CN105420183A - Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord Wharton jelly tissue - Google Patents
Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord Wharton jelly tissue Download PDFInfo
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Abstract
The invention provides a method for culturing umbilical cord mesenchymal stem cells in a separated mode from umbilical cord Wharton jelly tissue. The method includes the steps that umbilical cord tissue of a healthy newborn is used, washed, disinfected and mechanically cut into pieces to separate Wharton jelly, and the Wharton jelly is cultured in a suspension mode with a serum-free culture medium after being treated with erythrocyte lysate; medium change is carried out every three to five days, after the attachment rate in a plate reaches 30-70%, trypsinization is used, cell passage amplification is collected centrifugally, and confluence is carried out when the cell confluence rate reaches 80-90% to obtain the high-purity umbilical cord mesenchymal stem cells.
Description
Technical field
The present invention relates to the stable separating and extracting method of umbilical cord mesenchymal stem cells, particularly the method for separation and Culture mescenchymal stem cell from umbilical cord China Tong Shi glue tissue.
Background technology
Mescenchymal stem cell (mesenchymalstemcells, MSC) is the important member of stem cell line, derives from and grows early stage mesoderm and ectoderm, be present in whole body reticular tissue and organ interstitial.MSC in late 1970s Late Cambrian in marrow, but until eighties of last century nineties, after MSC multi-lineage potential is familiar with by people, this stem cell just receives increasing concern.
Mescenchymal stem cell belongs to multipotential stem cell, there are some researches show at present, and MSC can through being induced to differentiate into Various Tissues cell and the class cells such as fat, bone, bone ligament, nerve, liver, cardiac muscle, endothelium, pancreas islet; And find, mescenchymal stem cell all has obvious effect in immunosuppression, endocrine system adjustment, neural system adjustment and cardiovascular function improvement etc.
Also comparatively extensive to the extraction research of mescenchymal stem cell at present, comprise and extracting from the human multiple tissues such as fat, marrow, amniotic fluid, placenta, umbilical cord tissue, Cord blood, periodontal and organ.Wherein the mescenchymal stem cell in umbilical cord source because growing environment is single, vitro extraction is convenient, such that the mescenchymal stem cell purity extracted is high, quantity large, the ideal substitute of mesenchymal stem cells MSCs can be become, there is more wide clinical application potential.
In view of the multi-lineage potential of umbilical cord multipotential stem cell and the application potential in disease treatment field, the storage service of the umbilical cord mesenchymal stem cells of domestic market has broad prospects, therefore, the how separation and Extraction rate of the guarantee stem cell of efficiency standard, and ensure stem cell high-quality, thus meet the clinical demand of following stem cell transplantation, be current domestic stem cell storage service problem demanding prompt solution.
Current umbilical cord mesenchymal stem cells many employings enzyme digestion and organize adherent method to prepare, but in separation and Extraction process, erythrocytic interference is the important factor affecting umbilical cord mesenchymal stem cells quality and quantity, a large amount of erythrocytic existence makes the growing space of mescenchymal stem cell limited, affect the vigor state of stem cell simultaneously, very large impact is caused on umbilical cord mesenchymal stem cells separation and Extraction and exploitation.
Summary of the invention
The object of the invention is the needs for this area, a kind of umbilical cord mesenchymal stem cells separating and extracting method that can overcome the improvement of red corpuscle interference is provided.
Another object of the present invention is to provide the umbilical cord mesenchymal stem cells obtained by method of the present invention.
Specifically, 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 erythrocyte cracked liquid and take suspension culture to be separated primary mescenchymal stem cell from fresh umbilical cord China Tong Shi glue tissue, serum free culture system original cuiture line stabilization of going forward side by side is utilized to go down to posterity, to improve separation and Extraction success ratio and the cell purity of umbilical cord mesenchymal stem cells, for the clinical application of human umbilical cord mesenchymal stem cells provides possibility.
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 tissue China Tong Shi glue, it is a kind of method of separation and Culture umbilical cord mesenchymal stem cells, described method comprises: adopt the magnificent Tong Shi glue tissue that erythrocyte cracked liquid process is separated from umbilical cord, and adopt mesenchymal stem cell serum-free culture medium to cultivate.
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 0.1mMNa
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 mesenchymal stem cell serum-free culture medium that the present invention adopts 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).
