CN104212764A - Preparation method of clinical mesenchymal stem cells - Google Patents
Preparation method of clinical mesenchymal stem cells Download PDFInfo
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Abstract
The invention provides a preparation method of clinical mesenchymal stem cells. The method comprises screening puerperas before umbilical cord collection; taking healthy and mature volunteer puerperas as collecting targets; then performing separation, amplification and large-scale culture till the third generation, performing cryopreservation, and performing a series of quality detection on intermediate products generated during the separating process and the freezing process. When the frozen cells revive and are washed for standby, no obvious decrease of the viability occurs. The preparation method of the clinical mesenchymal stem cells aims at clinical, unified and systematic monitoring on the entire process from raw material collection to cell preparation accomplishment to achieve clinical standard product of the mesenchymal stem cells.
Description
Technical field
The invention belongs to cell biology, be specifically related to a kind of preparation method of clinical mescenchymal stem cell.
Background technology
Stem cell (Stem Cells, SC) is the multipotential cell that a class has the of self-replication capacity (self-renewing), is the cell of original and non-specialization.Under certain condition, it can be divided into several functions cell, has the potential function of the various histoorgan of regeneration and human body.Stem cell exists in all multicellular tissues, can split into multiple specialized cell, and can utilize self to provide more stem cells via mitotic division and differentiation.The source of stem cell has a lot, comprises umbilical cord, Cord blood and marrow.
The blood be retained in after baby due in placenta and umbilical cord is the important sources of stem cell.Hemopoietic stem cell in Cord blood can be used for treating multiple disease in the blood system and disease of immune system, comprise Malignancy (as: acute leukemia, chronic leukemia, multiple myeloma, marrow abnormality proliferation syndromes and lymphoma etc.), hemoglobinopathy (as: Thalassemia), marrow hematopoiesis function failure (as: aplastic anemia), congenital metabolic disease, congenital immune deficiency illness, autoimmune conditions, some noumenal tumour (as: small cell lung cancer, neuroblastoma, ovarian cancer and progressive muscular dystrophy etc.).Root reaches syndromes, henry reaches syndromes and draw many children diseases such as syndromes and acute lymphoblastic leukemia to be just used for treatment from navel blood stem cell in 1988.By 2011, Cord blood not only can treat tens kinds of refractory diseases and multiple incurable disease effectively, and the kinds of Diseases that it can be treated also constantly are increasing.The Cord blood of autologous storage uses once needs, does not need distribution type, and cytoactive is strong, and without the danger of immunological rejection, transplanting success is high, and curative ratio is high, and medical expense is low.
In people's umbilical cord, there is a class to have the cell colony of cells and characteristic of stem, be called as human mesenchymal stem cell (MSCs).Human mesenchymal stem cell has the potential of self and Multidirectional Differentiation, can be divided into triploblastica cell under different inductive condition, as osteocyte, chondrocyte, myocyte, adipocyte and neurocyte etc.In addition, also express the factor required for the multiple hematopoietic growth such as IL6, G-CSF and SCF, can maintain the proliferation of hematopoietic progenitors of long-term cultivation, display MSC has hematopoiesis support and promotes the effect of Radiation in jury.Research simultaneously shows that MSC has immunoregulation effect, can suppress T lymphopoiesis in vitro, and interior animal experiment finds that MSCs transplants and alloimmune can be suppressed to react, and alleviates and transplants relevant rejection, and extend the survival time of allogeneic.Nearest clinical trial finds that MSCs combines hematopoietic stem cell transplantation and can alleviate GVHD and reduce graft failure rate.The Multidirectional Differentiation ability of MSC and immunoregulation capability determine it and have broad application prospects in the field such as cell therapy, organizational project.
The multiplex animal serum of current cellar culture mescenchymal stem cell, as chicken, pig, ox etc., wherein foetal calf serum is because amount to obtain is large, pollution is light and be widely used.But in recent years along with the increase of zoonosis sickness rate, the tissue cultivated with foreign sera is under suspicion to the security of clinical application, therefore autoserum is cultivated and is more and more come into one's own.
