CN1286970C - Method of prodcing megacaryocyle using umbilical blood CD 344+cell in vitro induction method - Google Patents
Method of prodcing megacaryocyle using umbilical blood CD 344+cell in vitro induction method Download PDFInfo
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Abstract
The present invention discloses a megakaryocyte generating method through the in vitro induction of CD34<+> cells of umbilical cord blood. The present invention comprises the steps that the in vitro amplification of CD34<+> cells of umbilical cord blood is realized in a culture medium containing fetal calf serum, human stem cell factors (SCF), human thrombopoietin (TPO) and/or flt-3 ligand; megakaryocyte generation is induced by a serum-free culture medium containing human thrombopoietin (TPO), human interleukin-3(IL-3) and/or human granulocyte macro-system colony stimulating factors (GM-CSF). Compared with other methods, the method of the present invention, which is used for megakaryocyte induction from CD34<+> cells of umbilical cord blood, results in higher multiple of cell amplification, similar proportion of megakaryocytes in culture substances, and identical cell phenotype; consequently, the induction efficiency is obviously increased when megakaryocytes are induced by the method.
Description
Technical field
The present invention relates to the Megakaryocytic method of a kind of external evoked generation, relate in particular to by bleeding of the umbilicus CD34
+Cell is in the Megakaryocytic method of external evoked generation.
Background technology
Macronucleus hematopoiesis is to be started by the pluripotency hemopoietic stem cell, by forming sophisticated megalokaryocyte, finally forms the biological procedures of hematoblastic complexity.
Megakaryocytic generation generally is divided into three etap: progenitor cell, prematurity megalokaryocyte (parent cell before the macronucleus) and ripe megalokaryocyte.Some mitotic division signals can impel megakaryoblast propagation as cytokine, extracellular matrix.Parent cell is a kind of transient state cell before the macronucleus, and caing be compared to is the bridge of frame between progenitor cell, mature cell and postmitotic cell.
Macronucleus hematopoiesis is the hematopoiesis of a uniqueness, from the hemopoietic progenitor cell pond grow megalokaryocyte not only needed the mitotic division of cell but also need to examine in mitotic division-nuclear duplicate.Sophisticated megalokaryocyte stops propagation, and cell no longer divides, but the mitotic division in its sophisticated process center still carries out, and dna content continues to increase, and forms the megalokaryocyte of various times of bodies, discharges thrombocyte at last.
Megalokaryocyte and thrombocyte have significant application value clinically as the important component of human body hemocyte.Very big to hematoblastic infusion demand at first clinically, for the aleucia that a variety of causes causes, platelet transfusion is the treatment plan of using always.At present radiotherapy chemotherapy still is the main means of cancer therapy in addition, but a lot of patients are accepting to put, aleucia usually taking place easily after the chemotherapy, and this is one of principal risk in the cancer therapy; The other malignant disease often need be transplanted the hemopoietic function that recovers the patient by hematopoietic stem, and hematopoietic stem transplants whether successfully depend on neutrophil leucocyte and hematoblastic time of recovery, there are some researches prove that hematopoietic stem can the hematopoiesis of fast reconstitution neutrophil leucocyte after transplanting, but make platelet recovery also need long time to corresponding horizontal, therefore, clinically this class patient is generally still needed to import a considerable amount of thrombocytes at present, the supportive treatment of visible thrombocyte after for tumor chemoradiotherapy is also significant.But platelet transfusion at present has the risk that produces isoantibody and concurrent platelet transfusion resistance is arranged, can regulate and control intravital macronucleus hematopoiesis though clone the thrombopoietin TPO that comes out several years ago, as de SauvageFJ, Hass PE, people such as Spencer SD 1994 report (Stimulation ofmegakaryocytopoiesis and thrombopoiesis by the c-MPL ligand.Nature 1994 at Nature; 369:533.), but up to the present, clinical trial shows that the application of TPO shortens the aleukia phase that causes behind the chemicotherapy unsatisfactorily.
