CN105062966A - Producing method of human embryo trophoblast cells and placental mesenchymal stem cells - Google Patents
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Abstract
The invention discloses a producing method of human embryo trophoblast cells and placental mesenchymal stem cells. The producing method comprises following steps: processed placenta tissue is obtained via placenta sample processing; a cell suspension is obtained via tissue digestion; trophoblast cells and placental mesenchymal stem cells are obtained via density gradient density separation; trophoblast cells are obtained; and culturing of placental mesenchymal stem cells is carried out. According to the producing method, HyQTase and DNAse I are both adopted for digesting tissue; large amounts of trophoblast cells and placental mesenchymal stem cells with ideal purity and activity are obtained via density gradient separation; digestion fragments, fibroblasts, and erythrocytes are removed; the trophoblast cells and the placental mesenchymal stem cells are separated; purity of obtained cytotrophoblasts can be as high as 90%; in culturing of the placental mesenchymal stem cells, under micro gravity conditions, mesenchymal stem cells are processed with electromagnetic field and via sonic treatment successively, an anti-oxidant is injected, and subcultring to the ninth or the tenth generation can be realized.
Description
Technical field
The present invention relates to field of biomedicine technology, specifically relate to the production method of a kind of Human embryo nurse cell and placenta mesenchyma stem cell.
Background technology
Conventional separation methods is difficult to obtain the higher nurse cell of a large amount of purity and placenta mesenchyma stem cell usually; Although the cell purity that application selected by flow cytometry apoptosis method or magnetic bead sorting method obtain is good, cost is very high.
Nurse cell is cultivated in vitro and is gone down to posterity more difficult, the scheme that Many researchers directly processes after all adopting isolated cell or intervenes, and the nurse cell that therefore disposable acquisition is a large amount of is very favorable to experiment.Placenta mesenchyma stem cell is low in the abundance of placenta tissue, wants that the primary cell obtaining some amount also needs a lot of placenta tissues.
In prior art, the cellular constituent of placenta is more complicated, wherein comprised nurse cell plays an important role in Maternal-placental immune toler ance process, placenta mesenchyma stem cell has multi-lineage potential and suppresses lymphopoietic characteristic, conventionally is usually difficult to obtain a large amount of and that purity is higher above-mentioned two kinds of cells from method.
Summary of the invention
The technical problem that the present invention solves is the defect existed for prior art, the production method of a kind of Human embryo nurse cell and placenta mesenchyma stem cell is provided, can reaches that single job can obtain in a large number simultaneously, the Human embryo nurse cell of higher degree and the object of placenta mesenchyma stem cell.
The technical scheme that the present invention solves the problems of the technologies described above is:
A production method for Human embryo nurse cell and placenta mesenchyma stem cell, comprises the following steps:
(1) placenta sample disposal: placenta of giving birth under aseptic condition divests amnion, after wiping out maternal surface of placenta 2.0 ~ 3.0mm tissue, cut placental lobules, take 50g, be soaked in the conserving liquid containing antibacterial peptide, the described conserving liquid containing antibacterial peptide be 10 ~ 100 μ g/ml poly-lysines are joined in 10 ~ 100 μ g/ml gallic acid ester EGCG formulated, after soaking the scheduled time, the placental lobules operating scissors cut are shredded, repeatedly blood stains are rinsed with physiological saline, until washing fluid is close to colourless, obtain the placenta tissue after process;
(2) tissue digestion: use 15mmol/L Tutofusin tris, add Hartmann-solution D, regulate pH to 8.0, as digestion damping fluid, add the Digestive system that final concentration is 2.4g/LHyQTase (being purchased from HyClone company) and 300U/mLDNaseI composition, get Digestive system 320mL, placenta tissue after rinsing is digested several times, at every turn at 37 ± 0.2 DEG C, 180r/min constant temperature gas bath shaking table digestion 2min, after digestion, aseptic removal HyQTase solution, stops digestion reaction by new-born calf serum, obtains cell suspension;
