubator at 37° C. for 30 minutes to solidify the hydrogel.
筋芽細胞３．０×１０７ｃｅｌｌｓを、１．０×１０７ｃｅｌｌｓ／ｍＬとなるようにハイドロゲルと混合し、細胞を包埋したハイドロゲルを作製した。培養容器１００又は対照培養容器を底面とし、側面に型枠を設けて、そこに得られた細胞を包埋したハイドロゲルを流し込んだ。３７℃、３０分間、ＣＯ２インキュベータ内で静置し、ハイドロゲルを固化した。 (Preparation of hydrogel)
3.0×10 7 cells of myoblasts were mixed with the hydrogel at 1.0×10 7 cells/mL to prepare a hydrogel in which the cells were embedded. The
（培養）
次いで、増殖用培地（１０％ＦＢＳ ＤＭＥＭ＋１％Ｐｅｎｉｃｉｌｌｉｎ／Ｓｔｒｅｐｔｏｍｙｃｉｎ）を添加し、加えて３７℃、５％ＣＯ２で２日間培養した。
その後、培地を分化用培地（２％ＨＳ ＤＭＥＭ＋１％Ｐｅｎｉｃｉｌｌｉｎ／Ｓｔｒｅｐｔｏｍｙｃｉｎ＋１００μＭ）に交換し、さらに５日間培養を継続し、三次元筋組織を得た。培地は隔日で交換した。
培養の時間経過に伴い、三次元筋組織は凸部の短手方向に収縮（シュリンク）した。 (culture)
Next, a growth medium (10% FBS DMEM + 1% Penicillin/Streptomycin) was added and cultured for 2 days at 37° C. and 5% CO 2 .
Thereafter, the medium was replaced with a differentiation medium (2% HS DMEM + 1% Penicillin/Streptomycin + 100 μM), and culture was continued for an additional 5 days to obtain three-dimensional muscle tissue. The medium was changed every other day.
As the culture progressed over time, the three-dimensional muscle tissue contracted (shrinked) in the short direction of the convex part.
次いで、増殖用培地（１０％ＦＢＳ ＤＭＥＭ＋１％Ｐｅｎｉｃｉｌｌｉｎ／Ｓｔｒｅｐｔｏｍｙｃｉｎ）を添加し、加えて３７℃、５％ＣＯ２で２日間培養した。
その後、培地を分化用培地（２％ＨＳ ＤＭＥＭ＋１％Ｐｅｎｉｃｉｌｌｉｎ／Ｓｔｒｅｐｔｏｍｙｃｉｎ＋１００μＭ）に交換し、さらに５日間培養を継続し、三次元筋組織を得た。培地は隔日で交換した。
培養の時間経過に伴い、三次元筋組織は凸部の短手方向に収縮（シュリンク）した。 (culture)
Next, a growth medium (10% FBS DMEM + 1% Penicillin/Streptomycin) was added and cultured for 2 days at 37° C. and 5% CO 2 .
Thereafter, the medium was replaced with a differentiation medium (2% HS DMEM + 1% Penicillin/Streptomycin + 100 μM), and culture was continued for an additional 5 days to obtain three-dimensional muscle tissue. The medium was changed every other day.
As the culture progressed over time, the three-dimensional muscle tissue contracted (shrinked) in the short direction of the convex part.
（電気刺激）
培養５日目及び７日目に得られた三次元筋組織に対して、電気刺激培養システムＣ－Ｐａｃｅ（ＩｏｎＯｐｔｉｃ社製）を用いて、下記条件の電気刺激を与えた：頻度：１Ｈｚ、強さ：０．３Ｖ／ｍｍ、持続時間：４０ｍｓ。顕微鏡観察により、電気刺激に応答した収縮運動の有無を観察した。
培養容器１００を用いて得た三次元筋組織において、明確な収縮運動が観察された。一方、対照培養容器を用いて得た三次元筋組織においては、収縮運動は観察できなかった。 (electrical stimulation)
Electrical stimulation was applied to the three-dimensional muscle tissue obtained on the 5th and 7th day of culture using the electrical stimulation culture system C-Pace (manufactured by IonOptic) under the following conditions: frequency: 1 Hz, strong Power: 0.3V/mm, Duration: 40ms. The presence or absence of contraction movement in response to electrical stimulation was observed by microscopic observation.
