CN108504628A - The cultural method of human umbilical cord mesenchymal stem cells - Google Patents
The cultural method of human umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention belongs to technical field of cell culture, and in particular to a kind of cultural method of human umbilical cord mesenchymal stem cells.There is the problems such as residual cow's serum, Niu Yuan and pig source are viral for existing separation human umbilical cord mesenchymal stem cells, the present invention provides a kind of cultural method of human umbilical cord mesenchymal stem cells, and steps are as follows：A, human umbilical tissue is taken, using serum free medium original cuiture to P0 generations；B, by P0 for cell in culture dish secondary culture to P2 generations；C, it takes P2 for cell, expands culture in rolling bottle to the 5th generation, obtain the human umbilical cord mesenchymal stem cells after amplification cultivation.The method of the present invention whole process uses free serum culture mode, the cell factor manually prepared and chemotactic factor (CF) is added, culture medium sensitization is low, and potential hazard factor is few；Expanding culture using rolling bottle has advantages, the potential applicability in clinical practices such as fast, period short, at low cost, the amplifying cells considerable number of amplification wide.
Description
Technical field
The invention belongs to technical field of stem cell culture, and in particular to a kind of culture side of human umbilical cord mesenchymal stem cells
Method.
Background technology
Mescenchymal stem cell (MesenchymalStemCell, MSC), which is a group, has very strong proliferative capacity and multidirectional point
Change the stem cell of potential, the significant effect in terms of immunological regulation and anti-inflammatory response.Therefore, MSC is in treatment graft-versus-host
Extraordinary clinic is shown in terms of a variety of major diseases such as reaction (GVHD), autoimmune disease, severe liver diseases, diabetes
Foreground.
MSC's is tissue-derived extensive, such as MSC is rich in umbilical cord, marrow, fat Various Tissues.Wherein umbilical cord is to be connected in
Cord structures between embryo's umbilical region and placenta, rich in a variety of ancestral cells such as hematopoiesis, mesenchyma, nerve and endotheliums.And it is filled between umbilical cord
Matter stem cell detaches from neonatal umbilical cord and obtains, and compared with other tissue-derived MSC, it has more original, proliferation
The features such as ability is stronger and immunogenicity is lower.Source for mesenchymal stem cells simultaneously in umbilical cord is very abundant, and umbilical cord is as production
The Biohazard Waste of section has convenient material drawing, is not related to the limitations such as Medical Ethics, is that current mescenchymal stem cell is most important next
Source.Compared with the MSC of derived from bone marrow, the advantage of the mescenchymal stem cell in people's umbilical cord source is：Collection process is relatively easy,
Receptor is sent out after noninvasive, placental barrier makes umbilical cord be reduced transplanting by the primitiveness of virus and little bacterial contamination, bleeding of the umbilicus immune system
The probability of raw rejection, in vitro in cultivating system can fast breeding, can be proliferated within 3~5 days 4~5 times after 1 passage, and
Cell uniformity after passage is good.In addition, human umbilical cord mesenchymal stem cells have the stronger ability of going back to the nest and are easy to gene transfection
The advantages that become implement cell therapy ideal cell.
Applications of the MSC in clinical cytology treatment, gene therapy and organizational project is on condition that MSC can be realized in vitro
Large-scale production, prepared cell uniformity is good, and can keep the biological characteristics of mescenchymal stem cell.In order to realize navel
Large-scale production with MSC and commercial application need to accomplish that production technology whole process is stable, controllable, reproducible, amplification in vitro
It is efficient, at low cost.In order to reach above-mentioned target, establish in vitro a kind of human umbilical cord mesenchymal stem cells stable processing technique,
Efficient amplification, the scale manufacturing technique of cost-effective are very necessary.
The MSC separation methods in currently used umbilical cord source mainly have two kinds of enzyme digestion and tissue block adherent method：Tradition
Enzyme digestion it is of high cost, be easy caused by cell damage, or even generate cell mutation, there are greater risks for clinical application.
