CN105018422A - Large-scale preparation method of human umbilical cord-mesenchymal stem cell (hUC-MSC) - Google Patents

Abstract

The invention discloses a large-scale preparation method of human umbilical mesenchymal stem cell (hUC-MSC). The method comprises the following steps: collecting an umbilical cord and a placenta; separating and extracting the hUC-MSC from the umbilical cord, and carrying out amplification culture by using a traditional DMEM (dulbecco's modified eagle medium)/F12 culture solution containing fetal calf serum (FBS) in the early period; and carrying out hUC-MSC reculture by using the placental homogenate extraction (PHE) with the same source as the umbilical cord instead of the FBS in the later period, thereby obtaining the hUC-MSC raw material cells. On the premise of not using any commercial serum-free culture medium, the method can implement in-vitro large-scale preparation of the hUC-MSC, solves the problem of residues of bovine serum, and saves abundant cost.

Description

A kind of method prepared by human umbilical cord mesenchymal stem cells mass-producing

Technical field

The present invention relates to a kind of method prepared by human umbilical cord mesenchymal stem cells mass-producing.

Background technology

Mescenchymal stem cell (Mesenchymal Stem Cell, MSC) extensively distributes as a kind of adult stem cell and is in whole body Various Tissues, can mass propgation amplification in vitro, and under given conditions inwardly, in, the cytodifferentiation in outer three germinal layers source.MSC is the another scientific research focus after embryonic stem cell and hemopoietic stem cell, has become the practicality stem cell of 21st century treatment multiple systems disease.It has potential clinical value in immunomodulatory and tissue repair, is progressively applied to the treatment of the diseases such as systemic lupus erythematous, bone and muscle decline property disease, cardiovascular and cerebrovascular diseases, hepatopathy, brain and marrow nerve injury, senile dementia in recent years.

Mescenchymal stem cell (Mesenchymal Stem Cell, MSC) is the brand-new biological products based on stem-cell therapy.Domesticly at present there is no this type of launch, what abroad take the lead in developing this series products is Osiris company of the U.S., its product (trade(brand)name: Prochymal ^?) Canada and new southwestern government permission listing has successively been obtained in 2012.Prochymal ^?allosome mescenchymal stem cell (the Human Bone Marrow Mesenchymal Stem Cells deriving from people's marrow, hBM-MSC), be used for the treatment of graft versus host disease (GVH disease) (the Graft vs.Host Disease produced in hematopoietic stem cell transplantation, GvHD), one of its major function is immunoregulation effect.In addition, Prochymal ^?the II phase clinical study of type i diabetes was also first used for the treatment of in 2008.In addition, also there is similar product (trade(brand)name: Heaticellgram-AMI in Korea S ^?) in 2011 in Korean market, this product is autologous bone marrow mesenchymal stem cells preparation, and indication is acute myocardial infarction; Product (trade(brand)name: PluriPad is being ground by Pluristem company of Israel ^?) obtained FDA clinical study approval in 2011 in the U.S., for the repairing and treating of the acral tissue necrosis that diabetes cause, its composition is Human plactnta/umbilical cord mesenchymal stem cells.In addition, the clinical protocol of the relevant MSC cell therapy of FDA approval also comprises: 1. MSC venoclysis treatment Crohn is sick; 2. MSC topical application treatment periodontal disease; 3. MSC venoclysis treatment myocardial infarction; 4. the marrow MSC of MSC reparation this month plate and 5. G-CSF mobilization treats myocardial infarction etc.In domestic clinical study, the selection of MSC indication is comparatively extensive, intends being used for prevention or treatment GvHD, myocardial infarction, Cranial defect, diabetic foot necrotizing nasciitis, hepar damnification or necrosis of femoral head etc.

