CN105462919A - Method for separating and extracting hUC-MSC (human Umbilical Cord mesenchymal stem cells) from wharton jelly tissue of umbilical cord - Google Patents
Method for separating and extracting hUC-MSC (human Umbilical Cord mesenchymal stem cells) from wharton jelly tissue of umbilical cord Download PDFInfo
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Abstract
The invention provides a method for rapidly separating and extracting hUC-MSC (human Umbilical Cord mesenchymal stem cells). The method comprises the following steps: taking the freshly collected umbilical cord tissue of a healthy newborn baby, carrying out on-ice transportation on the freshly collected umbilical cord tissue in umbilical cord storage transportation liquid containing double antibodies, carrying out cleaning and disinfection by adopting 75% alcohol and normal saline, removing blood vessels, carrying out blunt dissection on wharton jelly, carrying out mechanical pulverization, treating the obtained product I by adopting red blood cell lysis buffer for 3 min, digesting the obtained product II by adopting IV collagenase, screening the obtained product III by adopting a 100-200-mesh sieve, carrying out suspension culture on the obtained product IV by adopting a serum-free medium, wherein the liquid is changed every 3-5 days, taking supernatant, detecting cell pollution, after the adherent rate in a plate reaches 30-70%, carrying out trypsinization, carrying out centrifugation, collecting cells, carrying out passage amplification, carrying out merging when the cell merging rate reaches 90% or above, collecting the cells, carrying out cryopreservation on the cells, and detecting the biological characteristics of hUC-MSC.
Description
Technical field
The present invention relates to the efficient sharp separation extracting method of umbilical cord mesenchymal stem cells, from fresh umbilical cord China Tong Shi glue tissue, particularly extract the method for mescenchymal stem cell.
Background technology
Mescenchymal stem cell (mesenchymalstemcells, MSC) derives to grow early stage mesoderm and ectoderm, has height self and multi-lineage potential, be extensively present in whole body reticular tissue and organ interstitial.There are some researches show at present, different cultural methods can make MSC show as neuronal phenotypes, insulin-like cell phenotype, endothelial cell phenotype, or expression cardiomyocyte markers, thus announcement MSC regulates in immunosuppression, endocrine system adjustment, neural system and has potential applicability in clinical practice widely in cardiovascular function improvement.Therefore MSC becomes the cell derived having practical value in cell therapy and field of gene gradually.Particularly compared with other MSC originated, the human umbilical cord mesenchymal stem cells (humanUmbilicalCordmesenchymalstemcells, hUC-MSCs) coming from people's umbilical cord have growing environment single, gather convenient, proliferation and differentiation ability is strong, immunogenicity is low, without plurality of advantages such as ethics problems.
In view of the multi-lineage potential of hUC-MSC and the application potential in disease treatment field, the storage service of the hUC-MSC of domestic market has broad prospects.But, how the guarantee stem cell of efficiency standard separation and Extraction rate and ensure stem cell high-quality, thus meeting the clinical demand of following stem cell transplantation, is the problem demanding prompt solution of current domestic stem cell storage service.
Current hUC-MSC many employings enzyme digestion and organize adherent method to prepare.But, the domestic and international separation about hUC-MSC at present and amplification method confusion, and synthesis steps is quite complicated, makes the large quantity of rapid extraction, high purity hUC-MSC still exists certain difficulty.
Summary of the invention
The object of the invention is the needs for this area, provide a kind of can efficiently, the method for separation and Extraction hUC-MSC rapidly.
Another object of the present invention is to provide the hUC-MSC obtained by method of the present invention.
Specifically, therefore, the present invention is directed to the extraction present situation of human umbilical cord mesenchymal stem cells both at home and abroad at present, utilize the red corpuscle that erythrocyte cracked liquid process is larger on the impact of hUC-MSC Primary Growth, utilize collagenase to carry out digestion to shorten the primary cell culture time simultaneously, thus realize extracting high purity hUC-MSC quickly and efficiently.Further, whole process adopts serum free culture system, is more conducive to the future clinical Transformation Application of stem cell.
Technical scheme of the present invention is as follows.
On the one hand, the invention provides the method for separation and extraction mescenchymal stem cell from fresh umbilical cord in vitro magnificent Tong Shi glue tissue, it is a kind of method of separation and Culture umbilical cord mesenchymal stem cells, and described method comprises: adopt erythrocyte cracked liquid and collagenase process China Tong Shi glue tissue.
The erythrocyte cracked liquid that the present invention adopts is for comprising NH
4cl and Na
2the aqueous solution of-EDTA, preferably comprises the NH of 1-20g/L
4cl and 0.05-0.2mMNa
2the aqueous solution of-EDTA, is more preferably the NH comprising 5-10g/L
4cl and 0.1mmol/LNa
2the aqueous solution of-EDTA, pH is 7.2-7.4.Before the use, cross 0.22 μm of microfiltration membrane, balance to room temperature.
The collagenase that the present invention adopts is IV Collagenase Type, preferably comprise the Digestive system of IV Collagenase Type, preferably comprise D-Hank ' the s liquid of IV Collagenase Type, Unidasa, DNA enzymatic and serum substitute, be more preferably D-Hank ' the s liquid comprising 1%IV Collagenase Type, 0.5% Unidasa, 300U/mlDNA enzyme and 2% serum substitute.Wherein percentage ratio percentage.
Method of the present invention specifically comprises: the umbilical cord of acquisition China Tong Shi glue tissue segmentation is become tissue block, adds the described erythrocyte cracked liquid of 1-3 times of tissue block volume, at room temperature process 2-5 minute; Then in treated tissue block, add the Digestive system comprising IV Collagenase Type again to process.
Preferably, described method also comprises: after collagenase digesting, adopts mesenchymal stem cell serum-free culture medium to cultivate, to obtain primary mescenchymal stem cell.
Mesenchymal stem cell serum-free culture medium comprises a-MEM/DMEM-F12, beta-mercaptoethanol, non-essential amino acid, recombination human basic fibroblast somatomedin (b-FGF) and serum substitute.
Preferably, described mesenchymal stem cell serum-free culture medium comprises the recombination human basic fibroblast somatomedin that the beta-mercaptoethanol of 0.05-0.2 parts by volume, the non-essential amino aqueous acid of 0.5-2 parts by volume, the serum substitute of 8-12 parts by volume, the a-MEM/DMEM-F12 of 85-95 parts by volume and final concentration are 5-15ng/ml, and it is respectively the glycine of 8-12mM, L-Ala, L-Tianmen acid amides, L-Aspartic acid, L-glutamic acid, proline(Pro) and Serine that wherein said non-essential amino aqueous acid comprises concentration; More preferably, described mesenchymal stem cell serum-free culture medium comprises the recombination human basic fibroblast somatomedin that the beta-mercaptoethanol of 0.1 parts by volume, the non-essential amino aqueous acid of 1 parts by volume, the serum substitute of 10 parts by volume, the a-MEM/DMEM-F12 of 89 parts by volume and final concentration are 10ng/ml; Most preferably, described mesenchymal stem cell serum-free culture medium is made up of described a-MEM/DMEM-F12, beta-mercaptoethanol, non-essential amino aqueous acid, recombination human basic fibroblast somatomedin and serum substitute.