Clear contraction movement was observed in the three-dimensional muscle tissue obtained using theculture vessel 100. On the other hand, no contraction movement could be observed in the three-dimensional muscle tissue obtained using the control culture vessel.
培養５日目及び７日目に得られた三次元筋組織に対して、電気刺激培養システムＣ－Ｐａｃｅ（ＩｏｎＯｐｔｉｃ社製）を用いて、下記条件の電気刺激を与えた：頻度：１Ｈｚ、強さ：０．３Ｖ／ｍｍ、持続時間：４０ｍｓ。顕微鏡観察により、電気刺激に応答した収縮運動の有無を観察した。
培養容器１００を用いて得た三次元筋組織において、明確な収縮運動が観察された。一方、対照培養容器を用いて得た三次元筋組織においては、収縮運動は観察できなかった。 (electrical stimulation)
Electrical stimulation was applied to the three-dimensional muscle tissue obtained on the 5th and 7th day of culture using the electrical stimulation culture system C-Pace (manufactured by IonOptic) under the following conditions: frequency: 1 Hz, strong Power: 0.3V/mm, Duration: 40ms. The presence or absence of contraction movement in response to electrical stimulation was observed by microscopic observation.
Clear contraction movement was observed in the three-dimensional muscle tissue obtained using the
（ＳＡＡ免疫染色）
７日間経過後、得られた三次元筋組織を４％ＰＦＡで固定し、Ｓａｒｃｏｍｅｒｉｃ α－ａｃｔｉｎｉｎ（ＳＡＡ）の免疫染色及びＨｏｅｃｈｓｔ３３３４２を用いた細胞核染色を行った。
顕微鏡観察の結果を図１１－１に示す。
培養容器１００を用いて得た三次元筋組織（左図）では、ＳＡＡで染色される筋管領域が多数観察され、また筋管領域は互いに配向していた。一方、対照培養容器を用いて得た三次元筋組織（右図）では、観察される筋管領域は少なく、かつ、配向性が乏しかった。 (SAA immunostaining)
After 7 days, the obtained three-dimensional muscle tissue was fixed with 4% PFA, and immunostaining for sarcomeric α-actinin (SAA) and cell nucleus staining using Hoechst 33342 were performed.
The results of microscopic observation are shown in Figure 11-1.
In the three-dimensional muscle tissue (left figure) obtained using theculture vessel 100, many myotube regions stained with SAA were observed, and the myotube regions were oriented with respect to each other. On the other hand, in the three-dimensional muscle tissue obtained using the control culture vessel (right figure), there were few myotube regions observed and poor orientation.
７日間経過後、得られた三次元筋組織を４％ＰＦＡで固定し、Ｓａｒｃｏｍｅｒｉｃ α－ａｃｔｉｎｉｎ（ＳＡＡ）の免疫染色及びＨｏｅｃｈｓｔ３３３４２を用いた細胞核染色を行った。
顕微鏡観察の結果を図１１－１に示す。
培養容器１００を用いて得た三次元筋組織（左図）では、ＳＡＡで染色される筋管領域が多数観察され、また筋管領域は互いに配向していた。一方、対照培養容器を用いて得た三次元筋組織（右図）では、観察される筋管領域は少なく、かつ、配向性が乏しかった。 (SAA immunostaining)
After 7 days, the obtained three-dimensional muscle tissue was fixed with 4% PFA, and immunostaining for sarcomeric α-actinin (SAA) and cell nucleus staining using Hoechst 33342 were performed.
The results of microscopic observation are shown in Figure 11-1.
In the three-dimensional muscle tissue (left figure) obtained using the
以上の結果から、培養容器の凸構造によって、三次元筋組織中の筋芽細胞から筋管への細胞分化及び筋管の配向が促されることが示唆された。
The above results suggested that the convex structure of the culture container promoted cell differentiation from myoblasts to myotubes in three-dimensional muscle tissue and the orientation of myotubes.