(3) density gradient density separation: successively spread the Percoll parting liquid into 7 density in 50mL centrifuge tube, each density 5mL, slowly add 5mL cell suspension again, the centrifugal 20min of 1200g under usage level whizzer room temperature, liquid more than careful reject centrifuge tube 20mL scale, collect the cellular layer of 12.5 ~ 20.0mL scale and the liquid of 12.5 ~ 7.5mL scale, the visible obvious white cloud cellular layer at centrifuge tube 15 ~ 20mL scale place, Percoll relative density corresponding is herein 1.046 ~ 1.059, is the region that nurse cell exists; Visible unconspicuous cellular layer near centrifuge tube 10mL scale, density is 1.072 herein, is the region that lymphocyte and placenta mesenchyma stem cell exist, puts in different centrifuge tube respectively, after diluting 5 times with D-Hank ' s liquid, the centrifugal 15min of 1000g under room temperature; Visible white cell mass at the bottom of centrifuge tube obtains the nurse cell after Graded Density separation and placenta mesenchyma stem cell;
(4) acquisition of nurse cell: be mixed with suspension with the bFGF containing DMEM/F12, pyruvate salt, 10mg/L Sodium Selenite Regular Insulin, 2mg/L thanomin and 20 milli μ g/mL that volume fraction is 10% foetal calf serum (FBS), nurse cell after density gradient centrifugation is carried out resuspended, remove inoblast with differential attachment method, obtain nurse cell; Through counting, the nurse cell amount of acquisition can reach (5.48 ± 1.98) × 108;
(5) cultivation of placenta mesenchyma stem cell: be mixed with suspension with the bFGF, the enhancer of proliferation that contain DMEM/F12, pyruvate salt, 10mg/L Sodium Selenite Regular Insulin, 2mg/L thanomin and 20 milli μ g/mL that volume fraction is 10% foetal calf serum (FBS), the placenta mesenchyma stem cell obtained after density gradient centrifugation is carried out resuspended, under microgravity environment, successively by electromagnetic field and sonic treatment mescenchymal stem cell, count and be inoculated in the culturing bottle of 75mm, supplement 4mg/ml Prostatropin (bFGF) in the medium, 10ng/mlN-ethanoyl-Cys, 100ng/ml calcium chloride, put into 37 DEG C, gas concentration lwevel is cultivate in the CO2 incubator of 0.5%, 2d changes liquid 1 time, after Growth of Cells to 90% merges, digestion is counting cells also, go down to posterity in 1: 4 ratio, after passage, the speed of growth is very fast, cellular form is more homogeneous, the change shape BMP4 process with 100ng/ml in obvious 2nd day, within 3rd day or 4, use the BMP4 process of 10ng/ml, BMP4 process with 1ng/ml in 5th day, when reaching for the 8th generation, cell starts to occur catabiosis, cell proliferation is slow, cell volume increases, a lot of black particle is there is in endochylema, now need to inject a kind of antioxidant, described antioxidant is polyoxyethylene glycol-put together catalase (PEG-catalase) or N-acetylcystein, or use polyphenoils, described polyphenoils is 1-500 μM of Pyruvic Acid Ethyl ester (EP), suppress the aging of mescenchymal stem cell, for increasing yield of dried cell, until reached for the 9th or ten generations.
Further, described enhancer of proliferation is the liquid component of the cord blood of condensation, with Promote cell's growth.
Further, described microgravity environment rotates formed stimulated microgravity by multiaxis, can obtain the mescenchymal stem cell with less average bubble size.
The invention has the beneficial effects as follows:
(1) the present invention adopts HyQTase and DNAseI jointly to digest tissue, after Graded Density is separated, can a large amount of nurse cell of simultaneously disposable acquisition and more placenta mesenchyma stem cell, little to cell injury, obtain cell purity and vigor is all more satisfactory;
(2) use the Percoll cellular segregation liquid of discontinuous gradient can remove digestion fragment, inoblast and red corpuscle, and nurse cell and placenta mesenchyma stem cell are made a distinction; The langhans cell obtained is after the differential velocity adherent choice specimen of calligraphy is except inoblast, and purity can reach 90%, and cost is lower.
(3) placenta mesenchyma stem cell obtained after density gradient separation, under training microgravity environment, successively by electromagnetic field and sonic treatment mescenchymal stem cell, and injects a kind of antioxidant, can be passaged to for the 9th or ten generations always.
