CN100453640C - Method of separating multipotent adult progenitor cells from umbilical cord blood - Google Patents
Method of separating multipotent adult progenitor cells from umbilical cord blood Download PDFInfo
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Abstract
The invention discloses the method for separating pluripotent ancestral cell from umbilical cord blood, comprising the following steps: using lymphoblast to separate umbilical cord blood monocyte, culturing for 3 days at culture medium with 8-15% bovine serum, throwing away cell which is not sticked on wall, replacing the culture medium with 2-5% bovine serum, bFGF, EGF, PDGF-BB, MCDB-201, ascorbic acid, anaflogistico and ITS addition agent, culturing, passage purifying, and separating. The method has the advantages of high separation rate. The cell phenotype, cell periodic time and evoked differentiation ability are the same.
Description
Technical field
The present invention relates to a kind of from Cord blood separating multipotent adult progenitor cells (multipotent adultprogenitor cells, method MAPC) relate in particular to external method by separating multipotent adult progenitor cells in the Cord blood.
Background technology
Stem Cell Engineering is as the fundamental research of organizational project, and the histoorgan reparation in future is had important role and far-reaching influence with substituting, and its key is to obtain being divided into the stem cell of required tissue.
According to the precedence difference that occurs in the ontogenetic process, stem cell can be divided into embryonic stem cell and adult stem cell.Owing at present embryonic stem cell differentiation and development regulatory mechanism is known little about it, embryonic stem cell source limited and transplant after have infinite multiplication and the danger of induced tumor, and the research and the application of embryonic stem cell related to reasons such as some ethics problems, embryonic stem cell is still remoter apart from clinical application.Along with stem cell biological is learned deepening continuously of research, in recent years find that there is the adult stem cell with multidirectional differentiation potential in some adult tissue, this class cell its particular organization of not only regenerating, and can under certain environment, change the cell that generates other tissue system, promptly interdepartmental system even stride the germinal layer differentiation and development.Because its source abundant (can derive from self), reason such as relate to and the ethics problem that exists is few, the clinical application that adult stem cell is transplanted more has the reality possibility.
The research majority of adult stem cell concentrates on the mescenchymal stem cell (MSC) of derived from bone marrow at present, MSC is verified can be divided into multiple human tissue cell, (Pittenger MF such as scleroblast, chondrocyte, adipocyte, myocyte, neurocyte for example, Mackay AM, Beck SC, et al.Multilineage potential of adult human mesenchymal stem cells.Science, 1999,284:143-147.).(Noort WA. is more and more paid attention in its effect at aspects such as hematopoiesis regulation and control, immunomodulatory, gene therapy and organizational projects, KruisselbrinkAB., in ' t Anker PS.Mesenchymal stem cells promote engraftment ofhuman umbilical cord blood-derived CD34+cells in NOD/SCID mice.Exp Hematol.2002; 30:870-878, Aggarwal S., Pittenger MF.Humanmesenchymal stem cells modulate allogeneic immune cell responses.Blood.2005; 105:1815-1822).But be used for the certain difficulty of cellular transplantation therapy existence owing to obtain a large amount of medullary cells, and marrow MSC number occurs with the increase at age and differentiation potential descends (D ' Ippolito G, Schiller PC, Ricordi C, et al.Age-related osteogenicpotential of mesenchymal stromal stem cells from human vertebral bonemarrow.J Bone Miner Res.1999; 14:1115-1122.), thereby be applied to the clinical certain difficulty that exists from now on.