And conventional tissue block adherent method obtains that the umbilical cord mesenchymal stem cells cell cycle is longer, and efficiency is low, the primary cell number of acquisition
Measure less, and purity is not high, cannot reach MSC criterion for clinical use, causes clinical application more difficult.
There are problems by MSC prepared by traditional plate culture, cell factory culture process, in preparation process
It may need to introduce a variety of allogenic materials, such as cow's serum, antibiotic, pig pancreatin, wherein cow's serum residue problem most represent
Property, Quality Control is more difficult in clinical application, has potential clinical application risk.As traditional MSC culture solutions usually all use base
Basal culture medium adds a certain proportion of fetal calf serum (FBS) and is used as complete culture solution.Be conducive to although FBS is added in traditional components
The adherent growth of MSC, but serum, which is added, also causes MSC to be easier to break up in incubation, and serum composition clinical risk is added
Bigger, whole Quality Control difficulty is big, limits the clinical application of MSC in this way.In addition, plate culture, cell factory cultivation
It can only generally accomplish 1 when MSC is passed in vitro:3-4 ratios are passed on, and MSC was normally applied no more than 5 generations, was also limited in this way
The larger scale clinical application of MSC is made.Therefore, urgent need is established a kind of whole using free serum culture mode, and whole process uses pancreatin
Substitute vitellophag can be passed on more, process stabilizing at high proportion in vivo, and the method that can prepare MSC on a large scale.
Invention content
The technical problem to be solved in the present invention is：The method of existing separation human umbilical cord mesenchymal stem cells easily causes cell damage
Wound is easy when culture to remain bovine serum albumin(BSA), there is the problems such as potential ox source, pig source virus are introduced into again.In addition, existing preparation
Method subculture in vitro separately it is less efficient, it is difficult to meet large-scale clinical application.
The present invention solve above-mentioned technical problem technical solution be：A kind of culture side of human umbilical cord mesenchymal stem cells is provided
Method includes the following steps：
A, human umbilical cord mesenchymal stem cells are detached
Human umbilical tissue is taken, collagenase digesting is added after broken, separation and Extraction umbilical cord mesenchymal stem cells are made unicellular
Suspension, used in culture dish MSCBM medium cultures to cell fusion degree for 80~90% when, obtain P0 for cell；
B, umbilical cord mesenchymal stem cells secondary culture
Take the P0 that step a is obtained for cell, using MSCBM medium cultures to P2 generations in culture dish；
C, umbilical cord mesenchymal stem cells expand culture
The P2 that step b is obtained is taken to be inoculated in rolling bottle, using MSCBM after being digested using trypsase substitute for cell
Culture medium is cultivated, and when cell fusion degree is 80~90%, obtains the human umbilical cord mesenchymal stem cells P3 after amplification cultivation
Generation；
D, step c is repeated, the human umbilical cord mesenchymal stem cells after amplification cultivation are obtained.
Wherein, in the cultural method of above-mentioned human umbilical cord mesenchymal stem cells, described in step a by umbilical cord tissue it is broken before
First carry out disinfection.Disinfection impregnates 2~3s using 75% ethyl alcohol, then is rinsed 2~3 times using the PBS buffer solution of sterilizing.
After umbilical cord tissue is cut into the segment of 2~3 ㎝ after rinsing, the DMEM culture mediums containing penicillin and streptomysin are added
Cleaning removes clot.
Wherein, in the cultural method of above-mentioned human umbilical cord mesenchymal stem cells, broken umbilical cord tissue described in step a is big
Small is 2~3mm3。
Wherein, in the cultural method of above-mentioned human umbilical cord mesenchymal stem cells, the volumetric concentration of clostridiopetidase A described in step a is
0.2~0.4%.
Wherein, in the cultural method of above-mentioned human umbilical cord mesenchymal stem cells, the addition of clostridiopetidase A described in step a is every
0.09~0.12g of clostridiopetidase A is added in the umbilical cord tissue of 10~15cm long.