Accompanying drawing explanation
Fig. 1 is the cell category figure of each concentration Percoll distributing position, relative density and correspondence in the embodiment of the present invention;
Fig. 2 is placenta mesenchyma stem cell growthhabit (× 100) figure, the A that in the embodiment of the present invention, Graded Density is separated rear inoculation culture: forth generation, the B: the eight generation, the C: the nine generation, the D: the ten generation;
Fig. 3 is Osteoblast Differentiation potential (× 100) figure, the A of placenta mesenchyma stem cell in the embodiment of the present invention: Induction experiments group, B: control group.
Embodiment
The concrete test carried out below in conjunction with laboratory is described in further detail technical scheme of the present invention, but the invention is not restricted to the following specific examples enumerated.
A production method for Human embryo nurse cell and placenta mesenchyma stem cell, comprises the following steps:
(1) placenta sample disposal: placenta of giving birth under aseptic condition divests amnion, after wiping out maternal surface of placenta 2.0mm tissue, cut placental lobules, take 50g, be soaked in the conserving liquid containing antibacterial peptide, the described conserving liquid containing antibacterial peptide be 10 μ g/ml poly-lysines are joined in 10 μ g/ml gallic acid ester EGCG formulated, after soaking the scheduled time, the placental lobules operating scissors cut are shredded, repeatedly blood stains are rinsed with physiological saline, until washing fluid is close to colourless, obtain the placenta tissue after process;
(2) tissue digestion: use 15mmol/L Tutofusin tris, add Hartmann-solution D, regulate pH to 8.0, as digestion damping fluid, add the Digestive system that final concentration is 2.4g/LHyQTase (being purchased from HyClone company) and 300U/mLDNaseI composition, get Digestive system 320mL, placenta tissue after rinsing is digested several times, at every turn at 36.8 DEG C, 180r/min constant temperature gas bath shaking table digestion 2min, after digestion, aseptic removal HyQTase solution, stops digestion reaction by new-born calf serum, obtains cell suspension;
(3) density gradient density separation: successively spread the Percoll parting liquid into 7 density in 50mL centrifuge tube, each density 5mL, as shown in table 1, slowly add 5mL cell suspension again, the centrifugal 20min of 1200g under usage level whizzer room temperature, liquid more than careful reject centrifuge tube 20mL scale, collect the cellular layer of 12.5 ~ 20.0mL scale and the liquid of 12.5 ~ 7.5mL scale, the visible obvious white cloud cellular layer at centrifuge tube 15 ~ 20mL scale place, Percoll relative density corresponding is herein 1.046 ~ 1.059, it is the region that nurse cell exists, visible unconspicuous cellular layer near centrifuge tube 10mL scale, density is 1.072 herein, is the region that lymphocyte and placenta mesenchyma stem cell exist, see Fig. 1, put in different centrifuge tube respectively, after diluting 5 times with D-Hank ' s liquid, the centrifugal 15min of 1000g under room temperature, visible white cell mass at the bottom of centrifuge tube obtains the nurse cell after Graded Density separation and placenta mesenchyma stem cell,
(4) acquisition of nurse cell: be mixed with suspension with the bFGF containing DMEM/F12, pyruvate salt, 10mg/L Sodium Selenite Regular Insulin, 2mg/L thanomin and 20 milli μ g/mL that volume fraction is 10% foetal calf serum (FBS), nurse cell after density gradient centrifugation is carried out resuspended, remove inoblast with differential attachment method, obtain nurse cell;
The qualification of nurse cell: the nurse cell that obtains gets part cell, washs 2 times with PBS, adjustment cell density to 1 × 109/L.Add 0.1mL cell suspension in 0.5mlEP pipe, the sheep anti mouse two being sequentially added into mouse anti human CK7 monoclonal antibody and FITC mark resists hatches, and arranges primary antibodie and FITC contrast, Flow cytometry 10000 cells.Through flow cytomery, CK7 positive rate is (90.00 ± 4.36) %.