According to the embodiment of the application, described non-essential amino aqueous acid can adopt Gibco company article No. to be the product of 11140.
According to the embodiment of the application, described serum substitute can adopt KnockOut
tMserumReplacement (Gibco Products, article No. 10828-010).
Preferably, described method comprises: the umbilical cord of acquisition China Tong Shi glue tissue is cut into 1-3mm
3the tissue block of size, adds the erythrocyte cracked liquid of 1.5-2 times of tissue block volume, processes 2-5 minute under room temperature; Then in treated tissue block, the Digestive system comprising IV Collagenase Type of 2-3 times of volume is added, at the CO of 37 DEG C and 5% concentration
2lower digestion 8-12 hour;
Preferably, described method also comprises: with 1-5 × 10
4cell/cm
2the cell obtained is inoculated in mesenchymal stem cell serum-free culture medium by the density of culture dish area, at the CO of 37 DEG C and 5% concentration
2lower cultivation, changes fresh culture in every 3-4 days.
Preferably, said method comprising the steps of:
(1) pre-treatment of umbilical cord tissue:
Fresh umbilical cord is divided into segment, removes blood vessel extravasated blood, longitudinally cut open, reject Umbilical artery and umbilical vein, blunt separation China Tong Shi glue, adds PBS buffer solution for cleaning;
(2) erythrocyte cracked liquid process:
The umbilical cord obtained through step (1) China Tong Shi glue tissue segmentation is become tissue block, adds erythrocyte cracked liquid process, the tissue block that collected by centrifugation is treated, add PBS buffer solution for cleaning;
(3) collagenase digesting:
Again processing adding the Digestive system comprising IV Collagenase Type in the tissue block obtained through step (2), then adding the dilution of PBS damping fluid, aseptic sieved filter collecting cell;
(4) original cuiture:
Mesenchymal stem cell serum-free culture medium is adopted to cultivate the cell obtained through step (3), to obtain primary mescenchymal stem cell;
Preferably, described method is further comprising the steps of:
(5) supernatant liquor detects:
Get the supernatant liquor of culturing cell in step (4), detect one or more in following items: hepatitis A, hepatitis B, the third liver, syphilis, human immunodeficiency virus, mycoplasma, chlamydozoan and intracellular toxin;
(6) Secondary Culture:
Getting test item in step (5) is negative culturing cell, centrifugal collecting cell after trysinization, Secondary Culture, collecting cell or continue to go down to posterity;
(7) cell detection:
Get cultured cells in step (6), detect one or more in following items: differentiation capability, cytoactive, cell purity, cell contamination and multiplication characteristic.
Preferably, described step (1) comprising: fresh umbilical cord is divided into the segment that 2-3cm is long, removes extravasated blood in blood vessel, longitudinally cut open, reject Umbilical artery and umbilical vein, blunt separation China Tong Shi glue, add the PBS damping fluid of 1.5-2 times of volume, slight wobble is cleaned;
Preferably, described step (2) comprising:
Umbilical cord China Tong Shi glue tissue through cleaning is cut into 1-3mm
3the tissue block of size, adds the erythrocyte cracked liquid of 1.5-2 times of tissue block volume, processes 2-5 minute under room temperature, collected by centrifugation tissue block, PBS buffer solution for cleaning 2-3 time; Wherein preferably, described centrifugal be 1000-1200rpm, at 4 DEG C centrifugal 6 minutes;
Preferably, described step (3) comprising:
The Digestive system comprising IV Collagenase Type of 2-3 times of volume is added, at the CO of 37 DEG C and 5% concentration in treated magnificent Tong Shi glue tissue block
2lower digestion 8-12 hour, then add PBS damping fluid dilution after through 100 orders or the aseptic sieved filter of 200 orders, collect filtered solution, PBS cleaning centrifugal; Wherein preferably, described centrifugal be 1000-1200rpm, at 4 DEG C centrifugal 6 minutes;
Preferably, described step (4) comprising:
With 1-5 × 10
4cell/cm
2the cell obtained through step (3) is inoculated in mesenchymal stem cell serum-free culture medium by the density of culture dish area, at the CO of 37 DEG C and 5% concentration
2lower cultivation, changes fresh culture in every 3-4 days;
Preferably, described step (5) comprising:
Get the supernatant liquor of culturing cell in step (4), that detects in following items is whole: hepatitis A, hepatitis B, the third liver, syphilis, human immunodeficiency virus, mycoplasma, chlamydozoan and intracellular toxin;
Preferably, described step (6) comprising:
Getting whole test item in step (5) is negative culturing cell, until attached cell converge rate reach 30-60% time, centrifugal collecting cell after trysinization, Secondary Culture reaches more than 90% to converging rate, and collecting cell is frozen or continue to go down to posterity; Wherein preferably, the working concentration of described pancreatin is mass percent is 0.125%, and digestion 1-2 minute, pats culture dish or culturing bottle sidewall in digestive process; And wherein preferably, described centrifugal be 1000-1200rpm, at 4 DEG C centrifugal 6 minutes;
Preferably, by collect cell with 2-3 × 10
6the density freezen protective of cell/ml is in-196 DEG C of liquid nitrogen; Or preferably, by collect cell with 1: 3-1: 4 ratio carry out passage;
Preferably, described step (7) comprising:
Get cultured cells in step (6), that detects in following items is whole: differentiation capability, cytoactive, cell purity, cell contamination and multiplication characteristic.
Preferably, before step (1), also carry out umbilical cord cleaning, preservation and use pre-treatment in described method, preferably include:
Gather spontaneous labor or caesarean delivered healthy newborn umbilical cord tissue under aseptic condition, after the cleaning of surface sterile physiological saline, put into umbilical cord and preserve transport liquid, preferably in 6 hours, be transported to clean Cell Lab on ice; Before use, fresh umbilical cord is rinsed 2-3 time with 75% aqueous ethanolic solution, then rinse 3-5 time by stroke-physiological saline solution;
Wherein preferably, described umbilical cord preserve transport liquid be comprise benzylpenicillin sodium for injection, Vetstrep, gentamicin and amphotericin B without calcium magnesium D-Hank ' s liquid; More preferably, the concentration of wherein said benzylpenicillin sodium, Vetstrep and gentamicin is 100-200U/mL, preferred 150U/ml; The concentration of amphotericin B is 200-400U/mL, preferred 300U/mL.