Clinical study finds that after the stem cell transplantation of routine, the same megalokaryocyte that is derived from body of infusion amplification in vitro can quicken the hematoblastic recovery of patient again.But in the peripheral blood and bleeding of the umbilicus of marrow, mobilization, Megakaryocytic quantity seldom, and according to the requirement of the clinical infusion of megalokaryocyte, the megalokaryocyte number of input will reach 1 * 10
5-2 * 10
6Cells/kg, a patient counts according to 50kg, needs total cell count of infusion will reach 5 * 10
6-1 * 10
8So, must provide a large amount of megalokaryocytes by the amplification in vitro technology, could satisfy requirements for clinical application.And import after the megakaryoblast to patient, the cell of input can further be bred in vivo, thereby increases platelet counts in the body, alleviates hematoblastic shortage.
The existing at present megalokaryocyte with amplification in vitro is used for clinical experiment, as gondola bone marrow transplantation center, from the peripheral blood of mobilizing, by external evoked Megakaryocytic generation, after megalokaryocyte is increased surplus in the of 50 times, apply it in the breast cancer disease human body of having accepted the transplanting of autologous peripheral blood progenitor cell, then patient no longer needs to import allotypic thrombocyte, and the patient of control group needs the platelet transfusion support.And the phenomenon of untoward reaction and infectation of bacteria do not appear in the patient of the outer cultured cells of receptor, and the clinical principium experiment shows that the megakaryoblast of amplification in vitro can be applied to clinicing aspect safely.Over past ten years clinically the demand to platelet transfusion approximately increased by 3 times, adopt alternate cell series products can effectively replenish the deficiency of present thrombocyte supply, thereby external evoked Megakaryocytic amplification research has important application value.
Vitro culture system been has has been researched and developed in some laboratories, from marrow, mobilizes and produces megakaryoblast blood transfusion product in peripheral blood and the bleeding of the umbilicus.People also mainly are devoted to seek the best of breed of cytokine at present, suitable medium and suitable culture systems (plastic containers, teflon surface container or automated cell perfusion bioreactor).1996, according to Schattner M, Lefebvre P, MingolelliSS, et al., Thrombopoietin-stimulated ex vivo expansion of human bone marrowmegakaryocytes[J] .Stem Cells.1996,14 (2): the 207-214. reported in literature, people such as Schattner adopt the IMDM that contains TPO, SCF, IL-3 and 1%HAS, 2.5%HS to cultivate BM CD34
+Or MNC, obtained the megalokaryocyte of larger amt; 1997, according to Bertolini F, BattagliaM, Pedrazzoli P, et al.Megakaryocytic progenitors can be generated ex vivo andsafely administered to autologous periipheral progenitor cell transplantrecipients[J] .Blood, 1997, the reported in literature of 89:2679～2685, people such as Bertolini adopt the serum-free culture system, at MGDF, SCF, IL-3, IL-6, IL-11, MIP-1a, under the effect of the FL various kinds of cell factor, cultivate the CD34 in the peripheral blood that comes from mobilization
+Cell, the MK cell that amplification in vitro was obtained on the 7th day has been used for clinical blood transfusion, is transplanted to and has quickened platelet recovery in the patient body.