(5) cultivation of placenta mesenchyma stem cell: with the DMEM/F12 containing volume fraction being 10% foetal calf serum (FBS), pyruvate salt, 10mg/L Sodium Selenite Regular Insulin, the bFGF of 2mg/L thanomin and 20 milli μ g/mL, enhancer of proliferation is mixed with suspension, described enhancer of proliferation is the liquid component of the cord blood of condensation, with Promote cell's growth, the placenta mesenchyma stem cell obtained after density gradient centrifugation is carried out resuspended, under rotating formed stimulated microgravity by multiaxis, successively by electromagnetic field and sonic treatment mescenchymal stem cell, to obtain the mescenchymal stem cell with less average bubble size, count and be inoculated in the culturing bottle of 75mm, supplement 4mg/ml Prostatropin (bFGF) in the medium, 10ng/mlN-ethanoyl-Cys, 100ng/ml calcium chloride, put into 37 DEG C, gas concentration lwevel is cultivate in the CO2 incubator of 0.5%, 2d changes liquid 1 time, after Growth of Cells to 90% merges, digestion is counting cells also, go down to posterity in 1: 4 ratio, after passage, the speed of growth is very fast, cellular form is more homogeneous, the change shape BMP4 process with 100ng/ml in obvious 2nd day, within 3rd day or 4, use the BMP4 process of 10ng/ml, BMP4 process with 1ng/ml in 5th day, when reaching for the 8th generation, cell starts to occur catabiosis, cell proliferation is slow, cell volume increases, a lot of black particle is there is in endochylema, now need to inject a kind of antioxidant, described antioxidant is polyoxyethylene glycol-put together catalase (PEG-catalase) or N-acetylcystein, or use polyphenoils, described polyphenoils is 1 μM of Pyruvic Acid Ethyl ester (EP), suppress the aging of mescenchymal stem cell, for increasing yield of dried cell, until reached for the 9th or ten generations, see Fig. 2.
The qualification of placenta mesenchyma stem cell:
Cell phenotype detect: get the 3rd generation cell, adjustment density to 1 × 109L
-1, often pipe adds 0.1mL cell suspension.Negative control pipe adds mouse IgG-FITC, IgG-PE; Other pipes add humanized murine antibodies CD14-PE respectively, CD45-PE, CD34-PE, CD29-FITC, CD44-FITC, HLA-DR-FITC, incubated at room 30min, Flow cytometry 10000 cells.Flow cytomery result shows, 3rd generation placenta mesenchyma stem cell surface marker more homogeneous, strongly expressed hyaluronic acid receptor CD44 and integrin family member CD29, positive rate is respectively (99.86 ± 0.05) %, (98.50 ± 1.26) %; Do not express hemopoietic stem cell mark CD34, CD45 and CD14, positive rate is respectively (1.62 ± 0.87) % (2.26 ± 1.95) %, (2.08 ± 0.97) %; Strongly expressed HLA-ABC, positive rate is (99.00 ± 1.58) %, not expression of HLA-DR, and positive rate is (1.66 ± 0.84) %.
Osteogenic potential detect: get the 3rd generation cell be inoculated on 12 orifice plates, each sample 3 hole, 2 holes are induced, and 1 hole is as negative control.Every hole adds 1mL perfect medium, containing 1 × 104 cell.Put into incubator to cultivate, every 2d changes liquid 1 time, until Growth of Cells starts induction after 70%-80% merges.Scleroblast inductive differentiation medium is contain the L-DMEM substratum that volume fraction is 10% foetal calf serum, 5mmol/L β-phospho-glycerol, 50mg/L vitamins C, 1nmol/L dexamethasone.Every 2d changes 1 inductive differentiation medium, and induction 20d, until clustering phenomena appears in cell.The placenta mesenchyma stem cell 1mLPBS of differentiation is washed 2 times; Every hole adds the neutral formalin that 1mL volume fraction is 10%, room temperature fixed cell 10min.Use sodium alizarinsulfonate (pH4.1) dyeing of 10g/L, room temperature 30min.Use deionized water eccysis sodium alizarinsulfonate again, until negative control hole background stainings basically eliminate.Induce 4 weeks hystazarin red colourings, placenta mesenchyma stem cell can be dyed to bright-coloured orange red, but negative control hole is not painted substantially, sees Fig. 3.
The above is the citing of best mode for carrying out the invention, and the part wherein do not addressed in detail is the common practise of those of ordinary skill in the art.Protection scope of the present invention is as the criterion with the content of claim, and any equivalent transformation carried out based on technology enlightenment of the present invention, also within protection scope of the present invention.