According to the specific embodiment of the present invention, the method for described separation and extraction mescenchymal stem cell from fresh umbilical cord China Tong Shi glue tissue comprises the following steps:
(1) collection of sample and transport: aseptic collection spontaneous labor or caesarean delivered healthy newborn umbilical cord tissue, puts into and preserves transport liquid, transport on ice;
(2) cleaning of umbilical cord tissue and sterilization: fresh umbilical cord sample is put into 50ml sterile centrifugation tube, 75% alcohol rinse 2-3 time, then rinse 3-5 time by stroke-physiological saline solution;
(3) pre-treatment of umbilical cord tissue: umbilical cord is cut into 2-3cm length left and right segment with eye scissors, with extravasated blood in tweezers removing blood vessel, longitudinally cut open with eye scissors for every section, two Umbilical artery and a umbilical vein is rejected with vascular clamp, blunt separation China Tong Shi glue, magnificent Tong Shi glue tissue block is placed in 50ml centrifuge tube, adds 1.5-2 times of volume PBS damping fluid, slight wobble is cleaned;
(4) erythrocyte cracked liquid process: the umbilical cord China Tong Shi glue tissue cleaned by PBS is transferred to the 100mm culture dish of cleaning sterile, is cut into 1-3mm
3size tissue block; Meat gruel shape umbilical cord China Tong Shi glue tissue block is transferred to centrifuge tube again, adds the erythrocyte cracked liquid of 1.5-2 times of tissue block volume; Room temperature treatment 2-5 minute, collected by centrifugation tissue block; PBS buffer solution for cleaning 2-3 time;
(5) IV Collagenase Type digestion: add tissue block volume 2-3 IV Collagenase Type solution doubly in centrifuge tube, move into CO
2concentration is in 37 DEG C of constant incubators of 5%, moves to clean bench after 8-12 hour from incubator, crosses the aseptic sieve of 100 or 200 order after adding the dilution of PBS damping fluid, and collect filtered solution in 50ml centrifuge tube, PBS buffer solution for cleaning is centrifugal, goes residual enzyme;
(6) hUC-MSC original cuiture: flick centrifuge tube floor cells group, add hUC-MSC serum free medium, make cell suspension, be transferred to 100mm culture dish, moves into CO
2concentration is in 37 DEG C of constant incubators of 5%, within every 3 days, changes fresh culture;
(7) get culture supernatant in step (6), detect following items: hepatitis A, hepatitis B, the third liver, syphilis, human immunodeficiency virus, mycoplasma, chlamydozoan, intracellular toxin;
(8) hUC-MSC Secondary Culture: attached cell converges rate when reaching about 60-80% in plate, trysinization, centrifugally remove supernatant collecting cell, be inoculated in T75 Tissue Culture Flask and proceed Secondary Culture, until converge rate to reach more than 90%, collecting cell is frozen or proceed Secondary Culture;
(9) for the hUC-MSC of step (8) gained, following items is detected: differentiation capability, cytoactive, cell purity, cell contamination, multiplication characteristic.
In aforesaid method, described sample is fresh umbilical cord tissue.
In aforesaid method, described umbilical cord preserve transport liquid be face with now join add benzylpenicillin sodium for injection, Vetstrep, gentamicin, amphotericin B without calcium magnesium D-Hank ' s liquid, wherein benzylpenicillin sodium, Vetstrep and the gentamicin final concentration in protection liquid is 100-200U/mL, and amphotericin B final concentration is 300U/ml; 40-60mL protects liquid to be loaded on aseptic specimen bottle, and sealed membrane seals.
In aforesaid method, described mescenchymal stem cell substratum is serum free medium, and comprising the beta-mercaptoethanol of 0.1 parts by volume, the non-essential amino acid of 1 parts by volume, the serum substitute of 10 parts by volume, the a-MEM/DMEM-F12 of 89 parts by volume and final concentration is the b-FGF of 10ng/ml.
Described erythrocyte cracked liquid is the NH comprising 5-10g/L
4cl and 0.1mmol/LNa
2the aqueous solution of-EDTA, pH is 7.2-7.4, crosses 0.22 μm of microfiltration membrane, balances to room temperature for subsequent use.
IV Collagenase Type Digestive system is D-Hank ' the s liquid containing 1% collagenase IV, 0.5% Unidasa, 300U/mlDNA enzyme, 2% serum substitute.
And in step (6), inoculum density is 2-3 × 10
4cell/cm
2.
In step (8), pancreas enzyme concentration is 0.125%, and digestion time is 1-2 minute, pats culture dish or culturing bottle sidewall in digestive process; The described ratio of going down to posterity is 1: 3-1: 4; Described frozen be every ml frozen storing liquid preserve 2-3 × 10
6individual cell.
On the other hand, the present invention also provides the hUC-MSC obtained by aforesaid method.
Preferably, described hUC-MSC has following characteristics:
(1) adhering to plastic culture vessel becomes fusiformis swirl shape to grow;
(2) the positive ratio of CD29, CD44, CD73, CD90, CD105 and HLA-ABC is greater than 99.0%; The positive ratio of CD45, CD34 and HLA-DR is less than 1.0%;
(3) externally scleroblast and stearoblast is induced to differentiate into;
(4) viable cell detected ratios reaches more than 99%;
(5) in typical " S type " growth curve characteristic;
(6) express versatility gene, described versatility gene be selected from SSEA-4, OCT-4, NANOG and SOX-2 one or more.
Another aspect, the invention provides erythrocyte cracked liquid as herein described, comprises the Digestive system of IV Collagenase Type and/or the purposes of described mesenchymal stem cell serum-free culture medium in the reagent for the preparation of separation and Culture mescenchymal stem cell.
Again on the one hand, the invention provides a kind of test kit for separating of cultivating mescenchymal stem cell, described test kit comprises erythrocyte cracked liquid as herein described, comprises the Digestive system of IV Collagenase Type and/or described mesenchymal stem cell serum-free culture medium.
The present invention proposes a kind of umbilical cord mesenchymal stem cells extracting method adopting erythrocyte cracked liquid to assist collagenase digesting, wherein can remove the red corpuscle larger to hUC-MSC primary cell growth effect with erythrocyte cracked liquid process, improve extracted hUC-MSCs purity to a great extent; And collagenase digesting significantly shorten the incubation time of primary cell, thus this extracting method achieves excellent results.Comprehensive, extracting method of the present invention just can gather in the crops primary cell at 6-8 days, and cell purity just can reach more than 99% after the s-generation.In addition, method of the present invention is simple, with low cost, the substratum serum-free component adopted, and form distinct, to avoid in culturing cell culturing process because serum batch difference causes the situation of cell growth process instability, also eliminate the possibility propagating the danger of xenogenesis pathogenic agent, serum-free culture based on this makes the security of hUC-MSC higher, has a good application prospect.
Through cells were tested by flow cytometry, through viability examination, differentiation capability qualification and versatility genetic analysis, the mescenchymal stem cell motility rate obtained by the inventive method is high, and purity is high, differentiation capability is strong, and the cell bank of foundation can be directly used in scientific research and clinical assisting therapy.