But the initiator cell that above-mentioned document adopts is CD34 in peripheral blood or the marrow
+Cell, the initiator cell that external evoked megalokaryocyte produces can be the hematopoietic stem that derives from marrow, mobilization peripheral blood and the bleeding of the umbilicus.Compare with marrow with becoming human peripheral, the bleeding of the umbilicus source is abundant, is rich in CD34
+Cell has the ability that high hyperplasia and long-term marrow hemopoiesis are rebuild; And the immunogenicity of umbilical hemopoietic ancestral cells undeveloped mature still, can reduce the occurrence probability of GVHD and reduce the danger of allos virus infection.Clinical data confirms that the umbilical cord blood transplantation that HLA mates wholly or in part all can be rebuild heredity and malignant disease patient's hemopoietic function of bone marrow.Compare marrow and mobilize peripheral blood, contained CD34 in the bleeding of the umbilicus
+Cell, be to be suitable for initiator cell (the Helen Tao that external evoked megalokaryocyte generates, Leonie Gaudry, Alison Rice, et al.Cord blood is better than bonemarrow for generating megakaryocytic progenitor cells[J] .ExperimentalHematology, 1999,27:293-301.) these methods all are the bleeding of the umbilicus CD34 that directly goes out from fresh separated
+Cell goes out to send to induce Megakaryocytic generation, because CD34 in the Cord blood
+The content of cell is limited, absolute quantity is few, as inducing Megakaryocytic initiator cell, the Megakaryocytic efficient of inducing of how many direct influences of its quantity, cause the Megakaryocytic limited amount that induces, can't satisfy clinical requirement, therefore press for and find a kind of method to improve Megakaryocytic amplification in vitro cell effect.
Summary of the invention
The technical issues that need to address of the present invention be disclose a kind of from cord blood CD 34
+Cells in vitro is induced and is generated Megakaryocytic method, to overcome the above-mentioned defective that prior art exists, satisfies the needs of clinical treatment.
Technical conceive of the present invention is such:
CD34 in the single part of bleeding of the umbilicus
+Absolute magnitude is few, is unfavorable for the external Megakaryocytic generation of inducing in a large number, and the contriver has designed a kind of sectional type training strategy for this reason, earlier at amplification in vitro CD34
+Cell makes it induce again and is divided into megalokaryocyte.Adopt this method to improve by CD34
+Cell sets out to induce and generates Megakaryocytic efficient.
Method of the present invention comprises amplification in vitro bleeding of the umbilicus CD34 in the substratum that contains foetal calf serum and human stem cell factor (SCF), human thrombopoietin (TPO) and/or flt-3 part earlier
+Cell; And then adopt in the serum free medium that contains human thrombopoietin (TPO), Ro 24-7472/000-3 (IL-3) and/or people's grain-huge assembly G-CSF (GM-CSF) and induce Megakaryocytic generation.
According to optimized technical scheme of the present invention:
(1) the isolating mononuclearcell that comes from Cord blood is suspended in the substratum, adds the CD34 antibody of magnetic bead link coupled mouse anti human, 4 ℃ were reacted 20～40 minutes, and collected CD34 then
+Cell is resuspended in the substratum again;
The said isolating mononuclearcell that comes from Cord blood refers to the mononuclearcell that Cord blood is collected after lymphocyte separation medium (Ficoll) density gradient centrifugation;
Said substratum can adopt conventional IMDM substratum, the preferred Lam AC that adopts, Li K, Zhang XB, Li CK, et al.Preclinical ex vivo expansion of cord bloodhematopoietic stem and progenitor cells:duration of culture; The media, serumsupplements, and growth factors used; And engraftment in NOD/SCID mice[J] .Transfusion.2001,41 (12): the IMDM substratum that the 1567-76. document is mentioned; Serum free medium is Qiu L, Meagher R, Welhausen S, et al.Ex vivo expansion of CD34
+Umbilicalcord blood cells in a defined serum-free medium (QBSF-60) with early effectcytokines[J] .J Hematother Stem Cell Res.1999,8 (6): the QBSF of the QualityBiological Inc. that the 609-18. document is mentioned
R-60 (serum free medium or the Lazzari L that contain L-glutaminate, Lucchi S, Porretti L, et al.Comparison of different serum-free media for ex vivoexpansion of HPCs from cord blood using thrombopoietin, Flt-3 ligand, IL-6, and IL-11[J] .Transfusion.2001, the 41:718-719 document is mentioned the serum free medium CellGro that hematopoietic stem is cultivated that is used for of CellGenix TechnologieTransfer GmbH
RThe stemline of SCGM or Sigma company
TMIn the hematopoietic stem cell expansion substratum any.