実施例２：培養筋組織の大きさ検討
（培養容器２００、培養容器３００、培養容器４００の作製）
実施例１における培養容器１００と同様にして、凸部の長手方向の長さ（Ｘ）：凸部の短手方向の長さ（Ｙ）が、４ｃｍ：３ｃｍである培養容器２００；４ｃｍ：８ｃｍである培養容器３００、１０ｃｍ：１０ｃｍである培養容器４００を作製した。 Example 2: Examination of the size of cultured muscle tissue (fabrication of culture vessels 200, 300, and 400)
In the same manner as theculture container 100 in Example 1, the culture container 200 has a length (X) of the convex part in the longitudinal direction: a length (Y) of the convex part in the short direction of 4 cm: 3 cm; 4 cm: 8 cm. A culture vessel 300 with a diameter of 10 cm and a culture vessel 400 with a diameter of 10 cm were produced.
（培養容器２００、培養容器３００、培養容器４００の作製）
実施例１における培養容器１００と同様にして、凸部の長手方向の長さ（Ｘ）：凸部の短手方向の長さ（Ｙ）が、４ｃｍ：３ｃｍである培養容器２００；４ｃｍ：８ｃｍである培養容器３００、１０ｃｍ：１０ｃｍである培養容器４００を作製した。 Example 2: Examination of the size of cultured muscle tissue (fabrication of
In the same manner as the
（ハイドロゲルの作製及び培養）
実施例１と同様にしてハイドロゲルを作製した。
得られた細胞を包埋したハイドロゲルを培養容器２００には３ｍＬ、培養容器３００には８ｍＬ、培養容器４００には２４ｍＬそれぞれ流し込み、３７℃、３０分間、ＣＯ２インキュベータ内で静置し、ハイドロゲルを固化した。
実施例１と同様の方法で、７日間培養し、三次元筋組織を得た。培養の時間経過に伴い、三次元筋組織は凸部の短手方向に収縮（シュリンク）した。
ハイドロゲルから三次元筋組織への培養中、凸部の短手方向の長さを経時的に測定し、収縮の程度（シュリンク量）を観察した。結果を図１２に示す。
７日間培養後、培養容器２００及び培養容器３００を用いた三次元筋組織ではシュリンク量は約２ｃｍであり、培養容器４００を用いた三次元筋組織ではシュリンク量は約５ｃｍであった。
このことから、三次元筋組織のシュリンク量は、培養容器の凸部の短手方向の長さの約２分の１であることが示唆され、シュリンク量を推定した大きさの培養容器を使用することで、任意の大きさの三次元筋組織の作製が可能であることが明らかになった。 (Preparation and culture of hydrogel)
A hydrogel was produced in the same manner as in Example 1.
3 mL of the resulting cell-embedded hydrogel was poured into the culture container 200, 8 mL into the culture container 300, and 24 mL into the culture container 400, and left standing in a CO 2 incubator at 37° C. for 30 minutes. The gel was solidified.
The cells were cultured for 7 days in the same manner as in Example 1 to obtain three-dimensional muscle tissue. As the culture progressed over time, the three-dimensional muscle tissue contracted (shrinked) in the short direction of the convex part.
During the culture of three-dimensional muscle tissue from the hydrogel, the length of the convex portion in the transverse direction was measured over time to observe the degree of contraction (shrink amount). The results are shown in FIG.
After culturing for 7 days, the shrinkage amount of the three-dimensional muscle tissue usingculture container 200 and culture container 300 was about 2 cm, and the shrinkage amount of the three-dimensional muscle tissue using culture container 400 was about 5 cm.
This suggests that the amount of shrinkage of three-dimensional muscle tissue is approximately half the length of the convex part of the culture container in the transverse direction, and a culture container of the size estimated for the amount of shrinkage was used. By doing this, it became clear that it was possible to create three-dimensional muscle tissue of any size.