Contain abundant hematopoietic stem/progenitor cells in the human Cord blood, after 1988 first routine cord blood stem cells were transplanted achieving success, cord blood stem cell was applied to clinical as a kind of important hemopoietic stem cell source.Studies show that in recent years, except hemopoietic stem cell, also exist the similar non-hematopoiesis of special and medulla mesenchyma cell to become body cell mass (Goodwin HS in the bleeding of the umbilicus, Bicknese AR, Chien SN, et al.Multilineage differentiation activity by cellsisolated from umbilical cord blood:expression of bone, fat, andneural markers.Biol Blood Marrow Transplant, 2001,7 (11): 581-8; Lee OK, Kuo TK, Chen WM, et al.Isolation of multipotent mesenchymalstem cells from umbilical cord blood.Blood.2004,103 (5): 1669-75;
G, Sensken S, Airey JA et.al.A Human Somatic Stem Cell fromPlacental Cord Blood with Intrinsic Pluripotent DifferentiationPotential.J Exp Med.2004,200 (2): 123-35), its outer appearnce is like inoblast, do not express hematopoietic cell mark (CD34, CD45, CD14, CD2, CD3, CD15, CD16, CD19, CD24, CD33, CD38, glycophorin A), express CD13, CD29, adhesion molecules such as CD44, people's mesenchyme cell sign SH1, the SH2 strong positive, the HLA-ABC positive and HLA-DR feminine gender.But different with mesenchymal stem cells MSCs is, its ratio is lower, per 10
6There is 0.05-2.8 in the Cord Blood Mononuclear Cell approximately, be lower than mesenchymal stem cells MSCs (per 10
6There is 2-5 in the mononuclearcell approximately, Koc, OH, Lazarus HM.Mesenchymal stem cells:heading into the clinic.BoneMarrow Transplant.2001; 27:235-239.), but this class cell may be more original, have more proliferation potential.Simultaneously, this class cell can be expressed the various kinds of cell factor, as SCF, and LIF, TGF-1b, M-CSF, GM-CSF etc. are to CD34
+Be better than marrow MSC (Kogler G on the amplification holding capacity of cell, RadkeTF, Lefort A, et al.Cytokine production and hematopoiesis supportingactivity of cord blood-derived unrestricted somatic stem cells.ExpHematol.2005; 33 (5): 573-83).What is more important, under specific inductive condition, this class cell has showed to multiple histiocytic differentiation potentials such as bone, cartilage, fat, nerve, liver, myocardial cells.In experimentation on animals, can promote mankind hemopoietic stem cell's the work of planting, repair tissue damage and improve disease symptoms.
Along with the foundation in cord blood stem cell storehouse in recent years, because Cord blood aboundresources, gather and simply and not can cause mother and neonatal damage, advantages such as cord blood stem cell has propagation and the self ability is strong, immunogenicity is weak, if the multipotent adult progenitor cells to the Cord blood source is developed and is used, can have far reaching significance to stem cell organizational project and application thereof for organizational project provides new seed cell.
The separating multipotent adult progenitor cells majority is still continued to use the adherent cultured method that goes down to posterity from Cord blood at present, is to carry out the sorting of immunomagnetic beads positive or negative by specific surface marker such as STRO-1, CD45 etc. but some are also arranged.Because this class cell content is few, the surface marker that lacks high degree of specificity, it is lower to be separated into power, as Bieback K, Kern S, Kluter H, Eichler H.Criticalparameters for the isolation of mesenchymal stem cells from umbilicalcord blood.Stem Cells.2004; 22 (4): 625-34. report, the condition of not adding somatomedin to contain 10% foetal calf serum is handled 59 cord blood samples only has 19 parts to separate successfully (28%).Domestic report similarly (29.17%, Zhu Meiling, Hu Yanfen, Wen Guanmei etc., people's placental blood interstital stem cell separation and Culture, Chinese Journal of Pathophysiology 2004,20 (9): 1743-1744), and there is the step complexity in the sorting that utilizes immunomagnetic beads to carry out, the cost problem of higher.Therefore press for and find a kind of simple mode to improve separating effect, provide more competent theoretical foundation and technological method for the Cord blood multipotent adult progenitor cells is applied to clinical treatment.
Summary of the invention
The technical issues that need to address of the present invention provide a kind of from Cord blood the method for separating multipotent adult progenitor cells, to overcome the above-mentioned defective that prior art exists, satisfy the needs of organizational project cell therapy.
Multipotent adult progenitor cells content in the Cord blood is less, and is lower with the success ratio that contains serum adherent culture partition method in-vitro separation merely commonly used, and cell breaks up easily under the condition of high serum.Simultaneously, the serum composition complexity though contain the favourable composition of many pair cells, also contains the deleterious composition of pair cell, the stdn difficulty that the mass discrepancy that exists between the different lot numbers also may make experiment and production.Be unfavorable for application from now on.But we find in previous experiments, reduce serum-concentration merely or carry out serum-free culture and be unfavorable for separation and Culture when not adding cell growth additive or cytokine.Therefore, we consider when reducing serum-concentration, adjust existing component of cultivating reagent, add suitable recombinant human cytokine and other growth additive, have to help reduce the disadvantageous effect of foreign sera and improve separating effect.