According to optimized technical scheme of the present invention, the concentration of mononuclearcell in the IMDM substratum is 5 * 10
7～1 * 10
8Cells/300uL, the add-on of the CD34 antibody of magnetic bead link coupled mouse anti human is 20～100uL.
Said collection CD34
+Cell refers to, will with the good cell of CD34 antibody response of magnetic bead link coupled mouse anti human, join the separator column that places magnetic field, with the PBS washing that contains EDTA, human serum albumin, cell suspension slowly passes through, non magnetic cell (non-CD34
+Cell), gives a baby a bath on the third day after its birth time with PBS (containing 2mM EDTA) by wash-out.Separator column is withdrawn magnetic field, with the PBS washing that contains EDTA, human serum albumin, CD34
+Cell is washed out.
(2) CD34 that separation is obtained
+Cell inoculation places 37 ℃, 5%CO in the substratum that contains foetal calf serum, human stem cell factor (SCF), human thrombopoietin (TPO) and flt-3 part
2, in the incubator of 100% humidity, cultivate collecting cell after 4～7 days, change and use serum free medium, and add thrombopoietin (TPO), interleukin 3 (IL-3) and/or grain-giant cells G CFS (GM-CSF), cultivated 7-10 days.
In the substratum, the concentration range of various cytokines is in the combination of cytokines that is adopted: human stem cell factor (SCF): 5～100ng/mL; Human thrombopoietin (TPO): 5～100ng/mL; Flt-3 part: 5～100ng/mL.
The concentration range of each cytokine is in the serum free medium: people's grain-huge assembly G-CSF (GM-CSF): 0～100ng/mL; Human thrombopoietin (TPO): 10～100ng/mL; Ro 24-7472/000-3 (IL-3): 1～20ng/mL.
Inoculum density is 1 * 10
4Cells/mL～1 * 10
5Cells/mL.
Said substratum can adopt conventional IMDM substratum, the preferred Lam AC that adopts, Li K, Zhang XB, Li CK, et al.Preclinical ex vivo expansion of cord bloodhematopoietiic stem and progenitor cells:duration of culture; The media, serumsupplements, and growth factors used; And engraftment in NOD/SCID mice[J] .Transfusion.2001,41 (12): the IMDM substratum that the 1567-76. document is mentioned;
Said serum free medium can adopt the conventional substratum that does not contain serum, preferably adopts Qiu L, Meagher R, Welhausen S, et al.Ex vivo expansion of CD34
+Umbilical cordblood cells in a defined serum-free medium (QB SF-60) with early effectcytokines[J] .J Hematother Stem Cell Res.1999,8 (6): the serum free medium QBSF that contains L-glutaminate of the QualityBiological Inc. that the 609-18. document is mentioned
R-60 or Lazzari L, Lucchi S, Porretti L, et al.Comparison of different serum-free media for ex vivoexpansion of HPCs from cord blood using thrombopoietin, Flt-3 ligand, IL-6, and IL-11[J] .Transfusion.2001, the 41:718-719 document is mentioned the serum free medium CellGro that hematopoietic stem is cultivated that is used for of CellGenix TechnologieTransfer GmbH
RSCGM or stemline
TMIn the hematopoietic stem cell expansion substratum any.