MSC's is extensively tissue-derived, as marrow, fat, umbilical cord.Wherein umbilical cord is connected in cord structures between embryo's umbilical region and placenta, is wherein rich in the multiple ancestral cells such as hematopoiesis, mesenchyme, nerve and endothelium.Compared with the MSC of derived from bone marrow, the advantage of people's umbilical cord derived mesenchymal stem cell is: collection process relatively simply, without wound, placental barrier makes umbilical cord be subject to virus and little bacterial contamination, the immune primitiveness of bleeding of the umbilicus to reduce the probability of transplanting rear acceptor generation rejection, can rise in value fast in culture system in vitro, the 4-5 that can rise in value for after going down to posterity 3-5 days doubly, in Secondary Culture 10 generation, above cell can increase 5-10 doubly, and speedup speed is without obvious reduction, go down to posterity stable.In addition, there is not ethnics Problem, stronger ability of going back to the nest and be easy to the advantages such as gene transfection and become and implement the desirable cell of cell therapy in human umbilical cord mesenchymal stem cells, and has mescenchymal stem cell and the unrivaled superiority of other adult stem cells of derived from bone marrow in the targeted therapy of tumour, organizational project and tissue repair.

The application prerequisite of MSC in cell therapy, gene therapy and organizational project is MSC mass-producing, standardization is cultivated.In order to realize mass-producing, industrialization; reach precise hard_drawn tuhes production process, reduce production cost, be convenient to downstream work and improve the quality of biological products and security, make the cell culture process continuing to optimize cell culture technology, optimization and selection suitable seem particularly important.Therefore easy, efficient, the safe culture process of human umbilical cord mesenchymal stem cells is set up very necessary.

But hUC-MSC prepared by traditional culture process often exists problems, because need in preparation process to introduce multiple allogenic material.Therefore the various residue problems brought limit the exploitation of hUC-MSC clinical application product, and as bovine serum, microbiotic, pancreatin etc., wherein bovine serum residue problem most is representative.

Traditional MSC nutrient solution all adopts basic medium such as DMEM or α MEM to add certain proportion FBS as complete culture solution usually.Although these traditional components can maintain the stem cell properties of MSC vitro culture, the uncertainty of FBS composition and heterology limit downstream research and the treatment use of MSC.Therefore the residue problem of foetal calf serum is human umbilical cord mesenchymal stem cells clinical application problem demanding prompt solution.The report in document " the external preparation strategy of clinical study mescenchymal stem cell " such as Wu Zuze, Pei Xuetao uses serum free culture system to solve the residual problem of bovine serum.Had commercialization MSC serum free medium on sale on the market at present, but it is expensive, therefore, it is very undesirable that serum free medium is applied to MSC industrialization amplification.Simultaneously according to " human body cell Therapy study and quality of the pharmaceutical preparations control techniques governing principle " relevant regulations, as commodity in use substratum must provide its further elements in stem cell production process, but usually not informing further elements owing to relating to secret of the trade businessman, still there is hidden danger in the security of therefore its clinical application.In addition, have in many Research Literatures and mention that various bovine serum substitutes the factor, as the platelet lysates factor etc., but it belongs to blood based article and price limits the application in MSC industrialization equally.Human serum must not be contained in clear stipulaties cell culture fluid in " Chinese Pharmacopoeia " 2010 editions the 3rd " biological products production calibrating zooblast matrix composition and vertification regulation ".And for the medicine that FBS must be used as supplementary material, Chinese Pharmacopoeia all proposes correlated quality requirement, wherein important and have representative index to be bovine serum residual quantity most, usually using bovine serum albumin as Testing index.At present, there is no any laws and regulations and carry out quality standard specification to the bovine serum of cell series products is residual." Chinese Pharmacopoeia " 2010 editions the 3rd has made clear and definite specification of quality to vaccine product, requires that its bovine serum residual quantity must not more than 50ng/ agent.HUC-MSC clinical plan dosage reference: Jun Liang. Allogenic mesenchymal stem cells transplantation in refractory systemic lupus erythematosus:a pilot clinical study.Ann Rheum Dis 2010; 69:1423 – 1429, is generally 1 × 10 ⁶individual cell/kg.If in mean body weight 50kg, the cell concentration of potion MSC is 6 × 10 ⁷individual cell.With reference to vaccine bovine serum residual mass standard in " Chinese Pharmacopoeia ", then injection hUC-MSC bovine serum residual quantity must not more than 1ng/ 1,000,000 cell.The technique using traditional foetal calf serum to cultivate MSC is difficult to reach this specification of quality.The anaphylaxis that can directly cause in MSC clinical application that exceeds standard of bovine serum residual quantity, security can not be ignored.Therefore also effectively the preparation method of the human umbilical cord mesenchymal stem cells of control bovine serum residual quantity is imperative to set up a kind of mass-producing.