There is influence in the verified growth to marrow MSC of multiple somatomedin, because the multipotent adult progenitor cells in Cord blood source has cell phenotype similar with marrow MSC and differentiation potential, so we consider to select for use the suitable somatomedin that helps marrow MSC growth to promote the growth of the multipotent adult progenitor cells in Cord blood source.
Verified growth (the Chen Y that can promote cell of Urogastron (EGF) and human blood platelets deutero-somatomedin (PDGF), Rabinovitch PS.Platelet-derived growthfactor, epidermal growth factor, and insulin-like growth factor Iregulate specific cell-cycle parameters of human diploid fibroblastsin serum-free culture.J Cell Physiol.1989,140 (1): 59-67, LucarelliE, Beccheroni A, Donati D, et al.Platelet-derived growth factorsenhance proliferation of human stromal stem cells.Biomaterials.2003,24 (18): 3095-100.).The two pair cell kinetic parameter has similar influence, all acts on the early stage restriction point of G1, stimulates the expression of G1 early genes such as c-fos, c-myc, regulates the cell proportion that enters the cell cycle from stationary state.Gronthos S waits studies confirm that, when having only the EGF of existence and/or PDGF under serum-free condition, the growth of inoblast colony forming unit (CFU-F) just can be kept, and the colony size during the two coupling is greater than single usefulness (P＜0.05).Compare with the colony cultivation that contains 20% foetal calf serum, the CFU-F colony size that adds under the serum-free condition with EGF+PDGF obviously increases (P＜0.05) (Gronthos S, Simmons PJ The growth factorrequirements of STRO-1-Positive human bone marrow stromal precursorsunder serum-deprived conditions in vitro.Blood, 199585 (4): 929-940).The research of Prostatropin (bFGF) has been confirmed that also it is to keeping of mescenchymal stem cell propagation and differentiation potential (the Bianchi G that has vital role, Banfi A, Mastrogiacomo M et al.Ex vivo enrichment of mesenchymal cellprogenitors by fibroblast growth factor 2.Exp.CellRes.2003,287 (1): 98-105).
Except that cytokine, Gronthos S etc. find that simultaneously under serum-free condition, dexamethasone and xitix are one of key factors of CFU-F growth, if but only contain these two kinds of materials and do not add ectogenic somatomedin, can not promote the growth of CFU-F.So, reducing serum-concentration, add these materials when adding cytokine, help the growth of Cord blood multipotent adult progenitor cells.
To utilizing studies show that of the adherent separation and Culture marrow MSC that goes down to posterity, used kinds of culture medium, initial inoculum density, to change liquid time etc. for the first time all be the important factor (SotiropoulouPA that influences separating effect, Perez SA, Salagianni M, et al.Characterization of the optimalculture conditions for clinical scale production of humanmesenchymal stem cells.Stem Cells.2006,24 (2): 462-71.).Change the liquid overlong time for the first time, contact for a long time with hematopoietic cell and can suppress to have attached the multipotent adult stem cell and enter vegetative state, the number of the influence cell that obtains and colony formation ability.This may be due to the environmental degradation that causes owing to reasons such as nutrient consumption, necrocytosiss, also may promote that cytodifferentiation is relevant with hematopoietic cell excretory cytokine.And it is too short to change the liquid time for the first time, and cell fails fully to adhere to, also the final cell number that obtains of influence.Inoculum density influences complex interactions between the cell equally.When the first contact external environment of former generation cultured cells, influencing each other between the cell is very important, and cell may produce some growth-promoting activity materials and promote survival and growth mutually.In addition, cell density is excessive, and attached cell does not have enough spaces and stretches; Cell density is crossed when low, and cell forms the clone and reach time of the desired density that goes down to posterity very long again, causes primary cell " wear out ", makes its amplification ability and all obviously decline of differentiation potential after going down to posterity.