Method of the present invention specifically comprises: the magnificent Tong Shi glue tissue segmentation obtained 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 mesenchymal stem cell serum-free culture medium is adopted to cultivate the magnificent Tong Shi glue tissue block obtained, to obtain primary mescenchymal stem cell.
Preferably, described method comprises: the 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; Then the tissue block uniform spreading of acquisition is reached in culture dish the covering of 60-80%, add the mesenchymal stem cell serum-free culture medium of 3-5 times of tissue block volume, at the CO of 37 DEG C and 5% concentration
2lower cultivation, during 3-5 days, half amount changes fresh culture, during 5-15 days after evenly climbing out of cell under tissue block, removes tissue block, and full dose is changed fresh mesenchymal stem cell serum-free culture medium and continued to cultivate, and after this often within 3-4 days, carries out a fresh culture and changes.
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 tissue, adds PBS buffer solution for cleaning;
(2) erythrocyte cracked liquid process:
The magnificent Tong Shi glue tissue segmentation obtained through step (1) 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) original cuiture:
Mesenchymal stem cell serum-free culture medium is adopted to cultivate the magnificent Tong Shi glue tissue block obtained through step (2), to obtain primary mescenchymal stem cell;
Preferably, described method is further comprising the steps of:
(4) supernatant liquor detects:
Get the supernatant liquor of culturing cell in step (3), detect one or more in following items: hepatitis A, hepatitis B, the third liver, syphilis, human immunodeficiency virus, mycoplasma, chlamydozoan and intracellular toxin;
(5) Secondary Culture:
Getting test item in step (4) 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;
(6) cell detection:
Get cultured cells in step (5), 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 tissue, add the PBS damping fluid of 2-5 times of volume, slight wobble is cleaned;
Preferably, described step (2) comprising:
Magnificent 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 tissue block uniform spreading obtained through step (2) is reached in culture dish 60-80%, preferably 70% covering, add the mesenchymal stem cell serum-free culture medium of 3-5 times of tissue block volume, at the CO of 37 DEG C and 5% concentration
2lower cultivation, during 3-5 days, half amount changes fresh culture, when 5-15 days, preferably 7-10 days after evenly climbing out of cell under tissue block, removes tissue block, full dose is changed fresh mesenchymal stem cell serum-free culture medium and is continued to cultivate, and after this often within 3-4 days, carries out a fresh culture and changes;
Preferably, described step (4) comprising:
Get the supernatant liquor of culturing cell in step (3), 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 (5) comprising:
Getting whole test item in step (4) is negative culturing cell, until attached cell converge rate reach 30-80%, preferably 40-50% time, centrifugal collecting cell after trysinization, adopt mesenchymal stem cell serum-free culture medium Secondary Culture to reach 50-90%, preferably 80-90% to converging rate, 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 DEG C of liquid nitrogen; Or preferably, by collect cell with 1: 3-1: 4 ratio carry out passage;
Preferably, described step (6) comprising:
Get cultured cells in step (5), 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 in vitro tissue comprises the following steps:
(1) collection of sample and transport: aseptic collection spontaneous labor or caesarean delivered healthy newborn umbilical cord sample, puts into umbilical cord and preserves transport liquid, transport on ice;
(2) cleaning of sample 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 arteries and a vein is rejected with mosquito forceps, blunt separation China Tong Shi glue tissue is placed in 50ml centrifuge tube, add 2-5 times of volume PBS damping fluid, slight wobble is cleaned;
(4) erythrocyte cracked liquid process: magnificent Tong Shi glue is organized the 100mm culture dish being again transferred to cleaning sterile respectively, be cut into 1-3mm
3size tissue block; Meat gruel shape umbilical cord tissue block is transferred to centrifuge tube again, rejoins the erythrocyte cracked liquid of 1.5-2 times of tissue block volume respectively; Room temperature treatment 2 minutes, collected by centrifugation tissue block; PBS buffer solution for cleaning 2-3 time.