実施例１と同様にしてハイドロゲルを作製した。
得られた細胞を包埋したハイドロゲルを培養容器２００には３ｍＬ、培養容器３００には８ｍＬ、培養容器４００には２４ｍＬそれぞれ流し込み、３７℃、３０分間、ＣＯ２インキュベータ内で静置し、ハイドロゲルを固化した。
実施例１と同様の方法で、７日間培養し、三次元筋組織を得た。培養の時間経過に伴い、三次元筋組織は凸部の短手方向に収縮（シュリンク）した。
ハイドロゲルから三次元筋組織への培養中、凸部の短手方向の長さを経時的に測定し、収縮の程度（シュリンク量）を観察した。結果を図１２に示す。
７日間培養後、培養容器２００及び培養容器３００を用いた三次元筋組織ではシュリンク量は約２ｃｍであり、培養容器４００を用いた三次元筋組織ではシュリンク量は約５ｃｍであった。
このことから、三次元筋組織のシュリンク量は、培養容器の凸部の短手方向の長さの約２分の１であることが示唆され、シュリンク量を推定した大きさの培養容器を使用することで、任意の大きさの三次元筋組織の作製が可能であることが明らかになった。 (Preparation and culture of hydrogel)
A hydrogel was produced in the same manner as in Example 1.
3 mL of the resulting cell-embedded hydrogel was poured into the
The cells were cultured for 7 days in the same manner as in Example 1 to obtain three-dimensional muscle tissue. As the culture progressed over time, the three-dimensional muscle tissue contracted (shrinked) in the short direction of the convex part.
During the culture of three-dimensional muscle tissue from the hydrogel, the length of the convex portion in the transverse direction was measured over time to observe the degree of contraction (shrink amount). The results are shown in FIG.
After culturing for 7 days, the shrinkage amount of the three-dimensional muscle tissue using
This suggests that the amount of shrinkage of three-dimensional muscle tissue is approximately half the length of the convex part of the culture container in the transverse direction, and a culture container of the size estimated for the amount of shrinkage was used. By doing this, it became clear that it was possible to create three-dimensional muscle tissue of any size.
実施例３：食用ブタ血液を用いたハイドロゲルの作製
（培養容器５００の作製）
実施例１における培養容器１００と同様にして、凸部の長手方向の長さ（Ｘ）：凸部の短手方向の長さ（Ｙ）が、８ｃｍ：１２ｃｍである培養容器５００を作製した。 Example 3: Production of hydrogel using edible pig blood (production of culture container 500)
In the same manner as theculture container 100 in Example 1, a culture container 500 was produced in which the length of the protrusion in the longitudinal direction (X): the length of the protrusion in the transverse direction (Y) was 8 cm:12 cm.
（培養容器５００の作製）
実施例１における培養容器１００と同様にして、凸部の長手方向の長さ（Ｘ）：凸部の短手方向の長さ（Ｙ）が、８ｃｍ：１２ｃｍである培養容器５００を作製した。 Example 3: Production of hydrogel using edible pig blood (production of culture container 500)
In the same manner as the
（ハイドロゲル組成（２））
フィブリノーゲン（２５ｍｇ／ｍＬ） ４０００μＬ（終濃度４ｍｇ／ｍＬ）（Ｓｉｇｍａ社製、Ｆ８６３０）
食用ブタ血液 ５０００μＬ（終濃度２０％）
Ｔｈｒｏｍｂｉｎ（２００Ｕｎｉｔ／ｍＬ） ２５０μＬ（終濃度２Ｕｎｉｔ／ｍＬ）（Ｓｉｇｍａ社製、Ｔ４６４８－１０ＫＵ）
ＤＭＥＭ １５７５０μＬ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製、１１９６５１１８） (Hydrogel composition (2))
Fibrinogen (25mg/mL) 4000μL (final concentration 4mg/mL) (Sigma, F8630)
Edible pig blood 5000μL (final concentration 20%)
Thrombin (200 Unit/mL) 250 μL (final concentration 2 Unit/mL) (manufactured by Sigma, T4648-10KU)