Summary of the invention

The object of the invention is to solve the problem, provide a kind of can the method for bovine serum residual quantity effectively in control hUC-MSC raw cell.

To achieve these goals, the technical solution used in the present invention is as follows:

A method prepared by human umbilical cord mesenchymal stem cells mass-producing, comprises the following steps:

(1) umbilical cord tissue is got, with containing dual anti-D-hank ' s liquid soaking disinfection;

(2) umbilical cord is cut into 1 ~ 2 ㎝;

(3) adopt D-hank ' s liquid repetitive scrubbing again, remove remained blood;

(4) umbilical cord is continued subtract broken washing, to washings clarification without color;

(5) separation and Extraction hUC-MSC single cell suspension from umbilical cord;

(6) take DMEM/F12+10%FBS as complete culture solution, cultivate unicellular for hUC-MSC in 5%CO2,37 DEG C of incubators, and every 2 ~ 3 days change a nutrient solution;

(7) until cytogamy degree about 80% time, had digestive transfer culture;

(8) repeating step (6) ~ (7) several times;

(9) repeating step (8), wherein replaces DMEM/F12+10% FBS with DMEM/F12+5%FBS+5%PHE;

(10) repeating step (8) several times, wherein replace DMEM/F12+10% FBS with DMEM/F12+10% PHE;

(11) until cytogamy degree about 80% time, trysinization;

(12) collecting cell suspension, 2000 × g, 5min are centrifugal;

(13) abandon supernatant liquor, obtain hUC-MSC cell precipitation.

Wherein, FBS is foetal calf serum, and PHE is placenta homogenate.

The preparation method of above-mentioned placenta homogenate is as follows:

(1) get umbilical cord homology placenta, denuded amniotic membrane, collect chorionic villi;

(2) damping fluid fully washs remained blood in chorionic villi;

(3) chorionic villi is carried out homogenate, and dilute tissue homogenate with basic culture solution;

(4) frozen under the condition of-80 DEG C;

(5) thaw under the condition of 4 DEG C;

(6) repeating step (4) ~ (5), make tissue homogenate multigelation twice;

(7), after thawing under the condition of 4 DEG C, ultrasonic wave is adopted to carry out fragmentation;

(8) 12000 × g, 20min, 4 DEG C centrifugal, collects supernatant liquor, then filter supernatant liquor, collects filtrate, obtain placenta homogenate.

What deserves to be explained is, placenta homogenate need be preserved under the condition of-80 DEG C.

The present invention compared with prior art, has the following advantages and beneficial effect:

(1) mescenchymal stem cell prepared of method of the present invention, its bovine serum residual quantity meets the requirement of Chinese Pharmacopoeia to related preparations, and can preserve for a long time and not lose that it is active, and operation is simple.Through FCM analysis, and vitro differentiation experimental identification, it is consistent that its cellular form of hUC-MSC, Surface Antigen and one-tenth fat Osteoblast Differentiation potential that placenta homogenate replaces FBS to cultivate obtaining and FBS cultivate the MSC obtained, and all meets international cell therapy association to the regulation of the minimum standard that MSC identifies.Security aspect, checked by external tumorigenicity, result shows without considerable change.Method of the present invention has wide potential applicability in clinical practice.