In the preliminary experiment in early stage, we also inquire into the Correlative Influence Factors of separating the Cord blood multipotent adult progenitor cells under low serum condition, the result shows, under the low serum condition that is adopted, preferably change 72 hours liquid time for the first time, adopt the DMEM/F12 substratum, initial inoculation density is 1 * 10
6/ cm
2Group helps the growth of Cord blood multipotent adult progenitor cells.Although to studies show that of the MSC of derived from bone marrow, under the 10-20% serum-concentration, low density (1 * 10
3/ cm
2) or extra-low density (1 or 3 cell/cm
2) inoculation helps separation amplification (the Sotiropoulou PA of MSC cell, PerezSA, Salagianni M, et al.Characterization of the optimal cultureconditions for clinical scale production of human mesenchymal stemcells.Stem Cells.2006,24 (2): 462-71.Colter D.C., Class.R, DiGirolamo C.M.et al.Rapid expansion of recycling stem cells incultures of plastic-adherent cells from human bone marrow.Proc NatlAcad Sci USA, 2000; 97 (7): 3213-3218.).But we find in preliminary experiment, under the condition that reduces serum-concentration, cross low initial inoculation density and be unfavorable for the separation of the non-hematopoiesis adult stem cell of Cord blood multipotency, this research to Tamama etc. is similar, they find, marrow MSC cultivates hanging down stronger (the Tamama K of tolerance of serum environment than low density when high-density culture, Fan VH, Griffith LG, et al.Epidermal growth factor as a candidate for ex vivoexpansion of bone marrow-derived mesenchymal stem cells, Stem Cells, 2006,24 (3): 686-95.).Simultaneously we find, gather to disengaging time≤4 hour and MNC cell count 〉=1.2 * 10
8Cord blood sample separation success ratio higher.Therefore, we have adopted these above-mentioned optimization technique factors in the method for the invention.
Method of the present invention comprises optimized choice Cord blood sample, utilize lymphocyte separation medium to separate the Cord blood mononuclearcell, in the substratum that contains 8～15% foetal calf serums, cultivated 3 days, discard not adherent cell, then be replaced by in the substratum that contains 2～5% foetal calf serums, rh-bFGF (bFGF), human epidermal growth factor (EGF), human blood platelets deutero-somatomedin (PDGF-BB), MCDB-201, xitix, dexamethasone and ITS additive and continue adherent culture, the purifies and separates that goes down to posterity Cord blood multipotent adult progenitor cells.
Technical scheme of the present invention:
1, after times dilutions such as phosphate buffered saline buffer (PBS) of human cord blood with 0.01M PH7.4, utilize lymphocyte separation medium to isolate mononuclearcell;
Said Cord blood is defined as: gather the blood from the umbilical vein that links to each other with the human fetal placenta.The preferred employing divided in 4 hours puerperiums mononuclearcell number 〉=1.2 * 10
8Human cord blood.The lymphocyte separation medium density that is used in the cellular segregation of the present invention is 1.077g/cm
3
2, isolating Cord blood mononuclearcell is suspended in the DMEM/F12 substratum that contains 10% foetal calf serum, with 0.5-2 * 10
6/ cm
2Density, preferred 1 * 10
6/ cm
2, be inoculated in the culturing bottle (preferably utilizing fibronectin (fibronectin) to wrap by culturing bottle in advance); Place 37 ℃, 5%CO
2, 100% humidity incubator in cultivate.
3, cultivate 48-72 hour (preferred 72 hours) after, discard not attached cell, change cell culture medium for containing rh-bFGF (bFGF), human epidermal growth factor (EGF), human blood platelets deutero-somatomedin (PDGF-BB) (the recombinant human cell growth factor concentration of above interpolation is generally 1-10ng/ml, preferably adopts 10ng/ml), 40%MCDB-201,2% foetal calf serum, 1 * Regular Insulin-Transferrins,iron complexes-Sodium Selenite (ITS), 10
-4M xitix, 10
-8Continue in the DMEM/F12 substratum of M dexamethasone to cultivate, change liquid 2-3 time weekly.
When 4, treating that above-mentioned 3 first culture merges near 70-80%, this culturing bottle is handled with 0.25% trypsinase, reclaimed cell then, the cell that obtains is counted.