DMEM 15750 μL (manufactured by Thermo Fisher Scientific, 11965118)
フィブリノーゲン（２５ｍｇ／ｍＬ） ４０００μＬ（終濃度４ｍｇ／ｍＬ）（Ｓｉｇｍａ社製、Ｆ８６３０）
食用ブタ血液 ５０００μＬ（終濃度２０％）
Ｔｈｒｏｍｂｉｎ（２００Ｕｎｉｔ／ｍＬ） ２５０μＬ（終濃度２Ｕｎｉｔ／ｍＬ）（Ｓｉｇｍａ社製、Ｔ４６４８－１０ＫＵ）
ＤＭＥＭ １５７５０μＬ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製、１１９６５１１８） (Hydrogel composition (2))
Fibrinogen (25mg/mL) 4000μL (final concentration 4mg/mL) (Sigma, F8630)
Edible pig blood 5000μL (
Thrombin (200 Unit/mL) 250 μL (
DMEM 15750 μL (manufactured by Thermo Fisher Scientific, 11965118)
（ハイドロゲルの作製及び培養）
実施例１におけるハイドロゲル組成において、マトリゲルに替えて食用ブタ血液を用いた上記組成によりハイドロゲルを作製した。
筋芽細胞２．５×１０８ｃｅｌｌｓを、１．０×１０７ｃｅｌｌｓ／ｍＬとなるようにハイドロゲルと混合し、細胞を包埋したハイドロゲルを作製した。培養容器５００又は対照培養容器を底面とし、側面に型枠を設けて、そこに得られた細胞を包埋したハイドロゲルを流し込んだ。３７℃、３０分間、ＣＯ２インキュベータ内で静置し、ハイドロゲルを固化した。
次いで、培養期間を１４日間とする以外は実施例１と同様の方法で、１４日間培養し、三次元筋組織を得た。 (Preparation and culture of hydrogel)
In the hydrogel composition in Example 1, a hydrogel was produced using the above composition using edible pig blood instead of Matrigel.
2.5×10 8 myoblast cells were mixed with the hydrogel at a concentration of 1.0×10 7 cells/mL to produce a hydrogel in which the cells were embedded. The culture container 500 or the control culture container was used as the bottom, a mold was provided on the side, and the obtained hydrogel in which cells were embedded was poured into the mold. The hydrogel was left standing in a CO 2 incubator at 37° C. for 30 minutes to solidify the hydrogel.
Next, the cells were cultured for 14 days in the same manner as in Example 1 except that the culture period was changed to 14 days to obtain a three-dimensional muscle tissue.
実施例１におけるハイドロゲル組成において、マトリゲルに替えて食用ブタ血液を用いた上記組成によりハイドロゲルを作製した。
筋芽細胞２．５×１０８ｃｅｌｌｓを、１．０×１０７ｃｅｌｌｓ／ｍＬとなるようにハイドロゲルと混合し、細胞を包埋したハイドロゲルを作製した。培養容器５００又は対照培養容器を底面とし、側面に型枠を設けて、そこに得られた細胞を包埋したハイドロゲルを流し込んだ。３７℃、３０分間、ＣＯ２インキュベータ内で静置し、ハイドロゲルを固化した。
次いで、培養期間を１４日間とする以外は実施例１と同様の方法で、１４日間培養し、三次元筋組織を得た。 (Preparation and culture of hydrogel)
In the hydrogel composition in Example 1, a hydrogel was produced using the above composition using edible pig blood instead of Matrigel.
2.5×10 8 myoblast cells were mixed with the hydrogel at a concentration of 1.0×10 7 cells/mL to produce a hydrogel in which the cells were embedded. The culture container 500 or the control culture container was used as the bottom, a mold was provided on the side, and the obtained hydrogel in which cells were embedded was poured into the mold. The hydrogel was left standing in a CO 2 incubator at 37° C. for 30 minutes to solidify the hydrogel.
Next, the cells were cultured for 14 days in the same manner as in Example 1 except that the culture period was changed to 14 days to obtain a three-dimensional muscle tissue.