(2) in the present invention, foetal calf serum and umbilical cord homology placenta homogenate are combined; for the external mass-producing preparation of hUC-MSC; both ensure that security that placenta originates, cost-saving; and reach the object of the residual quantity reducing foreign protein foetal calf serum, for the clinical application of hUC-MSC provides possibility.

(3) placenta homogenate of the present invention contains abundant cytokine and chemokine, is enough to the proliferation and growth potential maintaining stem cell.Compared with FBS, placenta homogenate is source of the same race, and sensitization is lower.Compared with commercialization serum free medium, placenta homogenate is drawn materials natural, and economical.It is consistent that its cellular form of hUC-MSC, Surface Antigen and the one-tenth fat Osteoblast Differentiation potential that use technique after improving to cultivate to obtain and FBS cultivate the MSC obtained, and all meets international cell therapy association to the regulation of the minimum standard that MSC identifies.

Accompanying drawing explanation

Fig. 1 is the cellular form of hUC-MSC.Wherein, A is the MSC obtained with umbilical cord tissue block creep plate method; B is be the obtained hUC-MSC of DMEM/F12+10% PHE with the obtained hUC-MSC of DMEM/F12+5% FBS+5% PHE, D with the obtained hUC-MSC of DMEM/F12+10%FBS, C.

Fig. 2 (a) ~ Fig. 2 (c) is hUC-MSC surface antigen detected result.Wherein, A is be that the obtained P7 of DMEM/F12+10% PHE is for hUC-MSC with the obtained P5 of DMEM/F12+5% FBS+5% PHE for hUC-MSC, C with the obtained P4 of DMEM/F12+10%FBS for hUC-MSC, B.

Fig. 3 is hUC-MSC differentiation potential detected result.Wherein, A, D are with the obtained hUC-MSC alizarin red S of DMEM/F12+10%FBS and oil red O stain, B, E are with the obtained hUC-MSC alizarin red S of DMEM/F12+5% FBS+5% PHE and oil red O stain, and C, F are the obtained hUC-MSC alizarin red S of DMEM/F12+10% PHE and oil red O stain.

Fig. 4 is that clone's colony (CFU) of hUC-MSC is formed.

Fig. 5 is hUC-MSC bovine serum residue detection result.Wherein, A is the residual quantity of the hUC-MSC BSA obtained with DMEM/F12+10%FBS, and B is the obtained hUC-MSC BSA residual quantity of DMEM/F12+5% PHE, is the obtained hUC-MSC BSA residual quantity of DMEM/F12+10% PHE.

Fig. 6 (a), Fig. 6 (b) are hUC-MSC lymphocyte Dual culture (MLR) detected result.Wherein, Fig. 6 (a) is the hUC-MSC one-way MLR result after PHE cultivates; Fig. 6 (b) is the hUC-MSC two-way MLR result after PHE cultivates.

Fig. 7 is the external tumorigenicity inspection of hUC-MSC (soft-agar cloning forms test).Wherein, A is that negative control MRC-5 soft-agar cloning is formed; B is that positive control Hela soft-agar cloning is formed; C is that hUC-MSC soft-agar cloning prepared by FBS culture system is formed; D is that hUC-MSC soft-agar cloning prepared by FBS and PHE co-culture system is formed; E is that hUC-MSC soft-agar cloning prepared by PHE culture system is formed.

Embodiment

Below in conjunction with drawings and Examples, the invention will be further described, and embodiments of the present invention include but not limited to the following example.

Embodiment 1

Tissue block creep plate method extracts hUC-MSC:

Pluripara's informed consent, gathers the healthy fetal cord of mature Cesarean esction.Before umbilical cord acquisition, puerpera need do the detections such as antibody of AIDS virus, hepatitis B virus antibody, antibody of HCV, syphilis helicoid antibody, gpt, mycoplasma, all qualifiedly can gather after guaranteeing security.