Accompanying drawing explanation
Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:
Fig. 1 is the cytological map in substratum composition screening process, wherein Figure 1A is lower concentration serum substitute culture medium inoculated 48 hours later cell form, Figure 1B is high density serum substitute culture medium inoculated 24 hours later cell form, Fig. 1 C is lower concentration bFGF culture medium inoculated 24 hours later cell form, and Fig. 1 D is high density bFGF culture medium culturing cell cellular form after going down to posterity.
Fig. 2 is the cellular form of cultivated hUC-MSC, wherein Fig. 2 A be primary cell initially converge form, Fig. 2 B be after going down to posterity cultivate one-tenth threadiness cellular form, Fig. 2 C is the cellular form not using erythrocyte cracked liquid process.
Fig. 3 (3A to 3I) is the result of hUC-MSC through flow cytometry analysis cell surface molecule of acquisition, and show described hUC-MSC and express CD29, CD44, CD73, CD90, CD105, HLA-ABC, positive ratio is greater than 99.0%; Do not express CD45, CD34, HLA-DR, positive ratio is less than 1.0%.
Fig. 4 is Vi-Cell cell viability analyser to the cell viability of the hUC-MSC obtained, growth characteristics analysis, wherein Fig. 4 A is the real-time activity analysis of hUC-MSC, Fig. 4 B is the growth curve of hUC-MSC, Fig. 4 C is the diameter distribution profile of hUC-MSC, and Fig. 4 D is circularity distribution plan after the digestion of hUC-MSC.Result shows that the activity of hUC-MSC is more than 99.7%, and cell dia is distributed in 9-15 μm, and has the multiplication characteristic of latent period, increased logarithmic phase, plateau.
Fig. 5 be the hUC-MSC that obtains to osteoblastic Induction of committed differentiation, wherein Fig. 5 A is the scarlet compound that the calcium tubercle generation color reaction of sodium alizarinsulfonate and osteogenetic process produces, and that to be oil red O steep specificity to the fat of stearoblast to Fig. 5 B is painted.
Fig. 6 is that the hUC-MSC versatility gene obtained is analyzed at the RT-PCR of transcriptional level.Wherein M is nucleic acid molecular weight standard, and 1 is reference gene β-Actin, and 2 is NANOG, and 3 is OCT-4, and 4 is SOX-2, and 5 is SSEA-4.
Fig. 7 is the hUC-MSC versatility differential protein that immuning fluorescent dyeing analysis obtains.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Unreceipted actual conditions, carry out according to the normal condition in field belonging to the present invention or the suggestion condition of instrument reagent supplier; Unreceipted commercial source is the conventional products that can commercially availablely buy.
embodiment 1the screening of mesenchymal stem cell serum-free culture medium composition
(1) the content screening of serum substitute
Tested substratum: the beta-mercaptoethanol of 0.1 parts by volume, recombination human basic fibroblast somatomedin (the b-FGF of 10ng/ml, Peprotech company), the non-essential amino aqueous acid (11140 of 1 parts by volume, Gibco company), 1, the KnockoutFBS serum substitute (10828-028, Gibco company) of 2,5,8,10,12,15 or 20 parts by volume, the a-MEM of 89 parts by volume.
In Biohazard Safety Equipment, get the hUC-MSC in the 3rd generation being located away from spontaneous labor neonatal umbilical cord China general formula glue tissue, with 2 × 10
4individual cell/cm
2density is inoculated in T75 Tissue Culture Flask, adds 12-15ml conventional commercial culture medium culturing cell.Cultivate and observation of cell completely adherent after, change 15mL tested substratum.Observation of cell growing state.
Result: in the medium respectively containing in three concentration groups of 1,2,5 parts by volume serum substitutes, cell proliferation is slow, at inoculation observation of cell after 24 hours, the cell aggregation of hUC-MSC part, cell is flat, index difference, degree of converging reaches about 20%, inoculates observation of cell after 48 hours, and hUC-MSC cell becomes clear, reach about 60% converge after, substantially stop breed (see Figure 1A); In the medium respectively containing in three concentration groups of 8,10,12 parts by volume serum substitutes, cell growth state is good, at inoculation observation of cell after 24 hours, hUC-MSC is that fusiformis swirl shape is assembled, and range of extension is high, and cell becomes clear, degree of converging reaches 40-60%, inoculate observation of cell after 48 hours, hUC-MSC cell becomes clear, and reaches more than 90% and converges; In the medium respectively containing in two concentration groups of 15,20 parts by volume serum substitutes, occur the situation identical with low concentration group, Growth of Cells is slow, cell flattening, clear-cut (see Figure 1B).
(2) the content screening of recombination human basic fibroblast somatomedin
Tested substratum: the beta-mercaptoethanol of 0.1 parts by volume, 1,2,5,8,10,12,15,18 or the recombination human basic fibroblast somatomedin (b-FGF of 20ng/ml, Peprotech company), the non-essential amino aqueous acid (11140 of 1 parts by volume, Gibco company), the KnockoutFBS serum substitute (10828-028, Gibco company) of 10 parts by volume, the a-MEM of 89 parts by volume.
With reference to (one) part method, with same cell source, equal densities inoculation, add 12-15mL conventional commercial culture medium culturing cell.Cultivate and observation of cell completely adherent after, change 15mL tested substratum.Observation of cell growing state.
Result: in the medium respectively containing 1, in two concentration groups of 2ng/mlbFGF, cell proliferation is slow, and cell state is poor, presents under-nutrition state (see Fig. 1 C); In the medium respectively containing 5,8,10,12, in the concentration group of 15ng/mlbFGF, cell normal growth, brightness is high, grows; In the medium respectively containing 18, in the concentration group of 20ng/mlbFGF, cell proliferation is good, bright, but in repeatedly succeeding generations, cell easily breaks up, and cell can become bulk to collect, or feeler elongated (see Fig. 1 D).