Chinese invention patent ZL200610067537.5 discloses a kind of preparation method of umbilical cord mesenchymal stem cells, and its step comprises such as: people's umbilical cord is removed remained blood, shreds, with collagenase digesting; Add trysinization again; Digestive system screen filtration, removes and does not digest tissue; Digestive system phosphoric acid buffer after filtering is diluted; Centrifugal; And cell is inoculated in substratum cultivates etc.
Chinese invention patent application 200910194915.X discloses a kind of mesenchymal stem cells in umbilical cord blood and preparation method thereof, is separated and obtains the stem cell of the UCB-MSCs cell subsets with Osteoblast Differentiation potential.Its technique means relates generally to after the single cell suspension of the mescenchymal stem cell from bleeding of the umbilicus and the mixing of fluorescently-labeled CD105 monoclonal antibody, is obtained the mesenchymal stem cells in umbilical cord blood of the CD105 positive (CD105+) by selected by flow cytometry apoptosis.This stem cell is inoculated in medically acceptable Biodegradable material, defines bone graft.
But though disclosed all kinds of mescenchymal stem cell preparation method all can obtain mescenchymal stem cell at present, but all do not meet the standard of clinical application, the present invention intends until whole process prepared by cell carries out clinical grade, unification, system monitoring from feedstock capture, to realize the clinical criteria product of mescenchymal stem cell.
Summary of the invention
For prior art defect; the invention provides a kind of preparation method of clinical mescenchymal stem cell; preparation method of the present invention screens puerpera before umbilical cord acquisition; using qualified health full term puerpera volunteer as collection target; carry out separation amplification afterwards and carry out pilot scale culture until the third generation, carry out freezen protective, to sepn process and refrigerating process middle product carry out a series of quality examination; freezing rear cell recovery washing is stand-by, and motility rate is without obvious reduction.
The invention provides a kind of preparation method of clinical mescenchymal stem cell, described preparation method comprises the steps:
The screening of step one, umbilical cord and preservation: screen, gather health full term puerpera volunteer umbilical cord, be placed in umbilical cord preserving fluid 4 DEG C refrigeration, and carried out separating treatment in 24 hours;
Step 2, umbilical cord are separated: transferred to by umbilical cord in culture dish, and physiological saline cleans; Remove ligation position, two ends, divest umbilical vein and two Umbilical artery; Flatten, magnificent Tong Shi glue of tearing, remove amnion; The magnificent Tong Shi glue of tearing is placed in centrifuge tube shred to 1 ~ 4mm
3fritter;
Step 3, mesenchymal cell original cuiture: the magnificent Tong Shi glue shredded is gone to and is equipped with in the ventilating cover culturing bottle of perfect medium, at 37 DEG C, 5%CO
2, cultivate in saturated humidity environment; Cell density reaches about 1g/T75 bottle, within the 5th day, changes liquid first, within after this every 3 days, changes liquid once, to about the 14th day; When cytogamy degree reaches more than 80%, go down to posterity with tryptic digestion, stop by former culture supernatant;
Step 4, Secondary Culture: when going down to posterity, passage cell inoculum density is about 6000/cm
2; After 3 days, cell density reaches more than 80%, again goes down to posterity; Reach the third generation, cell count is about 3 ~ 5 × 10
9, collecting cell also counts and motility rate, by 2 × 10
7/ ml/ pipe carries out freezen protective; Cell conditioned medium is carried out Sterility testing, detection of mycoplasma, gets 1 × 10
7cell does cell surface Mark Detection;
Step 5, mescenchymal stem cell surface marker, differentiation detect: according to ISCT mescenchymal stem cell standard detection positive indication and negative indication; Analytical Chemical Experiment is carried out to third generation cell and detects differentiation of stem cells ability, respectively to one-tenth fat, skeletonization, three the direction differentiation of one-tenth cartilage, carry out oil red O stain, Alizarin red staining and alcian blue dyeing qualification; After mescenchymal stem cell surface marker, differentiation detect, freezen protective;
Described positive indication is as CD90, CD105, CD73 etc.; Described negative indication is as CD79-a, CD14, CD34, CD45, HLA-DR;
Stem cell use between step 6, clinical use: get frozen cell, with brine twice after recovery, statistics cell count and Cell viability, mix stand-by by clinical needs by cell and compound electrolyte solution according to count results; And keep sample and carry out intracellular toxin and Bacteria Detection.