Wherein, in the cultural method of above-mentioned human umbilical cord mesenchymal stem cells, the condition of culture in incubator described in step a is
36.5~37.5 DEG C of temperature, CO2Concentration 4.5~5.5%.
Wherein, it in the cultural method of above-mentioned human umbilical cord mesenchymal stem cells, was replaced every 2~3 days when being cultivated described in step a
Culture solution.
Wherein, in the cultural method of above-mentioned human umbilical cord mesenchymal stem cells, the composition of the MSCBM culture mediums includes：It is sweet
Propylhomoserin, alanine, isoleucine, methionine, ascorbic acid, biotin, choline chloride, folic acid, calcium chloride, magnesium sulfate, chlorine
Change at least one of potassium, sodium chloride, glucose, phenol red, Sodium Pyruvate or sodium bicarbonate.
Preferably, in the cultural method of above-mentioned human umbilical cord mesenchymal stem cells, the MSCBM culture mediums be glycine,
Alanine, isoleucine, methionine, ascorbic acid, biotin, choline chloride, folic acid, calcium chloride, magnesium sulfate, potassium chloride,
The mixture of sodium chloride, glucose, phenol red, Sodium Pyruvate and sodium bicarbonate.
It is furthermore preferred that in the cultural method of above-mentioned human umbilical cord mesenchymal stem cells, the composition of the MSCBM culture mediums is such as
Shown in table 1.
1 MSCBM culture mediums of table form
Further, it is 5~15% cell factors and chemotactic factor (CF) that the MSCBM culture mediums, which also contain total amount,.More into
One step, the cell factor and chemotactic factor (CF) are the UltraGRO of Helios producers productionTMProduct.
Wherein, in the cultural method of above-mentioned human umbilical cord mesenchymal stem cells, the inoculum concentration being inoculated with described in step c is 3 × 106
~5 × 106A cell/rolling bottle.
Wherein, in the cultural method of above-mentioned human umbilical cord mesenchymal stem cells, the condition of culture described in step c is temperature 36.5
~37.5 DEG C, CO2Concentration 4.5~5.5%, incubator rotating speed are 1~1.5 turn/min.
Beneficial effects of the present invention are：
The present invention provides a kind of preparation methods of human umbilical cord mesenchymal stem cells scale, in separating primary cells,
Using a concentration of 0.2~0.4% clostridiopetidase A, the method currently centrifuged is not used to obtain cell precipitation after the completion of digestion, and it is straight
It connects and continues downward passage cell with digestive juice, obtained cell activity is high, and proliferative capacity is strong and cellularity is stablized；
On the other hand, the cell factor manually prepared and chemotactic is added in culture medium when original cuiture of the present invention and expansion are cultivated
The factor, whole process culture are added without serum composition and pig source pancreatin, and sensitization is low, and potential hazard factor is few；Expanded using rolling bottle
Big culture, the period is short, (each spinner culture area can reach about 2000cm at low cost, amplifying cells considerable number2, stablize
Amplification, which passes on 1 time, can make cell number expand 5~8 times, and 1 umbilical cord can obtain 5 × 10 after the method for the present invention expands11~
1×1012Cell)；
In addition, the cellularity expanded by the production technology is stable, every check complies with standard.People's navel of preparation
Band mescenchymal stem cell, is free of cow's serum ingredient, avoids the introducing in pig source, ox source virus, and can preserve for a long time without losing
Its activity, has wide potential applicability in clinical practice.
Description of the drawings
Fig. 1 is the cellular morphology of HUMSC different times；A is spinner culture HUMSC cellular morphologies after 24 hours；B is to turn
Bottle culture HUMSC cellular morphologies after 48 hours；C is cellular morphologies of the spinner culture HUMSC after 72 hours.