embodiment 2erythrocyte cracked liquid assists collagenase to extract the method for hUC-MSC
The collection of sample and transport: under aseptic condition, gather spontaneous labor neonatal umbilical cord sample, the umbilical cord put into containing benzylpenicillin sodium, Vetstrep, gentamicin and amphotericin B preserves transport liquid, and (concentration of benzylpenicillin sodium, Vetstrep and gentamicin described in D-Hank ' s liquid is 150U/ml; The concentration of amphotericin B is 300U/mL) in, be transported to clean cell room in 6 hours on ice;
The cleaning of sample and sterilization: in biological safety cabinet, put into 50ml sterile centrifugation tube by fresh umbilical cord sample, 75% alcohol rinse 2 times, then rinse 3 times by stroke-physiological saline solution;
The pre-treatment of umbilical cord tissue: umbilical cord is cut into 2-3cm length left and right segment with eye scissors, with extravasated blood in tweezers removing blood vessel, longitudinally cut open with eye scissors for every section, two Umbilical artery and a umbilical vein is removed with vascular clamp, umbilical cord China Tong Shi glue tissue is placed in 50ml centrifuge tube, add 1.5-2 times of volume PBS damping fluid, slight wobble is cleaned;
Erythrocyte cracked liquid process: the umbilical cord China Tong Shi glue tissue cleaned by PBS is transferred to the 100mm culture dish of cleaning sterile, is cut into 1-3mm
3size tissue block; Meat gruel shape umbilical cord China Tong Shi glue tissue block is transferred to centrifuge tube again, adds the erythrocyte cracked liquid of 1.5-2 times of tissue block volume, room temperature treatment 3 minutes, then 1200rpm, at 4 DEG C centrifugal 6 minutes, abandon supernatant, collection organization's block; PBS buffer solution for cleaning 2 times; Wherein erythrocyte cracked liquid comprises the NH of 5g/L
4cl and 0.1mMNa
2-EDTA, pH are 7.2-7.4;
IV Collagenase Type digests: the Digestive system (comprising D-Hank ' the s liquid of 1%IV Collagenase Type, 0.5% Unidasa, 300U/mlDNA enzyme, 2% serum substitute) comprising IV Collagenase Type adding tissue block 2 times of volumes in centrifuge tube, moves into CO
2concentration is in 37 DEG C of constant incubators of 5%, digests and from incubator, shifts out clean bench after 8 hours, crosses the aseptic sieved filter of 200 order after adding the dilution of PBS damping fluid, collect filtered solution, put into 50ml centrifuge tube, 1200rpm after PBS cleaning, at 4 DEG C centrifugal 6 minutes, go residual enzyme;
HUC-MSC original cuiture: flick centrifuge tube floor cells group, add umbilical cord mesenchymal stem cells serum free medium, make cell suspension, with 3 × 10
4cell/cm
2be inoculated in 100mm culture dish, be total to obtain 6 wares, move in constant incubator, within every 3 days, change fresh culture; Described mesenchymal stem cell serum-free culture medium comprises beta-mercaptoethanol, the non-essential amino aqueous acid (11140, Gibco company) of 1 parts by volume, the Knockout of 10 parts by volume of 0.1 parts by volume
tMthe a-MEM/DMEM-F12 of serum substitute, 89 parts by volume and final concentration are the b-FGF of 10ng/ml;
Move in constant incubator and cultivate the 5th day, slowly draw culture supernatant with pipettor, detect following items: hepatitis A, hepatitis B, the third liver, syphilis, human immunodeficiency virus, mycoplasma, chlamydozoan, intracellular toxin;
Passage: move in constant incubator and cultivate, original cuiture the 6th day attached cell converges (Fig. 2 A) when rate reaches about 50%, after adding 0.125% trysinization 1-2 minute (patting culture dish or culturing bottle sidewall in digestive process), centrifugal 6 minutes of 1200rpm at 4 DEG C, abandon supernatant collecting cell, be inoculated in T75 Tissue Culture Flask with 1: 4 ratio again and proceed Secondary Culture, until converge rate to reach 90%, collecting cell is frozen or proceed Secondary Culture.
Detection is learnt, reaches cell purity after 2nd generation be greater than 99% until cell.The one-tenth threadiness cellular form of cultivating after going down to posterity is shown in Fig. 2 B.
embodiment 3erythrocyte cracked liquid assists collagenase to extract the method for hUC-MSC
The erythrocyte cracked liquid adopted comprises the NH of 10g/L
4cl and 0.1mMNa
2-EDTA, pH are 7.2-7.4.
The method of reference example 2 is carried out, and the Digestive system comprising IV Collagenase Type digests 12 hours, and primary cell spreads 8 culture dish altogether, and being cultured to the 2nd day has mescenchymal stem cell adherent, converges rate and reaches 60%, go down to posterity after trysinization at about the 7th day; After reaching the third generation, cell purity is greater than 99.2%, and vigor is 99.7%.
embodiment 4erythrocyte cracked liquid is not used to extract the method for umbilical cord mesenchymal stem cells
The collection of sample and transport, the pretreatment process reference example 2 of umbilical cord tissue, is cut into 1-3mm by magnificent Tong Shi glue tissue
3meat gruel shape tissue block, without erythrocyte cracked liquid process, only adopt the Digestive system digestion 8h comprising IV collagenase, be evenly seeded in 100mm sterile petri dish after crossing 200 mesh sieves, every ware adds 10ml mesenchymal stem cell serum-free culture medium, puts into CO
2concentration be 5% 37 DEG C of incubators cultivate.Moving into incubator has mescenchymal stem cell adherent on the 3rd day, but has a large amount of red corpuscle to be attached at the bottom of ware, occupies the adherent space of stem cell, cell growth condition not good (Fig. 2 C).
embodiment 5different concns erythrocyte cracked liquid extracts the method for mescenchymal stem cell
The collection of sample and transport, the pretreatment process reference example 2 of umbilical cord tissue, is cut into 1-3mm by magnificent Tong Shi glue tissue
3meat gruel shape tissue block, adds the NH containing 1,2,5,7,10,15 or 20g/L respectively
4cl and 0.1mMNa
2the erythrocyte cracked liquid (pH is 7.2-7.4) of-EDTA, process 2 minutes, adopt the Digestive system digestion 8h comprising IV collagenase, be evenly seeded in 100mm sterile petri dish after crossing 200 mesh sieves, every ware adds 10ml mesenchymal stem cell serum-free culture medium, puts into CO
2concentration be 5% 37 DEG C of incubators cultivate.Observation of cell growing state.
Result: through wherein NH
4cl concentration be 1 or 2g/L erythrocyte cracked liquid process after, still have obvious visible red corpuscle to affect mescenchymal stem cell at the bottom of plate adherent; Through wherein NH
4cl concentration be 5,7 or 10g/L erythrocyte cracked liquid process after, at the bottom of ware, red corpuscle is less, the mescenchymal stem cell adherent time short (2-3 days); Through wherein NH
4cl concentration be 15 or 20g/L erythrocyte cracked liquid process after, without red corpuscle at the bottom of plate, but mescenchymal stem cell number of adherent is few, a large amount of floating dead cell, adherent evening time (4-5 days).
embodiment 6different time erythrocyte cracked liquid process umbilical cord extracts the method for mescenchymal stem cell
The collection of sample and transport, the pretreatment process reference example 2 of umbilical cord tissue, is cut into 1-3mm by magnificent Tong Shi glue tissue
3meat gruel shape tissue block, adds the NH containing 5g/L
4cl and 0.1mMNa
2the erythrocyte cracked liquid (pH is 7.2-7.4) of-EDTA, process umbilical cord tissue block respectively after 1,2,5,7 or 10 minutes, adopt the Digestive system digestion 8h comprising IV collagenase, evenly be seeded in after crossing 200 mesh sieves in 100mm sterile petri dish, every ware adds 10ml mesenchymal stem cell serum-free culture medium, puts into CO
2concentration be 5% 37 DEG C of incubators cultivate.Observation of cell growing state.