Preferably, in step one, described umbilical cord preserving fluid is serum free medium, not containing microbiotic; Umbilical cord cells activity can be maintained and reach 24 ~ 48 hours, avoid microbiotic allergic problem during clinical application.
Preferably, in step one, the test item of described screening comprises and carries virus, hereditary family history etc.; Described virus is as hepatitis B, the third liver, AIDS, syphilis etc.;
Preferably, in step 3, described tryptic concentration is 0.05%, dilutes 5 times by 0.25% finished product pancreatin; For cell dissociation process, lower concentration digestion keeps cytoactive and stem cell dryness; Described per-cent is mass volume ratio.
Preferably, in step 3, described perfect medium is formed by basic medium and serum substitute and glutamine proportioning, serum substitute ratio 10%; Glutamine ratio 0.02mmol/ml; For cellular segregation, amplification cultivation and cryoprotection process, the potential risk avoiding not clear animal derived protein to enter human body causing; Described per-cent is mass volume ratio.
Preferably, in step 4, described Sterility testing comprises and carries out bacterium and fungal detection with hemoculture instrument; Described detection of mycoplasma comprises carries out detection of mycoplasma by PCR method.
Preferably, in step 4, described freezen protective cell freezing used protection liquid by perfect medium and dimethyl sulfoxide (DMSO) (DMSO) formulated, wherein dimethyl sulfoxide (DMSO) (DMSO) final concentration 10%; For the protection of the activity of frozen cell; Described per-cent is mass volume ratio.
Compared with prior art, the present invention has following beneficial effect:
1, whole process total quality monitoring, until the cell quality that is prepared into product and finished product in the middle of finished product is all controlled from the screening before umbilical cord acquisition;
2, serum free medium, does not use microbiotic, the disease avoiding heterologous protein and microbiotic to cause or irritated risk;
3, pilot scale culture, ensures cell uniformity and validity;
4, lower concentration trysinization, keeps the activity of cell and the dryness of stem cell;
5, the positive indication expression rate more than 99% of stem cell surface Mark Detection, negative indication less than 2%.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
embodiment 1
The present embodiment provides a kind of preparation method of clinical mescenchymal stem cell, specifically comprises the steps:
The screening of step one, umbilical cord and preservation: screen, gather health full term puerpera volunteer umbilical cord, be placed in umbilical cord preserving fluid 4 DEG C refrigeration, and carried out separating treatment in 24 hours; Described umbilical cord preserving fluid is serum free medium, not containing microbiotic; The test item of described screening comprises and carries virus, hereditary family history etc.; Described virus is as hepatitis B, the third liver, AIDS, syphilis etc.;
Gather the female blood of a pipe simultaneously and give over to reinspection, reinspection project is that the virological immunologies such as hepatitis B, the third liver, AIDS, syphilis detect and gene test.
Step 2, umbilical cord are separated: transferred to by umbilical cord in culture dish, and physiological saline cleans; Remove ligation position, two ends, divest umbilical vein and two Umbilical artery; Flatten, magnificent Tong Shi glue of tearing, remove amnion; The magnificent Tong Shi glue of tearing is placed in centrifuge tube shred to 1 ~ 4mm
3fritter; Scavenging solution supernatant in umbilical cord sepn process carries out Sterility testing, carries out bacterium and fungal detection with hemoculture instrument, carries out detection of mycoplasma by PCR method.
Step 3, mesenchymal cell original cuiture: the magnificent Tong Shi glue shredded is gone to and is equipped with in the ventilating cover culturing bottle of perfect medium, at 37 DEG C, 5%CO
2, cultivate in saturated humidity environment; Cell density reaches about 1g/T75 bottle, within the 5th day, changes liquid first, within after this every 3 days, changes liquid once, to about the 14th day; When cytogamy degree reaches more than 80%, go down to posterity with tryptic digestion, stop by former culture supernatant; Described tryptic concentration is 0.05%, dilutes 5 times by 0.25% finished product pancreatin; For cell dissociation process, lower concentration digestion keeps cytoactive and stem cell dryness; Described perfect medium is formed by basic medium and serum substitute and glutamine proportioning, serum substitute ratio 10%; Glutamine ratio 0.02mmol/ml.