5, with the cell that obtains with 5-8 * 10
3/ cm
2Density (preferred 8 * 10
3/ cm
2) be inoculated in the above-mentioned 3 described substratum and cultivate, the cultivation of when cell merges near 70-80%, going down to posterity;
6, carry out 5 operation repeatedly, the cultivation of going down to posterity;
The invention has the beneficial effects as follows:
The invention provides a kind of simple method, promptly under low serum condition, adopt the method that adds the adherent culture of isolated that goes down to posterity of cell growth additive from the Cord blood separating multipotent adult progenitor cells.Utilize present method can be effectively in the middle of the Cord blood separating multipotent adult progenitor cells, isolated cells can go down to posterity repeatedly external, has the identical immunophenotype of the non-hematopoiesis adult stem cell of Cord blood with report, the cell cycle detection shows the G0/G1 phase that is in more than 97%, and has the potential of multidirectional differentiation under suitable inductive condition.When the preferred Cord blood standard of utilizing the present invention to propose and isolation technique, can from 70% sample, isolate and multipotent adult progenitor cells, compare with other method to have and significantly be separated into the power advantage.Simultaneously, the application of low serum culture condition helps to reduce influence of serum, the stdn application that will be beneficial to from now on further experiment and will produce.
Embodiment
Of the present invention a kind of from Cord blood the method for separating multipotent adult progenitor cells, may further comprise the steps successively:
The first step: with doubly dilutions such as phosphate buffered saline buffers, utilize lymphocyte separation medium to separate mononuclearcell external Cord blood;
Second step: isolating Cord blood mononuclearcell was cultivated 48-72 hour in the substratum that contains 8～15% foetal calf serums, discarded not adherent cell;
The 3rd step: be replaced by in the substratum that contains 2～5% foetal calf serums, rh-bFGF bFGF, human epidermal growth factor EGF, human blood platelets deutero-somatomedin PDGF-BB, MCDB-201, xitix, dexamethasone and 1 * Regular Insulin-Transferrins,iron complexes-Sodium Selenite ITS additive and continue adherent culture;
The 4th step: the purifies and separates that goes down to posterity Cord blood multipotent adult progenitor cells.
Described external Cord blood is for gathering the blood from the umbilical vein that links to each other with the human fetal placenta, and preferred the employing divided in 4 hours puerperiums mononuclearcell number 〉=1.2 * 10
8Human cord blood.
Below in conjunction with embodiment the present invention is described in further detail:
Separation, purifying and the amplification cultivation of embodiment 1. Cord blood multipotent adult progenitor cells
Cord blood is collected mononuclearcell after lymphocyte separation medium density gradient centrifugation, and with DMEM/F12 substratum washing 2 times, with cell suspension in the DMEM/F12 substratum.
The mononuclearcell that separation is obtained is with 1 * 10
6/ cm
2Inoculum density be inoculated in the DMEM/F12 substratum that contains 10% foetal calf serum each T-25 culturing bottle 7ml substratum.Place 37 ℃, 5%CO
2, 100% humidity incubator in, cultivated 72 hours.Discard not attached cell, change cell culture medium for containing 40%MCDB-201,2% foetal calf serum, 10ng/ml bFGF, 10ng/ml EGF, 10ng/ml PDGF-BB, 10
-4M xitix, 10
-8Continue in the DMEM/F12 substratum of M dexamethasone, 1 * ITS to cultivate.Change liquid 2-3 time weekly.Cultivate the visible attached cell of Cord blood sample of back 70%, be dispersed at first, formed cell clone gradually in 12-14 days, be the spindle shape whirlpool shape growth of homogeneous.When treating that cell merges near 70%, this culturing bottle is handled with 0.25% trypsinase, reclaimed cell then, the cell that obtains is counted.Average acquisition 7.5 * 10 behind the cell dissociation of every bottle of individual layer fusion
5Individual multipotent adult progenitor cells.With the cell that obtains with 8 * 10
3/ cm
2Density be inoculated in the above-mentioned substratum and cultivate the cultivation of when cell merges near 70-80%, going down to posterity.
The surface antigen characteristic of embodiment 2. Cord blood multipotent adult progenitor cells and cell cycle are detected
Get the amplification Cord blood multipotent adult progenitor cells in 3 generations, remove substratum.PBS washes twice, with trypsin 0.25%) handle, reclaim cell then, the cell that obtains is counted.Make 5 * 10 with PBS
5The single cell suspension of/ml, each pipe add 500 μ l, add the anti-people CD3 of 10 μ l, CD34, CD11a, CD29, CD105, CD45, CD44, CD73, CD49d, CD49e, CD31, CD62L, CD14, HLA-DR, CD144, vWF and anti-mouse IgG respectively
1Homotype contrast fluorescence antibody.4 ℃ of lucifuges were hatched 30 minutes, added the 1mlPBS washing, abandoned supernatant, added the PBS that 300 μ l contain 1% Paraformaldehyde 96, and streaming detects.It is positive to find that CD29, CD44, CD105, CD49d, CD73 express, CD34, CD45, CD11a, CD14, CD31, HLA-DR, CD144, vWF, CD62L feminine gender.