(5) original cuiture: by the tissue block uniform spreading of magnificent Tong Shi glue tissue at 100mm culture dish, cover 70% ware floorage, every ware adds 10ml mesenchymal stem cell serum-free culture medium, puts into CO
2concentration be 5% 37 DEG C of constant incubators cultivate, shifted out from incubator by plate when being cultured to 3-5 days, half amount changes fresh culture, continues to cultivate; At 7-10 days, under treating tissue block, evenly climb out of cell, remove tissue block full dose and change liquid continuation cultivation; After this within every 3 days, once liquid is changed;
(6) get culture supernatant in step (5), detect following items: hepatitis A, hepatitis B, the third liver, syphilis, human immunodeficiency virus, mycoplasma, chlamydozoan, intracellular toxin;
(7) passage: attached cell converges rate when reaching about 30-70% in plate, trysinization, centrifugally removes supernatant collecting cell, is inoculated in Tissue Culture Flask and proceeds Secondary Culture, until converge rate to reach 80-90%, collecting cell is for subsequent use, frozen or proceed Secondary Culture;
(8) for the umbilical cord mesenchymal stem cells of step (7) 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 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 and uses.
And in step (7), described pancreas enzyme concentration is 0.125%, and digestion time is 1-2 minute, pats culture dish or culturing bottle sidewall in digestive process.
On the other hand, the present invention also provides the mescenchymal stem cell obtained by aforesaid method.
Preferably, 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.
Another aspect, the invention provides described erythrocyte cracked liquid 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 and/or described mesenchymal stem cell serum-free culture medium.
Cell viability state is the most important quality control index of mescenchymal stem cell.But, in the separation and Extraction process of umbilical cord mesenchymal stem cells, erythrocytic interference is the important factor affecting umbilical cord mesenchymal stem cells quality and quantity, a large amount of erythrocytic existence makes the growing space of mescenchymal stem cell limited, affect the vigor state of stem cell simultaneously, very large impact is caused on umbilical cord mesenchymal stem cells separation and Extraction and exploitation.
Erythrocyte cracked liquid is used on the extracting method of umbilical cord mesenchymal stem cells by the present invention, obviously can improve the red corpuscle interference in umbilical cord mesenchymal stem cells original cuiture.Particularly, in original cuiture process, erythrocyte cracked liquid is adopted to process, avoid the interference of red corpuscle to the shifting out of mescenchymal stem cell, adherent and propagation, ensure that mescenchymal stem cell Success rate of virus isolation, improve cell purity, while the clinical grade quantitative requirement ensureing umbilical cord mesenchymal stem cells, more ensure that the vigor state of cell, for later passages cultivation, cryopreservation resuscitation and even clinical application provide a large amount of and rich activated mescenchymal stem cell, be conducive to the future clinical Transformation Application of stem cell.
In addition, the use of erythrocyte cracked liquid combines with serum free culture system by the present invention, guarantees success rate of extracting and the cell purity of umbilical cord mesenchymal stem cells, thus for current umbilical cord mesenchymal stem cells store, follow-up clinical application work provides stable service.This substratum serum-free component, and form distinct, to avoid in culturing cell culturing process because serum batch difference causes the situation of cell growth process instability, also eliminates the possibility propagating the danger of xenogenesis pathogenic agent.
Further, operation is simple for method of the present invention, shortens the original cuiture time.
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 cultivated umbilical cord mesenchymal stem cells cellular form, wherein Fig. 2 A is that cell in primary cell tissue block just climbs out of form, Fig. 2 B is the one-tenth threadiness cellular form of cultivating after going down to posterity, Fig. 2 C for not use erythrocyte cracked liquid to carry out mescenchymal stem cell separation, cellular form when cell just climbs out of.
Fig. 3 (3A to 3I) is the result of umbilical cord mesenchymal stem cells through flow cytometry analysis cell surface molecule of acquisition, show described umbilical cord mesenchymal stem cells 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 umbilical cord mesenchymal stem cells obtained, growth characteristics analysis, wherein Fig. 4 A is the real-time activity analysis of described umbilical cord mesenchymal stem cells, Fig. 4 B is the growth curve of described umbilical cord mesenchymal stem cells, and Fig. 4 C is the diameter distribution profile of described umbilical cord mesenchymal stem cells.Result shows that the activity of above-mentioned umbilical cord mesenchymal stem cells is more than 99.7%, cell dia is distributed in 9-15 μm, after becoming the umbilical cord mesenchymal stem cells digestion of fusiformis swirl shape growth, there is complete circularity, and there is the multiplication characteristic of latent period, increased logarithmic phase, plateau.