培養７日目で実施例１と同様の方法で電気刺激を与えて、顕微鏡観察により、電気刺激に応答した収縮運動の有無を観察した。その結果、収縮運動が観察された。
１４日間培養により、約８ｃｍ×８ｃｍの三次元筋組織を得た。培養の時間経過に伴い、三次元筋組織は凸部の短手方向に約４ｃｍ収縮（シュリンク）した。
以上の結果より、マトリゲルを食用ブタ血液に変更しても収縮可能な組織が作製可能であること、組織のシュリンク量はマトリゲルと同等であることが明らかとなった。 On the 7th day of culture, electrical stimulation was applied in the same manner as in Example 1, and the presence or absence of contraction movement in response to the electrical stimulation was observed by microscopic observation. As a result, contraction movement was observed.
A three-dimensional muscle tissue of approximately 8 cm x 8 cm was obtained by culturing for 14 days. As the culture time progressed, the three-dimensional muscle tissue shrank by about 4 cm in the transverse direction of the convex portion.
The above results revealed that it is possible to create a contractible tissue even when Matrigel is replaced with edible pig blood, and that the amount of shrinkage of the tissue is equivalent to that of Matrigel.
１４日間培養により、約８ｃｍ×８ｃｍの三次元筋組織を得た。培養の時間経過に伴い、三次元筋組織は凸部の短手方向に約４ｃｍ収縮（シュリンク）した。
以上の結果より、マトリゲルを食用ブタ血液に変更しても収縮可能な組織が作製可能であること、組織のシュリンク量はマトリゲルと同等であることが明らかとなった。 On the 7th day of culture, electrical stimulation was applied in the same manner as in Example 1, and the presence or absence of contraction movement in response to the electrical stimulation was observed by microscopic observation. As a result, contraction movement was observed.
A three-dimensional muscle tissue of approximately 8 cm x 8 cm was obtained by culturing for 14 days. As the culture time progressed, the three-dimensional muscle tissue shrank by about 4 cm in the transverse direction of the convex portion.
The above results revealed that it is possible to create a contractible tissue even when Matrigel is replaced with edible pig blood, and that the amount of shrinkage of the tissue is equivalent to that of Matrigel.
実施例４：凸部の高さ検討
（培養容器の作製）
凸部１０の高さ以外は実施例１における培養容器１００と同様にして、凸部１０の高さが１００μｍである培養容器６００、凸部１０の高さが３００μｍである培養容器７００、及び凸部１０の高さが５００μｍである培養容器８００を作製した。
（ハイドロゲルの作製及び培養）
実施例１と同様にしてハイドロゲルを作製及び培養をした。 Example 4: Examination of height of convex portion (fabrication of culture container)
A culture container 600 in which the height of theprojection 10 is 100 μm, a culture container 700 in which the height of the projection 10 is 300 μm, and a culture container 700 in which the height of the projection 10 is 300 μm are prepared in the same manner as the culture container 100 in Example 1 except for the height of the projection 10. A culture container 800 in which the height of the portion 10 is 500 μm was produced.
(Preparation and culture of hydrogel)
A hydrogel was prepared and cultured in the same manner as in Example 1.
（培養容器の作製）
凸部１０の高さ以外は実施例１における培養容器１００と同様にして、凸部１０の高さが１００μｍである培養容器６００、凸部１０の高さが３００μｍである培養容器７００、及び凸部１０の高さが５００μｍである培養容器８００を作製した。
（ハイドロゲルの作製及び培養）
実施例１と同様にしてハイドロゲルを作製及び培養をした。 Example 4: Examination of height of convex portion (fabrication of culture container)
A culture container 600 in which the height of the
(Preparation and culture of hydrogel)
A hydrogel was prepared and cultured in the same manner as in Example 1.
（電気刺激）
培養１４日目に実施例３と同様にして電気刺激を行い、電気刺激に応答した三次元筋組織の収縮運動を顕微鏡にて観察及び動画撮影した。撮影した動画に基づき、収縮量として、収縮に伴う三次元筋組織の移動距離（図中、Ｄｉｓｔａｎｃｅ（μｍ））の経時変化をグラフ化した。
結果を図１３に示す。 (electrical stimulation)
On the 14th day of culture, electrical stimulation was performed in the same manner as in Example 3, and the contraction movement of the three-dimensional muscle tissue in response to the electrical stimulation was observed using a microscope and videotaped. Based on the captured video, changes over time in the moving distance of the three-dimensional muscle tissue due to contraction (Distance (μm) in the figure) were graphed as the amount of contraction.