(1) umbilical cord tissue is got, containing dual anti-medicining liquid dipping;

(2) umbilical cord is cut into 1 ~ 2 ㎝;

(3) adopt D-hank ' s liquid repetitive scrubbing umbilical cord, remove remained blood on umbilical cord and clarify without color to washing lotion;

(4) umbilical cord tissue is cut into suede velvet-like, inoculation culture bottle;

(5) inversion is put in 5%CO2,37 DEG C of incubators, leaves standstill for some time;

(6) add the DMEM/F12 containing 10%FBS, 10ml, cultivate in 5%CO2,37 DEG C of incubators, within every 2 ~ 3 days, change a nutrient solution;

(7) until cytogamy degree about 80% time, nutrient solution and umbilical cord tissue block is abandoned;

(8) add 0.25% trysinization, stop with the DMEM/F12 containing 10%FBS;

(9) collecting cell suspension, 1000rpm, 5min are centrifugal;

(10) abandon supernatant liquor, the DMEM/F12 re-suspended cell containing 10%FBS precipitates;

(11) be re-seeded into new culturing bottle with the ratio 1:4 that goes down to posterity, in 5%CO2,37 DEG C of incubators, continue nutrient solution;

(12) repeat (7) ~ (11), the expansion realizing human umbilical cord mesenchymal stem cells is cultivated again;

(13) when cell confluency degree reaches 80% (see Figure 1A), trysinization, collects single cell suspension.

Embodiment 2

Enzyme digestion extracts hUC-MSC:

(1) get umbilical cord tissue, soak with containing dual anti-thimerosal;

(2) umbilical cord is cut into 1 ~ 2cm;

(3) adopt D-hank ' s liquid to carry out repetitive scrubbing, remove remained blood to washing lotion clarification without color;

(4) umbilical cord tissue is shredded, add 0.2% collagenase 37 DEG C and carry out digestion 2hr;

(5) Digestive system is carried out filter (100 order);

(6) filtrate is collected, and under the condition of 200 × g, centrifugal 5min;

(7) abandoning supernatant, precipitates with the DMEM/F12 re-suspended cell containing 10%FBS;

(8) repeating step (6), (7);

(9) cell is placed in 5%CO2,37 DEG C of incubators and cultivates, within every 2 ~ 3 days, change a nutrient solution;

(10) until cytogamy degree about 80% time (see Figure 1B), carry out trysinization, obtain single cell suspension;

(11) to go down to posterity inoculation with 1:4;

(12) repeating step (9) ~ (11), the expansion realizing human umbilical cord mesenchymal stem cells is cultivated again.

Embodiment 3

Serum and placenta homogenate are mixed for hUC-MSC vitro culture:

In order to realize mass-producing, people's umbilical cord MSC to cultivate and still to use traditional nutrient solution and DMEM/F12+10%FBS early stage, but in order to reduce the residual of xenobiotic too bovine serum, the amplification later stage replaces FBS completely with PHE.But MSC is comparatively responsive to growing environment, in order to ensure that MSC does not occur unconformable situation because of the sharply change of culture condition, the present invention adopts the progressively method of substitution to carry out transition cultivation.Operate as follows:

(1) single cell suspension that embodiment 1 obtains is collected;

(2) 1000rpm, 5min are centrifugal;

(3) abandoning supernatant, precipitates with DMEM/F12+5%FBS+5%PHE re-suspended cell;

(4) be re-seeded into new culturing bottle with the ratio 1:4 that goes down to posterity, in 5%CO2,37 DEG C of incubators, continue nutrient solution;

(5) when cell confluency degree reaches 80% (see Fig. 1 C), trysinization, collects single cell suspension.

Embodiment 4

Placenta homogenate alternative serum is used for hUC-MSC vitro culture:

After mixed transition is cultivated, placenta homogenate replaces FBS to be used for people's umbilical cord MSC vitro culture completely.

(1) single cell suspension that embodiment 2 obtains is collected;

(2) 1000rpm, 5min are centrifugal;

(3) abandoning supernatant, precipitates with DMEM/F12+10% PHE re-suspended cell;

(4) be re-seeded into new culturing bottle with the ratio 1:4 that goes down to posterity, continue to cultivate in 5%CO2,37 DEG C of incubators;

(5) when cell confluency degree reaches 80% (see Fig. 1 D), trysinization, collects single cell suspension.