Fig. 5 is that the umbilical cord mesenchymal stem cells of acquisition is 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, Fig. 5 B is the differential expression of described umbilical cord mesenchymal stem cells skeletonization born of the same parents marker gene OPN before and after differentiation, wherein M swimming lane is nucleic acid molecular weight standard, 1,4 swimming lanes are reference gene β-Actin, and 2,3 swimming lanes are that skeletonization marker gene OPN changes in differentiation-inducing front and back.
Fig. 6 is the Induction of committed differentiation of mescenchymal stem cell to stearoblast of acquisition, wherein Fig. 6 A is that the fat bubble specificity of oil red O to stearoblast is painted, Fig. 6 B is that described umbilical cord mesenchymal stem cells becomes the differential expression of fat born of the same parents marker gene PPAR-γ before and after differentiation, wherein M swimming lane is nucleic acid molecular weight standard, 2,4 swimming lanes are reference gene β-Actin, and 1,3 swimming lanes change in differentiation-inducing front and back for becoming fat marker gene PPAR-γ.
Fig. 7 is that the umbilical cord mesenchymal stem cells versatility gene obtained is analyzed at the RT-PCR of transcriptional level, and 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.
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; For dated commercial source, be 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 2the method of erythrocyte cracked liquid assisted extraction umbilical cord mesenchymal stem cells
The collection of sample and transport: under aseptic condition, gather Cesarean esction neonatal umbilical cord sample, put into containing benzylpenicillin sodium, Vetstrep, gentamicin and amphotericin B umbilical cord preserve transport liquid (D-Hank ' s liquid benzylpenicillin sodium, Vetstrep and gentamicin protection liquid final concentration be 150U/mL, amphotericin B final concentration is 300U/ml), be transported to clean GMP Cell Lab in 2 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, blunt separation China Tong Shi glue is placed in 50ml centrifuge tube, add 2-5 times of volume PBS damping fluid, slight wobble is cleaned;
Erythrocyte cracked liquid process: magnificent Tong Shi glue is organized and is again transferred to clean sterile petri dish respectively, be cut into 1-3mm
3size tissue block; Meat gruel shape umbilical cord tissue block is transferred to centrifuge tube again, rejoins 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; Wherein erythrocyte cracked liquid comprises the NH of 5g/L
4cl and 0.1mMNa
2-EDTA, pH are 7.2-7.4;
Original cuiture: by meat gruel shape China Tong Shi glue tissue block uniform spreading in 100mm sterile petri dish, cover 70% ware floorage, every ware adds 4 times of about 10ml mesenchymal stem cell serum-free culture mediums, puts into CO
2concentration be 5% 37 DEG C of incubators cultivate, when being cultured to 3 days, plate is shifted out from incubator, adds 5ml fresh culture; At 7 days, evenly climb out of cell (Fig. 2 A), remove tissue block under treating tissue block, add the cleaning of 5mlPBS weak vibrations, inhale and abandon PBS, every ware adds 10ml fresh culture, within after this every 3 days, once changes liquid; The mesenchymal stem cell serum-free culture medium wherein adopted 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;
Passage: tissue block paves ware the 12nd day, attached cell converges rate when reaching about 50%, massfraction is after 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, adopt mesenchymal stem cell serum-free culture medium Secondary Culture, until converge rate to reach 80-90%, collecting cell or proceed Secondary Culture.The one-tenth threadiness cellular form of cultivating after going down to posterity is shown in Fig. 2 B.
embodiment 3the method of erythrocyte cracked liquid assisted extraction umbilical cord mesenchymal stem cells
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 being cultured to the 5th day has mescenchymal stem cell to climb out of at the bottom of tissue block, and at the 7th day, mesenchymal stem cells formed swirl shape cell mass between left and right, after removing tissue block replacing fresh culture, about the 12nd day, cell confluency reached 60%, goes down to posterity after trysinization; After reaching the third generation, cell purity is greater than 99%, and vigor is greater than 95%.
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, Direct Uniform is layered in 100mm sterile petri dish, and cover 70% ware floorage, every ware adds 10ml mesenchymal stem cell serum-free culture medium, puts into CO
2concentration be 5% 37 DEG C of incubators cultivate.At 9 days, climb out of cell under tissue block, but have a large amount of red corpuscle to be attached at the bottom of ware, occupy the adherent space of stem cell (Fig. 2 C), adherent weak effect, cell growth condition is not good.