The results are shown in FIG.
培養１４日目に実施例３と同様にして電気刺激を行い、電気刺激に応答した三次元筋組織の収縮運動を顕微鏡にて観察及び動画撮影した。撮影した動画に基づき、収縮量として、収縮に伴う三次元筋組織の移動距離（図中、Ｄｉｓｔａｎｃｅ（μｍ））の経時変化をグラフ化した。
結果を図１３に示す。 (electrical stimulation)
On the 14th day of culture, electrical stimulation was performed in the same manner as in Example 3, and the contraction movement of the three-dimensional muscle tissue in response to the electrical stimulation was observed using a microscope and videotaped. Based on the captured video, changes over time in the moving distance of the three-dimensional muscle tissue due to contraction (Distance (μm) in the figure) were graphed as the amount of contraction.
The results are shown in FIG.
いずれの場合においても、三次元筋組織の収縮運動が観察された。中でも、凸部１０の高さが１００μｍである場合に移動量（収縮量）が特に大きかった。
In both cases, contraction movement of three-dimensional muscle tissue was observed. Among these, the amount of movement (amount of contraction) was particularly large when the height of the convex portion 10 was 100 μm.
１００ 培養容器
１０ 凸部
２０ アンカー部
２００ 培養容器
３００ 培養容器
４００ 培養容器 100Culture container 10 Convex portion 20 Anchor portion 200 Culture container 300 Culture container 400 Culture container
１０ 凸部
２０ アンカー部
２００ 培養容器
３００ 培養容器
４００ 培養容器 100
Claims (7)
- 下記の工程（Ａ）及び（Ｂ）を含む三次元筋組織の製造方法：
（Ａ）筋芽細胞を含むハイドロゲルを培養容器へ供給する工程であって、
前記培養容器は、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器である工程、並びに、
（Ｂ）前記培養容器中で、筋芽細胞を筋管へと分化誘導する工程。 A method for producing three-dimensional muscle tissue including the following steps (A) and (B):
(A) A step of supplying a hydrogel containing myoblasts to a culture container,
The culture container is a culture container including a pair of anchor parts facing each other at the ends of the container, and a plurality of convex parts arranged between the pair of anchor parts, and
(B) A step of inducing differentiation of myoblasts into myotubes in the culture vessel. - 前記培養容器において、前記凸部の長手方向の長さ（Ｘ）が１ｃｍ以上、かつ、前記凸部の短手方向の長さ（Ｙ）が１ｃｍ以上である、請求項１に記載の三次元筋組織の製造方法。 The three-dimensional container according to claim 1, wherein in the culture container, the length (X) of the protrusion in the longitudinal direction is 1 cm or more, and the length (Y) of the protrusion in the lateral direction is 1 cm or more. Method of manufacturing muscle tissue.
- 前記培養容器において、前記凸部の長手方向の長さ（Ｘ）に対する前記凸部の短手方向の長さ（Ｙ）の比〔（Ｙ）／（Ｘ）〕が０．７以上である、請求項１に記載の三次元筋組織の製造方法。 In the culture container, the ratio [(Y)/(X)] of the length (Y) of the convex part in the transverse direction to the length (X) of the convex part in the longitudinal direction is 0.7 or more. The method for producing three-dimensional muscle tissue according to claim 1.
- 前記凸部が、高さ５０μｍ～１ｍｍである、請求項１に記載の三次元筋組織の製造方法。 The method for producing three-dimensional muscle tissue according to claim 1, wherein the convex portion has a height of 50 μm to 1 mm.
- 前記凸部の互いの間隔が、１０μｍ～１ｍｍである、請求項１に記載の三次元筋組織の製造方法。 The method for producing three-dimensional muscle tissue according to claim 1, wherein the distance between the convex portions is 10 μm to 1 mm.
- 請求項１～５のいずれか１項に記載の製造方法により得られる三次元筋組織。 Three-dimensional muscle tissue obtained by the manufacturing method according to any one of claims 1 to 5.