(6) 1000rpm, 5min are centrifugal;

(7) abandon supernatant liquor, obtain people's umbilical cord MSC.

For the hUC-MSC prepared according to aforesaid method of the present invention, the people MSC formulated according to " international cell therapy association " (ISCT) identifies minimum standard statement of requirement, should have following characteristics:

1. can adherent growth in frosting;

2. cell surface molecule mark CD73, CD90, CD105, its positive rate is not less than 95%; And the positive rate of CD34, CD45, CD19, CD14 and HLA-DR must not higher than 2%;

3. os osseum cell, adipocyte and chondrocyte can be divided into through induction.

The cellular form of the hUC-MSC increased under culture condition of the present invention.

1, morphological observation.

Utilize this method to cultivate acquisition MSC and there is the adherent property of frosting, CDU-F colony can be formed, and form is relatively homogeneous, in growth arranged in parallel or swirling growth fusiform cell, see Fig. 2 (a) ~ Fig. 2 (c), wherein, all X-coordinate numerals of three width figure are " 10 ¹, 10 ², 10 ³, 10 ⁴, 10 ⁵, 10 ⁶, 10 ^7.2".

2, cell doubling time measures.

Cell doubling time refers to that the cell of multiplication capacity makes cell double the required time by mitotic division.The doubling time of subculture in vitro separately culturing cell can be obtained by survey calculation.Cell doubling time is different because of cell category, is relative constancy for its doubling time of allogenic cell.Thus cell doubling time directly reflects the speed of cell proliferation, when environmental factors makes cell doubling time change, then means that cell cycle progression there occurs change.Therefore, cell doubling time is also the important parameter of observation of cell cycle progression change.Moreover the mensuration of this parameter letter and easy, is more of practical significance.

With patterson formulae discovery cell population doublings time (population doubling time, PDT), PDT=t × lg2/lg (Nt/No), t is that cell count increases to the Nt time used by No, in hour, No is the inoculation initial cell number that goes down to posterity, and Nt is the cell count of results after increasing the t time.Apply this formula we calculate umbilical cord source the cell doubling time of mescenchymal stem cell in culture system of the present invention be 23 hours.

In sum, the mescenchymal stem cell using this law to cultivate has stronger multiplication capacity.

3, Flow cytometry hUC-MSC surface molecular mark CD14, CD45, CD73, CD90, CD105, CD19, HLA-DR.

(1) respectively people's umbilical cord MSC suspension that embodiment 1,2,3,4 obtains is proceeded to 50ml centrifuge tube;

(2) 1000rmp, 5min are centrifugal;

(3) abandoning supernatant, resuspended with PBS;

(4) calculate, adjustment concentration;

(5) divide into groups: get EP pipe 9, and number, often pipe adds 100 μ l cell suspensions and (ensures that viable cell quantity is 10 ⁵left and right);

(6) CD14, CD45, CD73, CD90, CD105, CD19, HLA-DR fluorescent-labeled antibody is added respectively;

(7) under the condition of 4 DEG C, lucifuge hatches 30min;

(8) often pipe adds 1ml PBS, mixing, and 200 × g, 5min are centrifugal, abandon supernatant liquor;

(9) repeating step (8) once;

(10) often pipe adds 200 μ l PBS, and re-suspended cell precipitates;

(11) upper machine testing.Result is as Fig. 3.

4, hUC-MSC skeletonization and become the qualification of fat differentiation potential.

(1) Osteoinductive differentiation

Inductive differentiation medium is STEMPRO ^?osteogenesis Differentiation Kit, changed liquid 1 time every 2 days, cultivate 14 days, PBS washed cell 1 time, 4% paraformaldehyde fixes 30 minutes, the generation of alizarin red S dyeing qualification calcium tubercle.Basis of microscopic observation, has a large amount of doped calcium in alizarin red S dyeing visible cell slurry, shows that the human umbilical cord mesenchymal stem cells that the method obtains has Osteoblast Differentiation potential, see Fig. 4 A, B, C.