In the logarithmic phase of cell growth, peptic cell, 70% cold ethanol is fixed 30 minutes for 4 ℃, PBS liquid washed cell 2 times, cell suspension in 0.5mlPBS, is added 10mg/ml RNaseA 10 μ l, after 30min is hatched in 37 ℃ of water-baths, put into ice bath immediately, add 5 μ g/ml PI dyeing (4 ℃, 30 minutes), flow cytometer detects, the result shows that the cell above 97% is in G
0/ G
1Phase.
Embodiment 3. Cord blood multipotent adult progenitor cells induce differentiation characteristic
Digestion amplification in vitro the 3rd generation multipotent adult progenitor cells, with 5 * 10
3/ cm
2Density be inoculated in 6 orifice plates of placing cover glass in advance.When cell reached the 60%-70% fusion, every hole was changed and is contained 10
-7The dexamethasone of mol/L, 10mmol/L β-phospho-glycerol, 0.05mol/L the Osteoblast Differentiation induced liquid 2ml of the DMEM/F-12 of vitamins C and 10%FCS carries out 3 weeks of inducing culture, change liquid weekly 2 times, took out slide in 14,21 days respectively at inductive, fixing, observe morphological change and carry out histological stain.
Digestion amplification in vitro the 3rd generation multipotent adult progenitor cells, with 5 * 10
3/ cm
2Density be inoculated in 6 orifice plates of placing cover glass in advance.When cell reached the 60%-70% fusion, every hole was changed and is contained 10
-8The fat induction liquid 2ml that becomes of the DMEM/F-12 of mol/L dexamethasone, 10 μ g/ml Regular Insulin, 0.5mmol/L IBMX and 200 μ mol/L INDOMETHACIN and 10%FCS carries out inducing culture, changed liquid once in 3～4 days, when in observing cytoplasm, having fat vacuole to form, nutrient solution is removed in suction, the PBS washing once, 70% alcohol fixation 10min dyes with Oil Red dye liquor.
The Cord blood multipotent adult progenitor cells after 14 days, is observed sodium alizarinsulfonate dyeing and is shown red calcification apposition under the osteogenic induction environment under the mirror; Von Kossa dyeing shows the calcification matrix precipitation of black.Becoming under the fat induced environment Oil Red dye liquor stained positive.Show that cell has the potential to skeletonization and lipoblast differentiation.
Claims (10)
1, a kind of from Cord blood the method for separating multipotent adult progenitor cells, may further comprise the steps successively:
The first step: with doubly dilutions such as phosphate buffered saline buffers, utilize lymphocyte separation medium to separate mononuclearcell external Cord blood;
Second step: isolating Cord blood mononuclearcell was cultivated 48-72 hour in the substratum that contains 8～15% foetal calf serums, discarded not adherent cell;
The 3rd step: be replaced by in the substratum that contains 2～5% foetal calf serums, rh-bFGF bFGF, human epidermal growth factor EGF, human blood platelets deutero-somatomedin PDGF-BB, MCDB-201, xitix, dexamethasone and 1 * Regular Insulin-Transferrins,iron complexes-Sodium Selenite ITS additive and continue adherent culture;
The 4th step: the purifies and separates that goes down to posterity Cord blood multipotent adult progenitor cells.
2, according to claim 1 from Cord blood the method for separating multipotent adult progenitor cells, it is characterized in that described external Cord blood is for gathering the blood from the umbilical vein that links to each other with the human fetal placenta, adopt and divide in 4 hours puerperiums mononuclearcell number 〉=1.2 * 10
8Human cord blood.
3, according to claim 1 from Cord blood the method for separating multipotent adult progenitor cells, it is characterized in that described second the step be: isolating Cord blood mononuclearcell is suspended in the DMEM/F12 substratum that contains 10% foetal calf serum, with (0.5-2) * 10
6/ cm
2Density, be inoculated in the culturing bottle; Place 37 ℃, 5%CO
2, 100% humidity incubator in cultivated 72 hours.