Fig. 2 is spinner culture HUMSC surface antigen testing results；A-E indicates the positive marker expression efficiencies, wherein CD44
Expression efficiency is 99.71%, CD73 99.70%, CD90 98.79%, CD29 99.50%, CD105 99.93%；F-
J indicates that feminine gender marker expression efficiencies, wherein CD45 are 0.08%, CD14 3.7%, and CD11b 0.35%, HLA-DR are
0.71%, CD34 0.23%.The above results show that positive findings are all higher than 95%, and negative findings are respectively less than 5%, illustrate to expand
Cell after culture meets testing requirements.
Fig. 3 is tri- systems of spinner culture HUMSC induction differentiation testing result；A be HUMSC at chondrocyte induction differentiated result, B is
HUMSC Adipogenic induction differentiated results, C are HUMSC Osteoinductive differentiation results.
Fig. 4 is spinner culture HUMSC Clone formation results；A is HUMSC Clone formation figures, and B is formed for tumor cell clone
Figure.
Fig. 5 is to test the spinner culture HUMSC cell cycles；G1 phase cells account for 50.78%, S phase cells and account for 35.70%, G2
Phase cell accounts for 12.41%.
Fig. 6 is spinner culture HUMSC karyotyping results；After treatment for HUMSC cells, karyorrhexis, chromosome divides A
It dissipates；B is that software analyzes after stain colour solid arranging situation, as a result shows 44 autosome+XY.
Specific implementation mode
The present invention provides a kind of cultural methods of human umbilical cord mesenchymal stem cells.Detaching primary mescenchymal stem cell
When, using a concentration of 0.2~0.4% clostridiopetidase A, clostridiopetidase A addition be preferably every 10~15cm long umbilical cord tissue in plus
Enter 0.09~0.12g of clostridiopetidase A.Under this collagenase concentration, after the completion of digestion, no longer need to obtain cell using the method for centrifugation
Precipitation, and is directly inoculated with digestive juice with culture medium in proportion, and obtained cell activity is high, proliferative capacity by force and cellularity more
Stablize.
In above-mentioned human umbilical cord mesenchymal stem cells secondary culture, adds and cultivated containing the DMEM of penicillin and streptomysin
Base cleans, and removes clot.The composition of the DMEM culture mediums is shown in Table 2.
2 DMEM culture mediums of table form
In order to which by the control of fetal calf serum residual quantity, within the scope of States Pharmacopoeia specifications, the present invention does not add cow's serum in culture,
But the MSCBM basal mediums of serum-free are used, cell factor and chemotactic factor (CF) are added in basal medium, is capable of providing
Nutrition, sensitization is low, and potential hazard factor is few.
In the present invention, the composition of the MSCBM culture mediums includes：Glycine, isoleucine, methionine, resists alanine
Bad hematic acid, biotin, choline chloride, folic acid, calcium chloride, magnesium sulfate, potassium chloride, sodium chloride, glucose, phenol red, pyruvic acid
At least one of sodium or sodium bicarbonate.Further, the culture medium also contains total amount and is 5~15% cell factors and becomes
Change the factor.
The invention is enlarged culture using rolling bottle, and control incubator rotating speed is 1~1.5 turn/min, is compared
The cell factory culture of static, the spinner culture period is short, at low cost, amplifying cells considerable number, and each rolling bottle stablizes amplification
5~8 times of cell number, obtained cellularity are stablized, cellular morphology, Surface Antigen and latent at cartilage differentiation at fat skeletonization
It is consistent that the HUMSC obtained can be cultivated with FBS, meet regulation of the international cell therapy association to the HUMSC minimum standards identified.
The specific implementation mode of the present invention is further explained with reference to embodiment, but does not indicate that and sends out this
Bright protection domain is limited in range described in embodiment.
The umbilical cord used in embodiment comes from healthcare hospital for women ＆ children of Sichuan Province；
Collagenase type I described in embodiment and pancreatin substitute are purchased to the official websites gibco；
Remaining reagent and equipment are ordinary commercial products used in embodiment.
The primary separation of 1 HUMSC of embodiment and culture
Acquire mature Cesarean esction healthy fetus umbilical cord.Puerpera need to do antibody of AIDS virus, B-mode liver before umbilical cord acquisition
The detections such as scorching antiviral antibody, antibody of HCV, syphilis helicoid antibody can all adopt after qualified property to ensure safety
Collection.