Step 4, Secondary Culture: passage cell inoculum density is about 6000/cm
2; After 3 days, cell density reaches more than 80%, again goes down to posterity; Reach the third generation, cell count is about 3 ~ 5 × 10
9, collecting cell also counts and motility rate, by 2 × 10
7/ ml/ pipe carries out freezen protective; Cell conditioned medium is carried out Sterility testing, detection of mycoplasma; Described Sterility testing comprises and carries out bacterium and fungal detection with hemoculture instrument; Described detection of mycoplasma comprises carries out detection of mycoplasma by PCR method.
Step 5, mescenchymal stem cell surface marker, differentiation detect: get 1 × 10
7cell carry out stem cell surface Mark Detection, according to ISCT mescenchymal stem cell standard detection CD90, CD105, CD73 tri-positive indication and CD79-a, CD14, CD34, CD45, HLA-DR five negative indication; Analytical Chemical Experiment is carried out to third generation cell and detects differentiation of stem cells ability, respectively to one-tenth fat, skeletonization, three the direction differentiation of one-tenth cartilage, carry out oil red O stain, Alizarin red staining and alcian blue dyeing qualification; After mescenchymal stem cell surface marker, differentiation detect, freezen protective.
Stem cell use between step 6, clinical use: get frozen cell, with brine twice after recovery, statistics cell count and Cell viability, mix stand-by by clinical needs by cell and compound electrolyte solution according to count results; And keep sample and carry out intracellular toxin and Bacteria Detection.
embodiment 2
The present embodiment provides a kind of preparation method of clinical mescenchymal stem cell, specifically comprises the steps:
The screening of step one, umbilical cord and preservation: screen, gather health full term puerpera volunteer umbilical cord, be placed in umbilical cord preserving fluid 4 DEG C refrigeration, and carried out separating treatment in 24 hours; Described umbilical cord preserving fluid is serum free medium, not containing microbiotic; The test item of described screening comprises and carries virus, hereditary family history etc.; Described virus is as hepatitis B, the third liver, AIDS, syphilis etc.;
Gather the female blood of a pipe simultaneously and give over to reinspection, reinspection project is that the virological immunologies such as hepatitis B, the third liver, AIDS, syphilis detect and gene test.
Step 2, umbilical cord are separated: transferred to by umbilical cord in culture dish, and physiological saline cleans; Remove ligation position, two ends, divest umbilical vein and two Umbilical artery; Flatten, magnificent Tong Shi glue of tearing, remove amnion; The magnificent Tong Shi glue of tearing is placed in centrifuge tube shred to 1 ~ 4mm
3fritter; Scavenging solution supernatant in umbilical cord sepn process carries out Sterility testing, carries out bacterium and fungal detection with hemoculture instrument, carries out detection of mycoplasma by PCR method.
Step 3, mesenchymal cell original cuiture: the magnificent Tong Shi glue shredded is gone to and is equipped with in the ventilating cover culturing bottle of perfect medium, at 37 DEG C, 5%CO
2, cultivate in saturated humidity environment; Cell density reaches about 1g/T75 bottle, within the 5th day, changes liquid first, within after this every 3 days, changes liquid once, to about the 14th day; When cytogamy degree reaches more than 80%, go down to posterity with tryptic digestion, stop by former culture supernatant; Described tryptic concentration is 0.05%, dilutes 5 times by 0.25% finished product pancreatin; For cell dissociation process, lower concentration digestion keeps cytoactive and stem cell dryness; Described perfect medium is formed by basic medium and serum substitute and glutamine proportioning, serum substitute ratio 10%; Glutamine ratio 0.02mmol/ml.
Step 4, Secondary Culture: passage cell inoculum density is about 6000/cm
2; After 3 days, cell density reaches more than 80%, again goes down to posterity; Reach the third generation, cell count is about 3 ~ 5 × 10
9, collecting cell also counts and motility rate, by 2 × 10
7/ ml/ pipe carries out freezen protective; Cell conditioned medium is carried out Sterility testing, detection of mycoplasma; Described Sterility testing comprises and carries out bacterium and fungal detection with hemoculture instrument; Described detection of mycoplasma comprises carries out detection of mycoplasma by PCR method.