4, according to claim 3 from Cord blood the method for separating multipotent adult progenitor cells, it is characterized in that described culturing bottle is to utilize fibronectin to wrap the culturing bottle of quilt in advance.
5, according to claim 3 from Cord blood the method for separating multipotent adult progenitor cells, it is characterized in that described density is 1 * 10
6/ cm
2
6, according to claim 1 from Cord blood the method for separating multipotent adult progenitor cells, the cell culture medium that it is characterized in that described the 3rd step is for containing 40%MCDB-201,2～5% foetal calf serums, 1-10ng/ml rh-bFGF bFGF, 1-10ng/ml human epidermal growth factor EGF, 1-10ng/ml human blood platelets deutero-somatomedin PDGF-BB, 1 * Regular Insulin-Transferrins,iron complexes-Sodium Selenite ITS, 10
-4M xitix, 10
-8The DMEM/F12 substratum of M dexamethasone.
7, according to claim 1 from Cord blood the method for separating multipotent adult progenitor cells, it is characterized in that described the 3rd step changes substratum 2-3 time weekly.
8, according to claim 6 from Cord blood the method for separating multipotent adult progenitor cells, it is characterized in that described the 4th step comprises:
A. when the first culture in the 3rd step merges near 70-80%, this culturing bottle is handled with trypsinase, reclaimed cell then, the cell that obtains is counted;
B. the cell that a is obtained is with (5-8) * 10
3/ cm
2Density be inoculated into above-mentioned the 3rd step and cultivate the cultivation of when cell merges near 70-80%, going down to posterity in the described substratum; Carry out the operation of b repeatedly, the cultivation of going down to posterity.
9, according to claim 1 from Cord blood the method for separating multipotent adult progenitor cells, it is characterized in that described phosphate buffering liquid concentration is 0.01M, pH=7.4; Lymphocyte separation medium proportion is 1.077g/cm
3
10, according to claim 1 from Cord blood the method for separating multipotent adult progenitor cells, it is characterized in that foetal calf serum concentration is that foetal calf serum concentration is 2% in 10%, the three step in described second step.
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| CN102978156A (en) * | 2012-11-13 | 2013-03-20 | 湖州市中心医院 | Expansion in vitro purification culture method of mesenchymal stem cells and culture medium |
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| CN103409366A (en) * | 2013-02-20 | 2013-11-27 | 潘骏 | Culture medium of adult multipotent adult progenitor cells and method for culturing multipotent adult progenitor cells by using same |
| CN104611295A (en) * | 2015-02-06 | 2015-05-13 | 华南农业大学 | Method for constructing machin progenitor leydig cell immortal line |
| CN109536443A (en) * | 2018-12-10 | 2019-03-29 | 天津长和生物技术有限公司 | Mescenchymal stem cell, cell culture medium and cultural method and application |
| US20200246390A1 (en) * | 2019-02-01 | 2020-08-06 | Abt Holding Company | Multipotent adult projenitor cells for treatment of ich |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1287166A (en) * | 1999-09-08 | 2001-03-14 | 何清华 | Separation, extracorporeal culture, preparation and application of human primitive mesenchymal stem cell population |
| CN1630717A (en) * | 2002-02-19 | 2005-06-22 | 美迪宝斯特有限公司 | Isolation and culture-expansion methods of mesenchymal stem/progenitor cells from umbilical cord blood and differentation method of umbilical cord blood-derived mesenchymal stem/progenitor cells into |
| US20040203142A1 (en) * | 2003-04-14 | 2004-10-14 | Reliance Life Sciences Pvt. Ltd. | Growth of neural precursor cells using umbilical cord blood serum and a process for the preparation thereof for therapeutic purposes |
| WO2005038014A1 (en) * | 2003-10-17 | 2005-04-28 | Innovative Dairy Products Pty Ltd As Trustee For The Participants Of The Cooperative Research Centre For Innovative Dairy Products | Isolation of stem cell-like cells and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978156A (en) * | 2012-11-13 | 2013-03-20 | 湖州市中心医院 | Expansion in vitro purification culture method of mesenchymal stem cells and culture medium |
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