The composition of substratum induces differentiation that significant effects is arranged to Megakaryocytic, according to the literature, there is the various kinds of cell factor (IL-1, IL-3, IL-6, IL-11, Flt3, FL-Ligand, SCF, GM-CSF, TPO) can in external or body, regulate Megakaryocytic propagation and/or maturation.Thereby the growth that synergistic effect starts cell can take place when these cytokines are used together, and wherein IL-3, GM-CSF start megalokaryocyte amplification and differentiation, and IL-3 can increase the number of Megakaryocytic absolute value and megakaryocyte colony in the liquid culture.TPO works to the macronucleus hematopoiesis and the hematoblastic generation of all levels, and TPO is external in vivo all activity, can produce the macronucleus colony by the hemopoietic progenitor cell when semisolid is cultivated, and produces megalokaryocyte when liquid culture.SCF itself is to the no effect of the formation of macronucleus colony, but it can with other cytokine (IL-3, IL-6, G-CSF, GM-CSF) compound action, thereby strengthen the ability that these cytokine stimulating megakaryocytes form.And concerning the cultivation of the early stage cell of hematopoiesis, SCF, FL are absolutely necessary.The contriver is surprised to find that GM-CSF, IL-3 in experiment, three kinds of combination of cytokines of TPO can be effectively from bleeding of the umbilicus CD34
+The Megakaryocytic generation of cell induction can improve Megakaryocytic ratio in the nutrient solution, but these three kinds of combination of cytokines do not help the amplification of total cell, and therefore Megakaryocytic absolute quantity is restricted.
The contriver adopts three kinds of cytokines of SCF+FL+TPO to stimulate CD34 in the nutrient solution earlier for this reason
+The propagation of cell adopts GM-CSF+IL-3+TPO or IL-3+TPO combination of cytokines to impel the method for megalokaryocyte differentiation again after one week, can effectively improve bleeding of the umbilicus CD34
+The Megakaryocytic efficient of cell induction.
In addition, foetal calf serum is to early stage bleeding of the umbilicus CD34
+The cultivation of cell is vital, but certain the unknown composition that is present in the foetal calf serum suppresses Megakaryocytic generation, and consider from the angle of clinical application, the contriver adopts the serum-free culture technology to induce megalokaryocyte to generate, the present invention adopts earlier and cultivates the sectional type culture method that is replaced with serum free medium again with containing foetal calf serum, can increase Megakaryocytic number when guaranteeing total cell amplification.
Adopt W.Piacibello, L.Garetto, M.Aglietta Ex vivo expansion ofmegakaryocytes[J] Transfusion Science, 2000,22:107-110, PantepAngchaisuksiri, Shawn R.Grigus, Patricia L et al.Secretion of a unique peptidefrom interleukin-2-sitimulated natural killer cells that induces endomitosis inimmature human megakaryocytes[J] .Blood, 2002,99:130-136. the method for reported in literature is to the cell counting after cultivating, detect the density of CFU-MK, CD41
+The ratio of cell, analysis CD41
+The polyploid of cell distributes, and the result shows that cell can increase 269.4 times, CD41 in the culture
+The ratio of cell can reach 46.2%, wherein 30% CD41
+Cell is 〉=8 times of somatocyte; 10000 cultured cells can obtain 92 CFU-MK.
Adopt method of the present invention from bleeding of the umbilicus CD34
+The cell induction megalokaryocyte is compared with additive method, Megakaryocytic being in similar proportion in the culture, and cell phenotype is identical, but therefore the cells expanded height induces the generation megalokaryocyte can significantly improve preparation efficiency in this way.
Embodiment
Embodiment 1
Bleeding of the umbilicus collect mononuclearcell, and with IMDM substratum washed twice, in the substratum of IMDM, concentration is 1 * 10 with cell suspension after lymphocyte separation medium (Ficoll) density gradient centrifugation
8Cells/300uL adds the CD34 antibody of 50uL magnetic bead link coupled mouse anti human, 4 ℃ of reactions 30 minutes, with the PBS washing that contains 2mM EDTA, 0.5% human serum albumin, in magnetic field with the non-CD34 on the separator column
+Cell washes, and contains the PBS of 2mMEDTA, 0.5% human serum albumin with CD34 with using again under the condition that breaks away from magnetic field
+The cell wash-out is resuspended in the IMDM substratum.