(1) gnotobasis takes the umbilical cord that length is about 10~15cm, after being rinsed well repeatedly with the PBS buffer solution of sterilizing,
It is cut to the segment of 2~3cm long, is positioned in the culture dish for filling basal medium (added with 1% penicillin and streptomysin).
(2) blood vessel is rejected, and is washed in the medium to without apparent bloodstain.
(3) in a small amount of media environment, umbilical cord tissue is cut to small bulk, is cut to about 1mm × 1mm's with eye scissors
Fragment.
(4) fragment is moved in 50mL centrifuge tubes, final concentration of 0.3%I Collagenase Types digestion is added, and (basal medium is matched
System), it is placed on 37 DEG C of shaking tables, adjustment rotating speed is 100rpm/min, is digested 2 hours.
(5) in order to realize that scale, people's umbilical cord MSC original cuitures use serum free medium MSCBM+ cell factors, but
MSC is more sensitive to growing environment, in order to ensure that the case where can not adapting to is not occurred by the change dramatically of condition of culture in MSC,
Cell factor and chemotactic factor (CF) total amount adding proportion are 10% when original cuiture in this experiment.
(6) draw sticky digestive juice 2mL, by 1 ︰ 9 (volume ratio) be added 18mL serum free mediums (factor-containing with become
Change the factor), 37 DEG C are positioned over after rocking uniformly, is cultivated in 5%CO2 incubators, cell is P0 generations at this time.
2 HUMSC secondary cultures of embodiment
Specific operation process is as follows：
(1) P0 in example 1 to be performed for cell it is to be fused to 80%~90% when, with pancreatin substitute digest after and centrifuge
Remove supernatant；
(2) use serum free medium (factor-containing and chemotactic factor (CF) total amount be 10%) that cell precipitation is resuspended, by 1 ︰, 3 weights
It is newly inoculated in new culture dish, in 5%CO2, continue to cultivate in 37 DEG C of incubators, cell is P1 generations at this time；
(3) when P1 for cell confluency degree up to 80~90% when, repeat above-mentioned (1), (2) operation, obtain P2 for cell.
The reason of using culture dish herein：P0~P2 is gradually increased for cell purity, to P2 for when cell surface marker with
Examination criteria is consistent, before expanding culture with rolling bottle, first has to ensure cell purity.
3 HUMSC of embodiment expands culture
After the P2 that embodiment 2 is obtained is for cell dissociation, it is seeded in rolling bottle and carries out adhere-wall culture.
(1) in example 2 to be performed P2 for cell fusion to 80~90% when, pancreatin substitute digestion, and obtaining unicellular outstanding
Liquid and centrifuge remove supernatant；
(2) use serum free medium (factor-containing and chemotactic factor (CF) total amount 10%) that cell precipitation is resuspended；
(3) it is reinoculated in rolling bottle after counting cell, in 5%CO2, continue to cultivate in 37 DEG C of incubators, wait for that cell converges
It is right up to 80~90%, obtain P3 for cell；
(4) above-mentioned (1) (2) (3) step is repeated, until obtaining P5 for human umbilical cord mesenchymal stem cells.
After cell precipitation is resuspended, the simultaneously new rolling bottle of renewed vaccination is counted, passing on 2 times by amplification in vitro obtains P5 for thin
Born of the same parents, each rolling bottle amplification times are 5~8 times, obtain expanding the HUMSC after culture.
The amplification times are 5~8 times：P3 for amplification cultivation to P4 generation, P4 generation be expanded to P5 for when, often expand
A generation is cultivated, cell quantity expands 5~8 times.
The practical cell expanded after cultivating of embodiment 3 has following characteristics：
1, morphological observation.
Embodiment 3, which obtains MSC, has frosting adherence quality, can form CDU-F colonies, and form is relatively uniform, in flat
Row aligned growth or the fusiform cell of circinate growth, are shown in Figure 1A, B, C.