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, be then layered in 100mm sterile petri dish by cell Direct Uniform, cover 70% ware floorage, 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, and mescenchymal stem cell climbs out of the time short (5-7 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 climbs out of the time long (7-12 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, then cell Direct Uniform is layered in 100mm sterile petri dish, covers 70% ware floorage, 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 climbs out of the time short (5-7 days); Be in the group of 7 or 10 minutes in the treatment time, without red corpuscle at the bottom of plate, but mescenchymal stem cell climbs out of the time long (7-12 days), and cell concentration is few, and the mescenchymal stem cell amplification grown slowly.
embodiment 7umbilical cord mesenchymal stem cells Morphological Identification
By the separation and Culture of embodiment 2, umbilical cord mononuclearcell is paved plate at cell and is observed after the 3rd day, visible bright adherent round cell under microscope.Cultivate and can see bright adherent round cell under the microscope after 5 days and put out the feelers, the adherent shape in fusiformis, formed swirl shape cell mass at about 10 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 8the surface marker of flow cytometry analysis umbilical cord mesenchymal stem cells
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 ratio: CD29 > 99.0%, CD44 > 99.0%, CD73 > 99.0%, CD105 > 99.0%, CD90 > 99.0%, HLA-ABC99.0%;
Positive ratio: CD34 < 1.0%, CD45 < 1.0%, HLA-DR < 1.0%.
Result is see Fig. 3.
embodiment 9cell viability instrument analyzes cell viability, the growth characteristics of umbilical cord mesenchymal stem cells
Be inoculated in T25 culturing bottle by the s-generation cell of embodiment 2 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.Until the whole adherent growth of cell after 10 hours, collect 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, show that umbilical cord mesenchymal stem cells is active 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 umbilical cord mesenchymal stem cells digestion of fusiformis swirl shape growth.
embodiment 10the qualification of umbilical cord mesenchymal stem cells multi-lineage potential
Osteoinductive differentiation
By embodiment 2 separation and Culture the 4th generation umbilical cord multipotential stem cell 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.Identify the expression of skeletonization marker gene OPN at transcriptional level with RT-PCR simultaneously.
Result, see Fig. 5, shows the umbilical cord mesenchymal stem cells 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.
Adipogenic induction breaks up
By embodiment 3 separation and Culture the 4th generation umbilical cord multipotential stem cell 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; Identify the expression of skeletonization marker gene PPAR-γ at transcriptional level with RT-PCR simultaneously.
Result is see Fig. 6, and show the umbilical cord mesenchymal stem cells that obtains in the process of the present invention at adipogenic induction after two weeks, oil red O is obviously painted to stearoblast, becomes fat marker gene PPAR-γ also before and after induction, to go out differential expression in addition.
embodiment 11rT-PCR analyzes umbilical cord mesenchymal stem cells versatility gene
By embodiment 2 separation and Culture the 3rd generation umbilical cord multipotential stem cell with 5 × 10
5cell/cm
2density be inoculated in T25 Tissue Culture Flask, converge collecting cell, extracting RNA to cell 100% after 2-3 days, the RNA reverse transcription of extracting obtained cDNA sample, performing PCR of going forward side by side increase, move to after agarose gel electrophoresis gel imaging instrument observe.
Result, see Fig. 7, shows umbilical cord versatility marker gene NANOG, band that OCT4, SOX2 and SSEA4 have light levels different.
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 (11)
1. a method for separation and Culture umbilical cord mesenchymal stem cells, is characterized in that, described method comprises: adopt the magnificent Tong Shi glue tissue that erythrocyte cracked liquid process is separated from umbilical cord, and adopt mesenchymal stem cell serum-free culture medium to cultivate.
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.
3. method according to claim 1 and 2, it is characterized in that, described 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.
4. according to the method in any one of claims 1 to 3, it is characterized in that, described method comprises: the magnificent Tong Shi glue tissue segmentation obtained 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 mesenchymal stem cell serum-free culture medium is adopted to cultivate the magnificent Tong Shi glue tissue block obtained, to obtain primary mescenchymal stem cell;
Preferably, described method comprises: the 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; Then the tissue block uniform spreading of acquisition is reached in culture dish the covering of 60-80%, add the mesenchymal stem cell serum-free culture medium of 3-5 times of tissue block volume, at the CO of 37 DEG C and 5% concentration
2lower cultivation, during 3-5 days, half amount changes fresh culture, during 5-15 days after evenly climbing out of cell under tissue block, removes tissue block, and full dose is changed fresh mesenchymal stem cell serum-free culture medium and continued to cultivate, and after this often within 3-4 days, carries out a fresh culture and changes.