(2) adipogenic induction differentiation

Inductive differentiation medium is STEMPRO ^?adipogenesis Differentiation Kit.Working method reference reagent box working instructions: the mescenchymal stem cell aggregate collecting amplification cultivation, trysinization become unicellular after, 12 orifice plates are inoculated in proper density, when the DMEM/F12 nutrient solution of 10%10FBS is cultured to cell confluency degree about 80%, change induction liquid, changed liquid every 2 days later, cultivate 3 weeks.Oil red O stain qualification differentiated result.Under microscope, red color visible fat drips extensive distribution, shows that the human umbilical cord mesenchymal stem cells that the method obtains has into fat differentiation potential, sees Fig. 4 D, E, F.

5, hUC-MSC bovine serum protein residual content detects:

Adopt Immunoenzymetric Assay Kit for the Measurement of Bovine Serum Albumin(CYGNUS company) measure bovine serum protein residual content in sample with euzymelinked immunosorbent assay (ELISA), working method reference reagent box working instructions: adopt the trial-product diluted trial-product that test kit provides, trial-product arranges 2 extent of dilution and measures, and each extent of dilution does diplopore replicate(determination).The absorbancy of kit standard product, the linearly dependent coefficient of standard substance, the absorbancy that diplopore measures is all in test kit claimed range, and test effectively.Do straight-line regression with the concentration of standard solution to its corresponding absorbancy, the absorbancy of trial-product is substituted into linear regression equation, then is multiplied by extension rate, calculate BSA content in trial-product, result is as Fig. 5.

6, hUC-MSC immunogenicity detects (unidirectional MLR):

(1) get another uncorrelated donor peripheral blood and extract monokaryon lymphocyte (PBMC), purified T cell is as responsive cell;

(2) T cell is inoculated in 24 orifice plates, every hole 1 × 10 ⁶cell;

(3) irritation cell (all through gamma-ray irradiation process, dosage 50Gy) is added respectively: negative control adds homology PBMC; Positive control adds allos PBMC; Test group adds people's umbilical cord 2*105MSC; Blank only adds nutrient solution.Irritation cell add-on is every hole 1*106;

(4) be placed in 5%CO2,37 DEG C of incubators and cultivate 4 days;

(5) every hole adds BrdU, and activity is 10 μMs, continues cultivation 18 hours in 5% CO2,37 DEG C of incubators;

(6) collecting cell, and according to BrdU Flow Kits(BD) detect and analyze;

(7) calculate SI(SI=test group positive rate/negative control group positive rate, S.I. is less shows that immunogenicity is less), result is as Fig. 6 (a).

7, hUC-MSC immunosuppressive activity detects (two-way MLR):

(1) adopt two uncorrelated donor peripheral bloods and extract PBMCs;

(2) PBMCs is seeded to 24 orifice plates, every hole adds each donor PBMCs each 1 × 10 ⁶individual;

(3) blank group does not add cell; Test group adds MSC, and adding cell quantity is 5 × 10 ⁴, 1 × 10 ⁵, 2 × 10 ⁵/ hole;

(4) be placed in 5%CO2,37 DEG C of incubators and cultivate 4 days;

(5) every hole adds BrdU, and activity is 10 μMs, continues to cultivate, 18 hours in 5%CO2,37 DEG C of incubators;

(6) collecting cell, and according to BrdU Flow Kits(BD) detect and analyze;

(7) IR=(1-experiment group B rdu positive rate/blank group Brdu positive rate is calculated) × 100%, Fig. 6 (b).