The CD34 that separation is obtained
+Cell is with 5 * 10
4The inoculum density of cells/mL is inoculated in the IMDM substratum that contains 10% foetal calf serum, adds human stem cell factor (SCF), human thrombopoietin (TPO), each 50ng/mL of flt-3 part in the substratum, places 37 ℃, 5%CO
2, in the incubator of 100% humidity, cultivate collecting cell after 7 days, change stemline with serum free medium (Sigma)
TMThe hematopoietic stem cell expansion substratum also adds thrombopoietin (TPO) 20ng/mL, interleukin 3 (IL-3) lng/mL, and grain-giant cells G CFS (GM-CSF) 25ng/mL places 37 ℃, 5%CO
2, in the incubator of 100% humidity, cultivated 7 days.Density, the CD41 of cell counting, detection CFU-MK
+The ratio of cell, analysis CD41
+The polyploid of cell distributes.Cell can increase 269.4 times, CD41 in the culture
+The ratio of cell can reach 46.2%, wherein 30% CD41
+Cell is 〉=8 times of somatocyte; 10000 cultured cells can obtain 92 CFU-MK.
Embodiment 2
Bleeding of the umbilicus collect mononuclearcell, and with IMDM substratum washed twice, in the substratum of IMDM, concentration is 1.5 * 10 with cell suspension after lymphocyte separation medium (Ficoll) density gradient centrifugation
8Cells/300ul adds the CD34 antibody of 100ul magnetic bead link coupled mouse anti human, 4 ℃ of reactions 30 minutes, with the PBS washing that contains 2mM EDTA, 0.5% human serum albumin, in magnetic field with the non-CD34 on the separator column
+Cell washes, and contains the PBS of 2mMEDTA, 0.5% human serum albumin with CD34 with using again under the condition that breaks away from magnetic field
+The cell wash-out is resuspended in the IMDM substratum.
The CD34 that separation is obtained
+Cell inoculation adds human stem cell factor (SCF) 100ng/mL in the substratum in the IMDM substratum that contains 10% foetal calf serum, human thrombopoietin (TPO) 50ng/mL, flt-3 part 25ng/mL place 37 ℃, 5%CO
2, in the incubator of 100% humidity, cultivate collecting cell after 4 days, change the serum free medium CellGro that hematopoietic stem is cultivated that is used for serum free medium CellGenix Technologie TransferGmbH
RAmong the SCGM, and add thrombopoietin (TPO) 50ng/mL, interleukin 3 (IL-3) 5ng/mL places 37 ℃, 5%CO
2, in the incubator of 100% humidity, cultivate collecting cell after 7 days, density, the CD41 of cell counting, detection CFU-MK
+The ratio of cell, analysis CD41
+The polyploid of cell distributes.Cell can increase 219.8 times, CD41 in the culture
+The ratio of cell can reach 53.9%, and wherein 15.3% CD41+ cell is 〉=8 times of somatocyte; 10000 cultured cells can obtain 153 CFU-MK.
Embodiment 3
Bleeding of the umbilicus collect mononuclearcell, and with IMDM substratum washed twice, in the substratum of IMDM, concentration is 6 * 10 with cell suspension after lymphocyte separation medium (Ficoll) density gradient centrifugation
7Cells/300ul adds the CD34 antibody of 20uL magnetic bead link coupled mouse anti human, 4 ℃ of reactions 40 minutes, with the PBS washing that contains 2mM EDTA, 0.5% human serum albumin, in magnetic field with the non-CD34 on the separator column
+Cell washes, and contains the PBS of 2mMEDTA, 0.5% human serum albumin with CD34 with using again under the condition that breaks away from magnetic field
+The cell wash-out is resuspended in the IMDM substratum.
The CD34 that separation is obtained
+Cell inoculation is in the IMDM substratum that contains 10% foetal calf serum, add human stem cell factor (SCF) 100ng/mL, human thrombopoietin (TPO) 50ng/mL in the substratum, place 37 ℃, 5%CO2 in the incubator of 100% humidity, cultivates collecting cell after 7 days, change is with serum free medium and add thrombopoietin (TPO) 20ng/mL, interleukin 3 (IL-3) 1ng/mL, grain one giant cells G CFS (GM-CSF) 50ng/mL cultivates collecting cell after 10 days.Density, the CD41 of cell counting, detection CFU-MK
+The ratio of cell, analysis CD41
+The polyploid of cell distributes.Cell can increase 256.4 times, CD41 in the culture
+The ratio of cell can reach 39.8%, wherein 43.2% CD41
+Cell is 〉=8 times of somatocyte; 10000 cultured cells can obtain 167 CFU-MK.