5. method according to any one of claim 1 to 4, 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 tissue, adds PBS buffer solution for cleaning;
(2) erythrocyte cracked liquid process:
The magnificent Tong Shi glue tissue segmentation obtained through step (1) 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) original cuiture:
Mesenchymal stem cell serum-free culture medium is adopted to cultivate the magnificent Tong Shi glue tissue block obtained through step (2), to obtain primary mescenchymal stem cell;
Preferably, described method is further comprising the steps of:
(4) supernatant liquor detects:
Get the supernatant liquor of culturing cell in step (3), detect one or more in following items: hepatitis A, hepatitis B, the third liver, syphilis, human immunodeficiency virus, mycoplasma, chlamydozoan and intracellular toxin;
(5) Secondary Culture:
Getting test item in step (4) 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;
(6) cell detection:
Get cultured cells in step (5), detect one or more in following items: differentiation capability, cytoactive, cell purity, cell contamination and multiplication characteristic.
6. method according to any one of claim 1 to 5, 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 tissue, add the PBS damping fluid of 3-5 times of volume, slight wobble is cleaned;
Preferably, described step (2) comprising:
Magnificent 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 tissue block uniform spreading obtained through step (2) is reached in culture dish 60-80%, preferably 70% covering, add the mesenchymal stem cell serum-free culture medium of 3-5 times of tissue block volume, at the CO of 37 DEG C and 5% concentration
2lower cultivation, during 3-5 days, half amount changes fresh culture, when 5-15 days, preferably 7-10 days after evenly climbing out of cell under tissue block, removes tissue block, and full dose is changed fresh culture and continued to cultivate, and after this often within 3-4 days, carries out a fresh culture and changes;
Preferably, described step (4) comprising:
Get the supernatant liquor of culturing cell in step (3), 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 (5) comprising:
Getting whole test item in step (4) is negative culturing cell, until attached cell converge rate reach 30-80%, preferably 40-50% time, centrifugal collecting cell after trysinization, adopt mesenchymal stem cell serum-free culture medium Secondary Culture to reach 50-90%, preferably 80-90% to converging rate, 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 DEG C of liquid nitrogen; Or preferably, the cell of collection is carried out passage with the ratio of 1:3-1:4;
Preferably, described step (6) comprising:
Get cultured cells in step (5), that detects in following items is whole: differentiation capability, cytoactive, cell purity, cell contamination and multiplication characteristic.
7. method according to any one of claim 1 to 6, 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.
8. by the mescenchymal stem cell of the method acquisition according to any one of claim 1 to 7.
9. mescenchymal stem cell according to claim 8, 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.
10. erythrocyte cracked liquid according to claim 2 and/or the purposes of mesenchymal stem cell serum-free culture medium according to claim 3 in the reagent for the preparation of separation and Culture mescenchymal stem cell.