8, the external tumorigenicity inspection (soft-agar cloning forms test) of the hUC-MSC for preparing of the inventive method:

(1) prepare the LMP agar liquid glucose of 1.2% and 0.7% two concentration with distilled water respectively, after autoclaving, maintain in the baking oven of more than 40 DEG C and do not solidify;

(2) 2 × DMEM perfect medium is prepared: 2 × DMEM+20%FBS mixes, and balances to room temperature, for subsequent use;

(3) 1:1 makes agarose and the mixing of 2 × DMEM perfect medium of 1.2% by volume;

(4) get six orifice plates and add 1.2% agarose mixed solution, 2mL/ hole, put room temperature and treat that it is solidified as bottom-layer agar, for subsequent use;

(5) cell process: hUC-MSC test group, positive control (Hela), negative control (MRC-5) use perfect medium (being followed successively by DMEM/F12, RPMI1640, the MEM containing 10%FBS) to prepare single cell suspension respectively

(6) count respectively, and regulate cell concn to 2 × 10 with corresponding nutrient solution ³individual cell/ml;

(7) cell inoculation: each single cell suspension mixes with 0.7% agar liquid glucose by 1:1 by volume, is seeded in step 4) 6 well culture plates for subsequent use, 1ml/ hole, leave standstill a moment, this is top-layer agar;

(8) in 37 DEG C, 5%CO2 saturated humidity cultured continuously 3 weeks;

(9) positive control should Clone formation as seen, and negative control should without Clone formation, and test side is effective, and viewing test group is with or without Clone formation (the results are shown in Figure 7).

What deserves to be explained is, the present invention also has following characteristics: the recycling 1. realizing waste umbilical cord and placenta, and there is not dispute of ethic; 2. utilize umbilical cord homology placenta to prepare placenta homogenate, avoid the extra possibility increasing pathogen infection, related diseases substance one step detects and can put in place; 3. placenta homogenate is containing abundant nutritional factor, as Urogastron, Basic Fibroblast Growth Factor, eosinophil activation's chemokine, vascular epidermal growth factor, interleukin class, TTGF etc., and can provide closer to the nutritional condition at body environment; 4. amplification in early stage still adopts tradition to realize Large scale in vitro amplification containing the substratum of foetal calf serum; 5. the later stage replaces foetal calf serum with placenta homogenate, can abundant metabolism can reduce costs again as the foetal calf serum of foreign protein.

According to above-described embodiment, just the present invention can be realized well.What deserves to be explained is; under prerequisite based on said structure design, for solving same technical problem, even if some making on the invention are without substantial change or polishing; the essence of the technical scheme adopted is still the same with the present invention, therefore it also should in protection scope of the present invention.

Claims (4)

1. the method prepared of human umbilical cord mesenchymal stem cells mass-producing, is characterized in that, comprise the following steps:

(1) aseptically umbilical cord and placenta is collected respectively;

(2) from umbilical cord, separation and Extraction goes out hUC-MSC; Placenta is prepared into placenta homogenate;

(3) hUC-MSC uses the DMEM/F12 containing foetal calf serum to carry out some generation amplification in vitros;

(4) continue to cultivate some generations with the DMEM/F12 containing placenta homogenate;

(5) digest collecting cell suspension, centrifugal abandoning supernatant, namely obtain hUC-MSC raw cell.

2. the method prepared of a kind of human umbilical cord mesenchymal stem cells mass-producing according to claim 1, it is characterized in that, the preparation method of described placenta homogenate is as follows:

(1) get placenta, denuded amniotic membrane, collect chorionic villi, and clean;

(2) chorionic villi is carried out homogenate;

(3) the resuspended homogenate of damping fluid is utilized;

(4) frozen under the condition of-80 DEG C;

(5) multigelation under the condition of 4 DEG C, then carries out low temperature ultrasonic fragmentation;

(6) centrifugal, collect supernatant liquor, then supernatant liquor is filtered, and collect filtrate, obtain placenta homogenate.

3. the method prepared of a kind of human umbilical cord mesenchymal stem cells mass-producing according to claim 2, it is characterized in that, described damping fluid is DMEM/F12.

4. the method prepared of a kind of human umbilical cord mesenchymal stem cells mass-producing according to claim 3, is characterized in that, described placenta derives from same individuality with the umbilical cord extracting hUC-MSC.