Claims (7)
1. from bleeding of the umbilicus CD34
+Cells in vitro is induced and is generated Megakaryocytic method, it is characterized in that, comprises amplification in vitro bleeding of the umbilicus CD34 in the substratum that contains foetal calf serum and human stem cell factor, human thrombopoietin and flt-3 part earlier
+Cell; And then adopt the serum free medium that contains human thrombopoietin, Ro 24-7472/000-3 and people's grain-huge assembly G-CSF to induce Megakaryocytic generation.
2. method according to claim 1 is characterized in that, the isolating mononuclearcell that comes from Cord blood is suspended in the substratum, adds the CD34 antibody of magnetic bead link coupled mouse anti human, and CD34 is collected in reaction then
+Cell, amplification in vitro bleeding of the umbilicus CD34 in the substratum that contains foetal calf serum and human stem cell factor, human thrombopoietin and flt-3 part again
+Cell.
3. method according to claim 1 is characterized in that, the substratum that contains foetal calf serum and human stem cell factor, human thrombopoietin and flt-3 part adopts the IMDM substratum of the foetal calf serum that contains 10-20%; Serum free medium adopts the stemline of Sigma company
TMHematopoietic stem cell expansion substratum or Quality Biological Inc. contain the serum free medium QBSF of L-glutaminase
R-60 or CellGenix Technologie Transfer GmbH be used for the serum free medium CellGro that hemopoietic stem cell and progenitor cell are cultivated
RAmong the SCGM any.
4. method according to claim 2 is characterized in that, the concentration of mononuclearcell in the IMDM substratum is 5 * 10
7～1 * 10
8Cell/300 microlitres, the add-on of the CD34 antibody of magnetic bead link coupled mouse anti human is 20～100 microlitres.
5. method according to claim 2 is characterized in that, the CD34 that separation is obtained
+Cell in the substratum that contains foetal calf serum and human stem cell factor, human thrombopoietin and flt-3 part, 37 ℃, 5%CO
2, cultivated under 100% humidity 4～7 days.
6. method according to claim 1 is characterized in that, cultivates 7-10 days in serum free medium.
7. according to each described method of claim 1～6, it is characterized in that, contain in the substratum of foetal calf serum and human stem cell factor, human thrombopoietin and flt-3 part, the concentration range of various cytokines is in the combination of cytokines that is adopted: human stem cell growth: 5-100ng/mL; Human thrombopoietin: 5-100ng/mL; Flt-3 part: 5-100ng/mL.
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| CN112574951B (en) * | 2020-12-08 | 2023-04-18 | 苏州方舟生物科技有限公司 | Method and system for in vitro production of human megakaryocytes and platelets |
| CN113249320B (en) * | 2021-05-26 | 2022-12-16 | 中国人民解放军海军军医大学第一附属医院 | Application of abscisic acid in promoting in-vitro differentiation of human induced pluripotent stem cells into megakaryocytes and platelets |
| CN113502263B (en) * | 2021-06-09 | 2023-05-23 | 华南理工大学 | Differentiation-promoting culture medium and method for promoting differentiation of CD34 positive cells into platelets |
| CN115612666B (en) * | 2021-07-16 | 2024-11-05 | 河络新图生物科技(上海)有限公司 | Method for preparing platelets in vitro and culture medium thereof |
| CN114634906B (en) * | 2022-01-26 | 2024-02-09 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | In vitro efficient megakaryocyte progenitor cell differentiation induction system with definite components and definite effects |
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