11. 1 kinds of test kits for separating of cultivation mescenchymal stem cell, it is characterized in that, described test kit comprises erythrocyte cracked liquid according to claim 2 and/or mesenchymal stem cell serum-free culture medium according to claim 3.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN107189982A (en) * | 2017-06-12 | 2017-09-22 | 北京焕生汇生物技术研究院 | A kind of cultural method of human umbilical cord mesenchymal stem cells |
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| CN110804585A (en) * | 2019-11-19 | 2020-02-18 | 武汉济源高科技有限公司 | Separation method of umbilical cord mesenchymal stem cells |
| JP2020191861A (en) * | 2019-05-27 | 2020-12-03 | フォシャン ジンヂュン ジエン ヘルス マネジメント コンサルティング カンパニー リミテッドＦｏｓｈａｎ Ｊｉｎｇｚｈｕｎ Ｊｉｅｎ Ｈｅａｌｔｈ Ｍａｎａｇｅｍｅｎｔ Ｃｏｎｓｕｌｔｉｎｇ Ｃｏ．， Ｌｔｄ． | Umbilical cord mesenchymal stem cells mscs and culture methods and applications thereof |
| CN112292447A (en) * | 2019-02-28 | 2021-01-29 | 京东方科技集团股份有限公司 | Umbilical cord mesenchymal stem cell and preparation method of cell membrane thereof |
| CN112522791A (en) * | 2020-12-09 | 2021-03-19 | 河北医科大学 | Construction method of human umbilical cord mesenchymal stem cell bank |
| CN112545976A (en) * | 2020-12-25 | 2021-03-26 | 北京亦科诺生物科技有限公司 | Preparation method of vitality-enhancing anti-aging essence |
| CN114196626A (en) * | 2022-02-16 | 2022-03-18 | 北京国卫生物科技有限公司 | Umbilical cord mesenchymal stem cells and isolated culture and amplification method thereof |
| CN115044545A (en) * | 2022-07-11 | 2022-09-13 | 睿博斯(北京)生物科技有限公司 | Mesenchymal stem cell and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101629165A (en) * | 2009-07-24 | 2010-01-20 | 中国人民解放军海军总医院 | Preparation method of umbilical cord Huatong glue mesenchymal stem cells and product thereof |
| US20130302285A1 (en) * | 2011-11-09 | 2013-11-14 | National University Of Singapore | Wharton's Jelly Mesenchymal Stem Cells and Uses Thereof |
| CN103589683A (en) * | 2013-08-12 | 2014-02-19 | 北京东方华辉生物医药科技有限公司 | Separation method and culture method for umbilical cord mesenchymal stem cells |
| CN103589686A (en) * | 2013-11-06 | 2014-02-19 | 四川新生命干细胞科技股份有限公司 | Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells |
| CN104212764A (en) * | 2014-09-22 | 2014-12-17 | 上海华颜干细胞科技有限公司 | Preparation method of clinical mesenchymal stem cells |
-
2015
- 2015-12-11 CN CN201510918205.2A patent/CN105420183A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101629165A (en) * | 2009-07-24 | 2010-01-20 | 中国人民解放军海军总医院 | Preparation method of umbilical cord Huatong glue mesenchymal stem cells and product thereof |
| US20130302285A1 (en) * | 2011-11-09 | 2013-11-14 | National University Of Singapore | Wharton's Jelly Mesenchymal Stem Cells and Uses Thereof |
| CN103589683A (en) * | 2013-08-12 | 2014-02-19 | 北京东方华辉生物医药科技有限公司 | Separation method and culture method for umbilical cord mesenchymal stem cells |
| CN103589686A (en) * | 2013-11-06 | 2014-02-19 | 四川新生命干细胞科技股份有限公司 | Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells |
| CN104212764A (en) * | 2014-09-22 | 2014-12-17 | 上海华颜干细胞科技有限公司 | Preparation method of clinical mesenchymal stem cells |
Non-Patent Citations (4)
| Title |
|---|
| VIKRAM SABAPATHY ET AL.: "Human Wharton’s Jelly Mesenchymal Stem Cells Plasticity Augments Scar-Free Skin Wound Healing with Hair Growth", 《PLOS ONE》 * |
| 张卫东等: "红细胞裂解液法体外分离培养兔骨髓间充质干细胞", 《中国组织工程研究》 * |
| 李康智等: "脐带血有核细胞不同提取和保存方法的比较", 《华夏医学》 * |
| 闫俊卿等: "人脐带间充质干细胞生物学特性及向类肝细胞的分化", 《世界华人消化杂志》 * |
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| CN110804585A (en) * | 2019-11-19 | 2020-02-18 | 武汉济源高科技有限公司 | Separation method of umbilical cord mesenchymal stem cells |
| CN112522791A (en) * | 2020-12-09 | 2021-03-19 | 河北医科大学 | Construction method of human umbilical cord mesenchymal stem cell bank |
| CN112545976A (en) * | 2020-12-25 | 2021-03-26 | 北京亦科诺生物科技有限公司 | Preparation method of vitality-enhancing anti-aging essence |
| CN114196626A (en) * | 2022-02-16 | 2022-03-18 | 北京国卫生物科技有限公司 | Umbilical cord mesenchymal stem cells and isolated culture and amplification method thereof |
| CN115044545A (en) * | 2022-07-11 | 2022-09-13 | 睿博斯(北京)生物科技有限公司 | Mesenchymal stem cell and preparation method thereof |
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