2, the cell surface marker that flow cytomery culture obtains
(1) the HUMSC suspensions that embodiment 3 obtains are transferred to 50ml centrifuge tubes；
(2) it is grouped：Take streaming pipe 14, and number, often pipe be added 100 μ l cell suspensions (ensure living cells quantity be 4 ×
104 or so)；
(3) positive marker detections are 5 following：CD44、CD105、CD29、CD73、CD90；
Negative marker detections are 5 following：CD45、CD11b、CD14、CD34、HLA-DR；
(4) testing result such as Fig. 2.As a result show that positive marker expression rates are all higher than 95%, negative marker expression rates
Respectively less than 5%, illustrate to expand the cellularity stabilization after technique culture, meets testing requirements.
3, HUMSC inductions differentiation obtained by spinner culture
Using LONZA stem cells three it is induction agent box by cell acquired in embodiment 3, to it respectively at fat,
Skeletonization breaks up at chondrocyte induction.Differentiated result is shown in Fig. 3.As a result after being shown as chondrocyte induction, blue dyeing is formed under microscope；
After Adipogenic induction, it can be seen that red fat drips are formed under microscope；After osteogenic induction, visible change tubercle under microscope.As a result
It is mescenchymal stem cell to illustrate the cell really.
4, HUMSC Clone formations obtained by spinner culture
(1) in Example 3 exponential phase single-layer culturing cell, pancreatin substitute is digested to individual cells.
(2) cell suspension is made into the dilution of gradient multiple, is inoculated in culture dish with cell density appropriate.Be placed in 37 DEG C,
5%CO2And in the environment of saturated humidity, stationary culture 2~3 weeks；
(3) often observation terminates culture when occurring macroscopic clone in culture dish.After fixation and dye observation.
(4) plate is inverted and is superimposed a transparent film with grid, with the naked eye directly counted Clone formation, as a result see
As a result Fig. 4 shows that HUMSC can form normal cell clone.
5, the HUMSC cell cycles obtained by spinner culture are detected
(1) cell cultivated in embodiment 3 is collected, cell quantity is 0.5~2 × 106It is a；Cell is collected after fixation, and
Corresponding coloring agent is added and is protected from light dyeing, upper machine testing after dyeing.
(2) red fluorescence is detected at excitation wavelength 488nm wavelength with flow cytometer, count 2~3 × 104A cell,
As a result software ModFit analyses are fitted with the cell cycle.It when analysis, is shown using FL2-w and FL2-A, removes conjuncted cell.Knot
Fruit sees Fig. 5, as a result shows the HUMSC cell cycles in normal range (NR).
6, MSC karyotypings obtained by spinner culture
(1) the good HUMSC of growth conditions is chosen, passes in 37 DEG C of incubators of postposition and cultivates 72h.
(2) cultivate the cell after 72h fixed through colchicine processing, Hypotonic treatment, cell, film-making, dyeing etc. it is a series of
After operation, then it is placed in microscopically observation.
(3) microscopy：The human mesenchymal stem cell load prepared is put under microscope low power lens, finds well dispersed, dye
The moderate division phases of color, conversion oil mirror examine, and selective staining body is clear, and the cell of good dispersion degree carries out caryogram point
Analysis.The results are shown in Figure 6, and after karyorrhexis, visible stain body is uniformly dispersed under the microscope, and software analysis result shows chromosome
It it is 46, including 44 autosomes and 2 sex chromosome, it is Humsc to illustrate the cell really.
For the HUMSC prepared according to the above method of the present invention, formulated according to " international cell therapy association (ISCT) "
HUMSC identifies minimum standard statement of requirement, should have following characteristics：
It (1) can adherent growth in frosting；
(2) cell surface molecule marks CD73, CD90, CD105, positive rate to be not less than 95%；And CD34, CD45,
The positive rate of CD19, CD14 and HLA~DR must not be higher than 2%；
(3) os osseum cell, adipocyte and cartilage cell can be divided into through induction.