Result: be in the group of 1 minute in the treatment time, still have obvious visible red corpuscle to affect mescenchymal stem cell at the bottom of plate adherent; Be that in the group of 2 or 5 minutes, at the bottom of ware, red corpuscle is less in the treatment time, mescenchymal stem cell number of adherent is many, and cell becomes clear, quick wall attaching (2-3 days); Be in the group of 7 or 10 minutes in the treatment time, without red corpuscle at the bottom of plate, mescenchymal stem cell quantity is few, and adherent growth of mesenchymal stem cells is slow.
embodiment 7the Digestive system of different concns IV Collagenase Type extracts the method for mescenchymal stem cell
The collection of sample and transport, the pretreatment process reference example 2 of umbilical cord tissue, is cut into 1-3mm by magnificent Tong Shi glue tissue
3meat gruel shape tissue block, adds the NH containing 5g/L
4cl and 0.1mMNa
2the erythrocyte cracked liquid (pH is 7.2-7.4) of-EDTA, process 2 minutes, collected by centrifugation tissue block, add in centrifuge tube tissue block 2 times of volumes comprise IV Collagenase Type Digestive system (comprise 0.1%, 0.5%, 1%, 2% or 5%IV Collagenase Type, 0.5% Unidasa, 300U/mlDNA enzyme, 2% serum substitute D-Hank ' s liquid), move into CO
2concentration is in 37 DEG C of constant incubators of 5%, digests and from incubator, shifts out clean bench after 8 hours.Cross the aseptic sieve of 200 order.Add umbilical cord mesenchymal stem cells serum free medium, make cell suspension, with 3 × 10
4cell/cm
2be inoculated in 100mm culture dish, put into CO
2concentration be 5% 37 DEG C of incubators cultivate.Observation of cell growing state.
Result: after wherein IV Collagenase Type concentration is the Digestive system process 8h of 0.1% or 0.5%, still have a large amount of tissue block not digest completely in Digestive system, 200 mesh sieve difficulties, easily block; After wherein IV Collagenase Type concentration is the Digestive system process 8h of 1%, fully, a small amount of remnant tissue block, easily sieves in tissue block digestion, after paving ware, and most cells adherent growth, dead cell is few; After wherein IV Collagenase Type concentration is the Digestive system process 8h of 2% or 5%, tissue block digestion fully, is crossed 200 mesh sieves with after equal densities paving ware, is had a large amount of cell dissociation dead, can not adherent growth.
embodiment 8the Digestive system of different time IV Collagenase Type extracts the method for mescenchymal stem cell
Implementation method is with reference to the condition of embodiment 5, the Digestive system of IV Collagenase Type is D-Hank ' the s liquid comprising 1%IV Collagenase Type, 0.5% Unidasa, 300U/mlDNA enzyme, 2% serum substitute, the Digestive system treatment time is respectively 2,4,6,8,10,12,16,20,24h, other conditions are identical, observation of cell growing state.
Result: the treatment time be 2,4 or 6h group in, digest insufficient, still have in Digestive system a large amount of tissue block remain, 200 mesh sieves difficulty, easily block, cell quantity is few, and the primary cell culture time is long; The treatment time be 8,10 or 12h group in, fully, a small amount of residual tissue block, easily sieves in tissue block digestion, after paving ware, most cells adherent growth, dead cell is few, and Growth of Cells is quick; The treatment time be 16,20 or 24h group in, tissue block digestion fully, crosses 200 mesh sieves with after equal densities paving ware, can not adherent dead cell more, adherent cell growth is slow.
embodiment 9hUC-MSC Morphological Identification
By the separation and Culture of embodiment 3, in cultivation after 2 days, visible bright adherent round cell under microscope.Cultivate and can see bright adherent round cell under the microscope after 3 days and put out the feelers, the adherent shape in fusiformis, formed swirl shape cell mass at about 7 days and occur.In had digestive transfer culture culturing process, cellular form is homogeneous, and propagation is fast, and succeeding generations cell state is stablized.
embodiment 10the surface marker of flow cytometry analysis hUC-MSC
Example 3 separation and Culture the 3rd generation cell, after Growth of Cells to 90% converges, 2mL0.125% trysinization, then centrifugal 6 minutes of 1200rpm at 4 DEG C, abandons supernatant collecting cell, and PBS cleaning twice, by cell often pipe 1 × 10
5be transferred to streaming pipe, add 5 μ LCD34-PE, CD45-FITC, CD29-FITC, CD44-PE, CD73-PE, CD105-PE, CD90-FITC, HLA-ABC-FITC, HLA-DR-PE, IgG1-PE (Isotype control) and IgG1-FITC (Isotype control) antibody respectively, at mixing 4 DEG C, lucifuge hatches 30 minutes, PBS cleaning once, centrifugally remove supernatant, add the resuspended mixing of 500 μ LPBS damping fluid, upper machine testing (flow cytometer XL, Beckman company), each sample collection 1 × 10
4cell.
Immunophenotyping is as follows:
Positive expression: CD29 > 99.0%, CD44 > 99.0%, CD73 > 99.0%, CD105 > 99.0%, CD90 > 99.0%, HLA-ABC99.0%;
Negative expression: CD34 < 1.0%, CD45 < 1.0%, HLA-DR < 1.0%.
Result is see Fig. 3.
embodiment 11cell viability instrument analyzes cell viability, the growth characteristics of hUC-MSC
Be inoculated in T25 culturing bottle by the s-generation cell of embodiment 3 separation and Culture, reach after 95%-100% converges until cell, 0.125% trysinization, collecting cell is with 1 × 10
5/ hole density is inoculated in two 6 orifice plates.After cell is all adherent and some growth after 10 hours, collects two porocytes and add 500 μ LPBS and make cell suspension, upper machine analysis (cell viability analyser Vi-CellXR, Beckman company).After this every 12 hours sampling analysis, draw growth curve.
Result is see Fig. 4, and show that hUC-MSC activity is more than 99.7%, cell dia is distributed in 9-12 μm, has complete circularity, and have the multiplication characteristic of latent period, increased logarithmic phase, plateau after becoming the hUC-MSC digestion of fusiformis swirl shape growth.
embodiment 12the qualification of hUC-MSC multi-lineage potential
1) Osteoinductive differentiation
By the 4th generation hUC-MSC of embodiment 2 separation and Culture with 3 × 10
4cell/cm
2be seeded to 6 porocyte culture plates, after 24 hours, freshly prepared people UCMSC Osteoinductive differentiation substratum (HUXUC-90021 is added in every hole, match industry product) 2mL, after this within every 3 days, fresh Osteoblast Differentiation inducing culture is changed, after 2 weeks, paraformaldehyde is fixed, Alizarin red staining 3-5 minute.