- 三次元筋組織を製造するための培養容器であって、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる、培養容器。
A culture vessel for producing three-dimensional muscle tissue, the culture vessel comprising a pair of anchor parts facing each other at the ends of the vessel, and a plurality of convex parts arranged between the pair of anchor parts.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2022-122160 | 2022-07-29 | ||
| JP2022122160A JP2024018683A (en) | 2022-07-29 | 2022-07-29 | Method for producing three-dimensional muscle tissue |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2024024929A1 true WO2024024929A1 (en) | 2024-02-01 |
Family
ID=89706686
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2023/027702 WO2024024929A1 (en) | 2022-07-29 | 2023-07-28 | Method for producing three-dimensional muscle tissue |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP2024018683A (en) |
| WO (1) | WO2024024929A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113508172A (en) * | 2019-03-04 | 2021-10-15 | 日清食品控股株式会社 | Three-dimensional muscle tissue and method for producing same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008289375A (en) * | 2007-05-22 | 2008-12-04 | Dainippon Printing Co Ltd | Cell culture support for forming string-like myocardial cell aggregate |
| JP2020141573A (en) * | 2019-03-04 | 2020-09-10 | 日清食品ホールディングス株式会社 | Three-dimensional muscle tissue and producing method thereof |
| WO2021132478A1 (en) * | 2019-12-26 | 2021-07-01 | 国立大学法人東京大学 | Three-dimensional tissue complex, and method for producing three-dimensional tissue complex |
-
2022
- 2022-07-29 JP JP2022122160A patent/JP2024018683A/en active Pending
-
2023
- 2023-07-28 WO PCT/JP2023/027702 patent/WO2024024929A1/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008289375A (en) * | 2007-05-22 | 2008-12-04 | Dainippon Printing Co Ltd | Cell culture support for forming string-like myocardial cell aggregate |
| JP2020141573A (en) * | 2019-03-04 | 2020-09-10 | 日清食品ホールディングス株式会社 | Three-dimensional muscle tissue and producing method thereof |
| WO2021132478A1 (en) * | 2019-12-26 | 2021-07-01 | 国立大学法人東京大学 | Three-dimensional tissue complex, and method for producing three-dimensional tissue complex |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113508172A (en) * | 2019-03-04 | 2021-10-15 | 日清食品控股株式会社 | Three-dimensional muscle tissue and method for producing same |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2024018683A (en) | 2024-02-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bomkamp et al. | Scaffolding biomaterials for 3D cultivated meat: prospects and challenges | |
| JP7033095B2 (en) | Three-dimensional muscle tissue and its manufacturing method | |
| AU2016204474B2 (en) | Engineered Comestible Meat | |
| WO2024024929A1 (en) | Method for producing three-dimensional muscle tissue | |
| JP2023093403A (en) | Cell culturing method using animal muscle tissue extract | |
| JP7240028B2 (en) | Apparatus and method for producing tissue from cells | |
| Kumar et al. | Technological and structural aspects of scaffold manufacturing for cultured meat: recent advances, challenges, and opportunities | |
| EP4410959A1 (en) | Cell culture gel, method for producing cell culture gel with cells, method for producing cells, three-dimensional muscle tissue produced by same, and cultured meat | |
| WO2023127970A1 (en) | Cell proliferation medium for producing cultured meat | |
| WO2023047832A1 (en) | Cell culture component, cell culture medium, method for producing serum, and method for producing cells | |
| US20240026260A1 (en) | Device for forming three-dimensional muscle tissue and method of producing three-dimensional muscle tissue | |
| RU2778255C2 (en) | Compositions out of cultured meat | |
| Dominko | From Farm to Lab: The Potential of Decellularized Plant Leaves for Sustainable Meat Production | |
| JP2009022264A (en) | Medium for cell culture containing fish serum | |
| IL283011A (en) | Method for inducing hypertrophic muscle fibers for industrial meat production | |
| Soccol et al. | Cultivated Meat: Technologies, Commercialization and Challenges |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23846659 Country of ref document: EP Kind code of ref document: A1 |