It can be obtained by embodiment result：A kind of method of rolling bottle prepare with scale human umbilical cord mesenchymal stem cells of the present invention
Easy to operate, cytotostatic is uniform, at low cost, can carry out pilot scale culture, and obtained mescenchymal stem cell expands multiple energy
Reach 5~8 times, and remained without fetal calf serum, the introducing without potential pig source virus, ox source virus, disclosure satisfy that ISCT is formulated
People MSC identify that minimum standard statement of requirement, remarkable in economical benefits are worth of widely use.
Claims (10)
1. the cultural method of human umbilical cord mesenchymal stem cells, which is characterized in that include the following steps：
A, human umbilical cord mesenchymal stem cells are detached
Human umbilical tissue is taken, collagenase digesting is added after broken, single cell suspension is made in separation and Extraction umbilical cord mesenchymal stem cells,
Used in culture dish MSCBM medium cultures to cell fusion degree for 80~90% when, obtain P0 for cell；
B, human umbilical cord mesenchymal stem cells secondary culture
Take the P0 that step a is obtained for cell, using MSCBM culture mediums secondary culture to P2 generations in culture dish；
C, umbilical cord mesenchymal stem cells expand culture
The P2 that step b is obtained is taken to be inoculated in rolling bottle, cultivated using MSCBM after being digested using trypsase substitute for cell
Base is cultivated, and when cell fusion degree is 80~90%, obtains human umbilical cord mesenchymal stem cells P3 generations；
D, step c is repeated, the human umbilical cord mesenchymal stem cells after amplification cultivation are obtained.
2. the cultural method of human umbilical cord mesenchymal stem cells according to claim 1, it is characterised in that：Described in step a
Umbilical cord tissue it is broken before first carry out disinfection；Disinfection impregnates 2~3s, then the PBS buffer solution drift using sterilizing using 75% ethyl alcohol
It washes 2~3 times.
3. the cultural method of human umbilical cord mesenchymal stem cells according to claim 1, it is characterised in that：Described in step a
The volumetric concentration of clostridiopetidase A is 0.2~0.4%.
4. the cultural method of human umbilical cord mesenchymal stem cells according to claim 1, it is characterised in that：Described in step a
0.09~0.12g of clostridiopetidase A is added in the umbilical cord tissue that the addition of clostridiopetidase A is every 10~15cm long.
5. the cultural method of human umbilical cord mesenchymal stem cells according to claim 1, it is characterised in that：It is trained described in step a
It is 36.5~37.5 DEG C of temperature, CO to support the condition of culture in case2Concentration 4.5~5.5%.
6. the cultural method of human umbilical cord mesenchymal stem cells according to claim 1, it is characterised in that：Step a, in b, c
The composition of the MSCBM culture mediums includes：Glycine, alanine, isoleucine, methionine, ascorbic acid, biotin, choline
In chloride, folic acid, calcium chloride, magnesium sulfate, potassium chloride, sodium chloride, glucose, phenol red, Sodium Pyruvate or sodium bicarbonate extremely
Few one kind.
7. the cultural method of human umbilical cord mesenchymal stem cells according to claim 6, it is characterised in that：The MSCBM
It is 5~15% cell factors and chemotactic factor (CF) that culture medium, which also contains total amount,.
8. the cultural method of human umbilical cord mesenchymal stem cells according to claim 7, it is characterised in that：The cell because
Son and the UltraGRO that chemotactic factor (CF) is the production of Helios producersTMProduct.
9. the cultural method of human umbilical cord mesenchymal stem cells according to claim 1, it is characterised in that：It is connect described in step c
The inoculum concentration of kind is 3 × 106~5 × 106A cell/rolling bottle.
10. the cultural method of human umbilical cord mesenchymal stem cells according to claim 1, it is characterised in that：Described in step c
Condition of culture is 36.5~37.5 DEG C of temperature, CO2Concentration 4.5~5.5%, incubator rotating speed are 1~1.5 turn/min.
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