Result, see Fig. 5 A, shows the hUC-MSC that obtains in the process of the present invention at osteogenic induction after two weeks, the calcium tubercle generation scarlet color reaction of sodium alizarinsulfonate and osteogenetic process, and skeletonization marker gene OPN also goes out differential expression before and after induction in addition.
2) adipogenic induction differentiation
By the 4th generation hUC-MSC of embodiment 2 separation and Culture with 2 × 10
4cell/cm
2be seeded to 6 porocyte culture plates, until cell reach 100% converge after, every hole is added adipogenic induction division culture medium A liquid (HUXUC-90031, match industry product) and is started induction, change adipogenic induction division culture medium B liquid after 3 days into and carry out maintenance 24 hours, so circulate.When fat ooze now more but less time, maintain 7 days with adipogenic induction liquid B, induction terminate after 4% paraformaldehyde fix, oil red O stain.
Result is see Fig. 5 B, and show the hUC-MSC that obtains in the process of the present invention at adipogenic induction after two weeks, oil red O is obviously painted to stearoblast.
embodiment 13rT-PCR analyzes hUC-MSC versatility gene
By the 3rd generation hUC-MSC of embodiment 2 separation and Culture with 5 × 10
5the density of cell is inoculated in T25 Tissue Culture Flask, collecting cell is converged to cell 100% after 2-3 days, by total RNA extraction reagent box (R6834-01, OMRGA Products) extracting RNA, by the RNA of extracting Reverse Transcription box (RR014A, TAKARA product) reverse transcription obtains cDNA sample, and performing PCR of going forward side by side increases, and moves to gel imaging instrument and observe after agarose gel electrophoresis.
Result, see Fig. 6, shows umbilical cord versatility marker gene NANOG, band that OCT4, SOX2 and SSEA4 have light levels different.
embodiment 14immuning fluorescent dyeing analysis hUC-MSC specific proteins
By the 3rd generation hUC-MSC of embodiment 2 separation and Culture with 5 × 10
3the density of cell per well is inoculated in 24 porocyte culture plates, treat that Growth of Cells to 30% ~ 50% converges, with 0.25%TritonX-100 punching process 20 minutes after fixing 15 minutes with 4% paraformaldehyde, lowlenthal serum adds the mouse-anti people primary antibodie (anti-SOX2 antibody, anti-OCT4 antibody, anti-NANOG antibody and anti-NANOG antibody) of having diluted after closing, lucifuge of spending the night at 4 DEG C is hatched; The goat against murine two adding FITC mark afterwards resists, and at room temperature lucifuge hatches 2 hours; Then contaminate core with DAPI/PI, at room temperature lucifuge hatches 20 minutes, fluorescence microscopy Microscopic observation.
Result, see Fig. 7, shows that the hUC-MSC of separation and Extraction in the process of the present invention expresses SOX-2, OCT-4, NANOG and SSEA-4 specific proteins.
Specific description of embodiments of the present invention does not above limit the present invention, and those skilled in the art can make various change or distortion according to the present invention, only otherwise depart from spirit of the present invention, all should belong to the scope of claims of the present invention.
Claims (12)
1. a method of separation and Extraction hUC-MSC, is characterized in that, described method comprises: adopt erythrocyte cracked liquid and collagenase process umbilical cord China Tong Shi glue tissue.
2. method according to claim 1, is characterized in that, described erythrocyte cracked liquid is for comprising NH
4cl and Na
2the aqueous solution of-EDTA, preferably comprises the NH of 1-20g/L
4cl and 0.05-0.2mMNa
2the aqueous solution of-EDTA, is more preferably the NH comprising 5-10g/L
4cl and 0.1mMNa
2the aqueous solution of-EDTA, pH is 7.2-7.4;
Preferably, described collagenase is IV Collagenase Type, preferably comprise the Digestive system of IV Collagenase Type, preferably comprise D-Hank ' the s liquid of IV Collagenase Type, Unidasa, DNA enzymatic and serum substitute, be more preferably D-Hank ' the s liquid comprising 1%IV Collagenase Type, 0.5% Unidasa, 300U/mlDNA enzyme and 2% serum substitute.
3. method according to claim 1 and 2, is characterized in that, described method comprises: the umbilical cord of acquisition China Tong Shi glue tissue segmentation is become tissue block, adds the described erythrocyte cracked liquid of 1-3 times of tissue block volume, process 2-5 minute under room temperature; Then the Digestive system comprising IV Collagenase Type described in adding in treated tissue block processes again.
4. according to the method in any one of claims 1 to 3, it is characterized in that, described method also comprises: after collagenase digesting, adopts mesenchymal stem cell serum-free culture medium to cultivate, to obtain primary mescenchymal stem cell; Wherein said mesenchymal stem cell serum-free culture medium comprises a-MEM/DMEM-F12, beta-mercaptoethanol, non-essential amino acid, recombination human basic fibroblast somatomedin (b-FGF) and serum substitute;
Preferably, described mesenchymal stem cell serum-free culture medium comprises the recombination human basic fibroblast somatomedin that the beta-mercaptoethanol of 0.05-0.2 parts by volume, the non-essential amino aqueous acid of 0.5-2 parts by volume, the serum substitute of 8-12 parts by volume, the a-MEM/DMEM-F12 of 85-95 parts by volume and final concentration are 5-15ng/ml, and it is respectively the glycine of 8-12mM, L-Ala, L-Tianmen acid amides, L-Aspartic acid, L-glutamic acid, proline(Pro) and Serine that wherein said non-essential amino aqueous acid comprises concentration;
More preferably, described mesenchymal stem cell serum-free culture medium comprises the recombination human basic fibroblast somatomedin that the beta-mercaptoethanol of 0.1 parts by volume, the non-essential amino aqueous acid of 1 parts by volume, the serum substitute of 10 parts by volume, the a-MEM/DMEM-F12 of 89 parts by volume and final concentration are 10ng/ml;
Most preferably, described mesenchymal stem cell serum-free culture medium is made up of described a-MEM/DMEM-F12, beta-mercaptoethanol, non-essential amino aqueous acid, recombination human basic fibroblast somatomedin and serum substitute.
5. method according to any one of claim 1 to 4, is characterized in that, described method comprises: the umbilical cord of acquisition China Tong Shi glue tissue is cut into 1-3mm
3the tissue block of size, adds the erythrocyte cracked liquid of 1.5-2 times of tissue block volume, processes 2-5 minute under room temperature; Then in treated tissue block, the Digestive system comprising IV Collagenase Type described in 2-3 times of volume is added, at the CO of 37 DEG C and 5% concentration
2lower digestion 8-12 hour;
Preferably, described method also comprises: with 1-5 × 10
4cell/cm
2the cell obtained is inoculated in mesenchymal stem cell serum-free culture medium by the density of culture dish area, at the CO of 37 DEG C and 5% concentration
2lower cultivation, changes fresh culture in every 3-4 days.