Claims (3)
1. a production method for Human embryo nurse cell and placenta mesenchyma stem cell, is characterized in that comprising the following steps:
(1) placenta sample disposal: placenta of giving birth under aseptic condition divests amnion, after wiping out maternal surface of placenta 2.0 ~ 3.0mm tissue, cut placental lobules, take 50g, be soaked in the conserving liquid containing antibacterial peptide, the described conserving liquid containing antibacterial peptide be 10 ~ 100 μ g/ml poly-lysines are joined in 10 ~ 100 μ g/ml gallic acid ester EGCG formulated, after soaking the scheduled time, the placental lobules operating scissors cut are shredded, repeatedly blood stains are rinsed with physiological saline, until washing fluid is close to colourless, obtain the placenta tissue after process;
(2) tissue digestion: use 15mmol/L Tutofusin tris, add Hartmann-solution D, regulate pH to 8.0, as digestion damping fluid, add the Digestive system that final concentration is 2.4g/LHyQTase and 300U/mLDNaseI composition, get Digestive system 320mL, placenta tissue after rinsing is digested several times, at every turn at 37 ± 0.2 DEG C, 180r/min constant temperature gas bath shaking table digestion 2min, after digestion, aseptic removal HyQTase solution, stops digestion reaction by new-born calf serum, obtains cell suspension;
(3) density gradient density separation: successively spread the Percoll parting liquid into 7 density in 50mL centrifuge tube, each density 5mL, slowly add 5mL cell suspension again, the centrifugal 20min of 1200g under usage level whizzer room temperature, liquid more than careful reject centrifuge tube 20mL scale, collect the cellular layer of 12.5 ~ 20.0mL scale and the liquid of 12.5 ~ 7.5mL scale, put in different centrifuge tube respectively, after diluting 5 times with D-Hank ' s liquid, the centrifugal 15min of 1000g under room temperature, obtains the nurse cell after Graded Density separation and placenta mesenchyma stem cell;
(4) acquisition of nurse cell: be mixed with suspension with the bFGF containing DMEM/F12, pyruvate salt, 10mg/L Sodium Selenite Regular Insulin, 2mg/L thanomin and 20 milli μ g/mL that volume fraction is 10% foetal calf serum, nurse cell after density gradient centrifugation is carried out resuspended, remove inoblast with differential attachment method, obtain nurse cell;
(5) cultivation of placenta mesenchyma stem cell: with the DMEM/F12 containing volume fraction being 10% foetal calf serum, pyruvate salt, 10mg/L Sodium Selenite Regular Insulin, the bFGF of 2mg/L thanomin and 20 milli μ g/mL, enhancer of proliferation is mixed with suspension, the placenta mesenchyma stem cell obtained after density gradient centrifugation is carried out resuspended, under microgravity environment, successively by electromagnetic field and sonic treatment mescenchymal stem cell, count and be inoculated in the culturing bottle of 75mm, put into 37 DEG C, gas concentration lwevel is cultivate in the CO2 incubator of 0.5%, 2d changes liquid 1 time, after Growth of Cells to 90% merges, digestion is counting cells also, go down to posterity in 1: 4 ratio, after passage, the speed of growth is very fast, cellular form is more homogeneous, the change shape BMP4 process with 100ng/ml in obvious 2nd day, within 3rd day or 4, use the BMP4 process of 10ng/ml, BMP4 process with 1ng/ml in 5th day, when reaching for the 8th generation, cell starts to occur catabiosis, cell proliferation is slow, cell volume increases, a lot of black particle is there is in endochylema, now need to inject a kind of antioxidant, described antioxidant is polyoxyethylene glycol-put together catalase or N-acetylcystein, suppress the aging of mescenchymal stem cell, for increasing yield of dried cell, until reached for the 9th or ten generations.
2. the production method of a kind of Human embryo nurse cell as claimed in claim 1 and placenta mesenchyma stem cell, is characterized in that described enhancer of proliferation is the liquid component of the cord blood of condensation.
3. the production method of a kind of Human embryo nurse cell as claimed in claim 1 and placenta mesenchyma stem cell, is characterized in that described microgravity environment rotates formed stimulated microgravity by multiaxis.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108611317A (en) * | 2018-04-25 | 2018-10-02 | 北京市农林科学院 | N-acetylcystein is used to prepare the application in pig placental trophoblasts in vitro culture agent |
| CN112126622A (en) * | 2020-08-27 | 2020-12-25 | 陕西佰傲干细胞再生医学有限公司 | Umbilical cord mesenchymal stem cell primary isolation culture method capable of improving yield |
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