6. method according to any one of claim 1 to 5, is characterized in that, said method comprising the steps of:
(1) pre-treatment of umbilical cord tissue:
Fresh umbilical cord is divided into segment, removes blood vessel extravasated blood, longitudinally cut open, reject Umbilical artery and umbilical vein, blunt separation China Tong Shi glue, adds PBS buffer solution for cleaning in amnion tissue;
(2) erythrocyte cracked liquid process:
The umbilical cord tissue obtained through step (1) is divided into tissue block, adds erythrocyte cracked liquid process, the tissue block that collected by centrifugation is treated, add PBS buffer solution for cleaning;
(3) collagenase digesting:
Again processing adding the Digestive system comprising IV Collagenase Type in the tissue block obtained through step (2), then adding the dilution of PBS damping fluid, aseptic sieved filter collecting cell;
(4) original cuiture:
Mesenchymal stem cell serum-free culture medium is adopted to cultivate the cell obtained through step (3), to obtain primary mescenchymal stem cell;
Preferably, described method is further comprising the steps of:
(5) supernatant liquor detects:
Get the supernatant liquor of culturing cell in step (4), detect one or more in following items: hepatitis A, hepatitis B, the third liver, syphilis, human immunodeficiency virus, mycoplasma, chlamydozoan and intracellular toxin;
(6) Secondary Culture:
Getting test item in step (5) is negative culturing cell, centrifugal collecting cell after trysinization, Secondary Culture, and collecting cell is for subsequent use, frozen or continue to go down to posterity;
(7) cell detection:
Get cultured cells in step (6), detect one or more in following items: differentiation capability, cytoactive, cell purity, cell contamination and multiplication characteristic.
7. method according to any one of claim 1 to 6, it is characterized in that, described step (1) comprising: fresh umbilical cord is divided into the segment that 2-3cm is long, remove extravasated blood in blood vessel, longitudinally cut open, reject Umbilical artery and umbilical vein, blunt separation China Tong Shi glue, in amnion tissue, add the PBS damping fluid of 1.5-2 times of volume, slight wobble is cleaned;
Preferably, described step (2) comprising:
Umbilical cord China Tong Shi glue tissue through cleaning is cut into 1-3mm
3the tissue block of size, adds the erythrocyte cracked liquid of 1.5-2 times of tissue block volume, processes 2-5 minute under room temperature, collected by centrifugation tissue block, PBS buffer solution for cleaning 2-3 time; Wherein preferably, described centrifugal be 1000-1200rpm, at 4 DEG C centrifugal 6 minutes;
Preferably, described step (3) comprising:
The Digestive system comprising IV Collagenase Type of 2-3 times of volume is added, at the CO of 37 DEG C and 5% concentration in treated magnificent Tong Shi glue tissue block
2lower digestion 8-12 hour, cross 100 orders or the aseptic sieved filter of 200 orders after then adding the dilution of PBS damping fluid, collect filtered solution, PBS cleaning is centrifugal; Wherein preferably, described centrifugal be 1000-1200rpm, at 4 DEG C centrifugal 6 minutes;
Preferably, described step (4) comprising:
With 1-5 × 10
4cell/cm
2the cell obtained is inoculated in mesenchymal stem cell serum-free culture medium by the density of culture dish area, at the CO of 37 DEG C and 5% concentration
2lower cultivation, changes fresh culture in every 3-4 days;
Preferably, described step (5) comprising:
Get the supernatant liquor of culturing cell in step (4), that detects in following items is whole: hepatitis A, hepatitis B, the third liver, syphilis, human immunodeficiency virus, mycoplasma, chlamydozoan and intracellular toxin;
Preferably, described step (6) comprising:
Getting whole test item in step (5) is negative culturing cell, until attached cell converge rate reach 30-60% time, centrifugal collecting cell after trysinization, Secondary Culture reaches more than 90% to converging rate, and collecting cell is for subsequent use, frozen or continue to go down to posterity; Wherein preferably, the working concentration of described pancreatin is mass percent is 0.125%, and digestion 1-2 minute, pats culture dish or culturing bottle sidewall in digestive process; And wherein preferably, described centrifugal be 1000-1200rpm, at 4 DEG C centrifugal 6 minutes;
Preferably, by collect cell with 2-3 × 10
6the density freezen protective of cell/ml is in-196 liquid nitrogen; Or preferably, by collect cell with 1: 3-1: 4 ratio carry out passage;
Preferably, described step (7) comprising:
Get cultured cells in step (6), that detects in following items is whole: differentiation capability, cytoactive, cell purity, cell contamination and multiplication characteristic.
8. method according to any one of claim 1 to 7, is characterized in that, also carries out umbilical cord cleaning, preservation and use pre-treatment in described method before step (1);
Preferably, described process comprises:
Gather spontaneous labor or caesarean delivered healthy newborn umbilical cord tissue under aseptic condition, after the cleaning of surface sterile physiological saline, put into umbilical cord and preserve transport liquid, preferably in 6 hours, be transported to clean Cell Lab on ice; Before use, fresh umbilical cord is rinsed 2-3 time with 75% aqueous ethanolic solution, then rinse 3-5 time by stroke-physiological saline solution;
Wherein preferably, described umbilical cord preserve transport liquid be comprise benzylpenicillin sodium for injection, Vetstrep, gentamicin and amphotericin B without calcium magnesium D-Hank ' s liquid; More preferably, the concentration of wherein said benzylpenicillin sodium, Vetstrep and gentamicin is 100-200U/mL, preferred 150U/ml; The concentration of amphotericin B is 200-400U/mL, preferred 300U/mL.
9. by the hUC-MSC of the method acquisition according to any one of claim 1 to 8.
10. hUC-MSC according to claim 9, is characterized in that, described mescenchymal stem cell has following characteristics:
(1) adhering to plastic culture vessel becomes fusiformis swirl shape to grow;
(2) the positive ratio of CD29, CD44, CD73, CD90, CD105 and HLA-ABC is greater than 99.0%; The positive ratio of CD45, CD34 and HLA-DR is less than 1.0%;
(3) externally scleroblast and stearoblast is induced to differentiate into;
(4) viable cell detected ratios reaches more than 99%;
(5) in typical " S type " growth curve characteristic;
(6) express versatility gene, described versatility gene be selected from SSEA-4, OCT-4, NANOG and SOX-2 one or more.
11. erythrocyte cracked liquids according to claim 2, the Digestive system comprising IV Collagenase Type and/or the purposes of mesenchymal stem cell serum-free culture medium according to claim 4 in the reagent for the preparation of separation and Culture mescenchymal stem cell.
12. 1 kinds for separating of the test kit cultivating mescenchymal stem cell, it is characterized in that, described test kit comprises erythrocyte cracked liquid according to claim 2, comprises the Digestive system of IV Collagenase Type and/or mesenchymal stem cell serum-free culture medium according to claim 4.
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