Step 5, mescenchymal stem cell surface marker, differentiation detect: get 1 × 10
7cell carry out stem cell surface Mark Detection, according to ISCT mescenchymal stem cell standard detection CD90, CD105, CD73 tri-positive indication and CD79-a, CD14, CD34, CD45, HLA-DR five negative indication; Analytical Chemical Experiment is carried out to third generation cell and detects differentiation of stem cells ability, respectively to one-tenth fat, skeletonization, three the direction differentiation of one-tenth cartilage, carry out oil red O stain, Alizarin red staining and alcian blue dyeing qualification; After mescenchymal stem cell surface marker, differentiation detect, freezen protective.
Stem cell use between step 6, clinical use: get frozen cell, with brine twice after recovery, statistics cell count and Cell viability, mix stand-by by clinical needs by cell and compound electrolyte solution according to count results; And keep sample and carry out intracellular toxin and Bacteria Detection.
embodiment 3
The present embodiment provides a kind of preparation method of clinical mescenchymal stem cell, specifically comprises the steps:
The screening of step one, umbilical cord and preservation: screen, gather health full term puerpera volunteer umbilical cord, be placed in umbilical cord preserving fluid 4 DEG C refrigeration, and carried out separating treatment in 24 hours; Described umbilical cord preserving fluid is serum free medium, not containing microbiotic; The test item of described screening comprises and carries virus, hereditary family history etc.; Described virus is as hepatitis B, the third liver, AIDS, syphilis etc.;
Gather the female blood of a pipe simultaneously and give over to reinspection, reinspection project is that the virological immunologies such as hepatitis B, the third liver, AIDS, syphilis detect and gene test.
Step 2, umbilical cord are separated: transferred to by umbilical cord in culture dish, and physiological saline cleans; Remove ligation position, two ends, divest umbilical vein and two Umbilical artery; Flatten, magnificent Tong Shi glue of tearing, remove amnion; The magnificent Tong Shi glue of tearing is placed in centrifuge tube shred to 1 ~ 4mm
3fritter; Scavenging solution supernatant in umbilical cord sepn process carries out Sterility testing, carries out bacterium and fungal detection with hemoculture instrument, carries out detection of mycoplasma by PCR method.
Step 3, mesenchymal cell original cuiture: the magnificent Tong Shi glue shredded is gone to and is equipped with in the ventilating cover culturing bottle of perfect medium, at 37 DEG C, 5%CO
2, cultivate in saturated humidity environment; Cell density reaches about 1g/T75 bottle, within the 5th day, changes liquid first, within after this every 3 days, changes liquid once, to about the 14th day; When cytogamy degree reaches more than 80%, go down to posterity with tryptic digestion, stop by former culture supernatant; Described tryptic concentration is 0.05%, dilutes 5 times by 0.25% finished product pancreatin; For cell dissociation process, lower concentration digestion keeps cytoactive and stem cell dryness; Described perfect medium is formed by basic medium and serum substitute and glutamine proportioning, serum substitute ratio 10%; Glutamine ratio 0.02mmol/ml.
Step 4, Secondary Culture: passage cell inoculum density is about 6000/cm
2; After 3 days, cell density reaches more than 80%, again goes down to posterity; Reach the third generation, cell count is about 3 ~ 5 × 10
9, collecting cell also counts and motility rate, by 2 × 10
7/ ml/ pipe carries out freezen protective; Cell conditioned medium is carried out Sterility testing, detection of mycoplasma; Described Sterility testing comprises and carries out bacterium and fungal detection with hemoculture instrument; Described detection of mycoplasma comprises carries out detection of mycoplasma by PCR method.
Step 5, mescenchymal stem cell surface marker, differentiation detect: get 1 × 10
7cell carry out stem cell surface Mark Detection, according to ISCT mescenchymal stem cell standard detection CD90, CD105, CD73 tri-positive indication and CD79-a, CD14, CD34, CD45, HLA-DR five negative indication; Analytical Chemical Experiment is carried out to third generation cell and detects differentiation of stem cells ability, respectively to one-tenth fat, skeletonization, three the direction differentiation of one-tenth cartilage, carry out oil red O stain, Alizarin red staining and alcian blue dyeing qualification; After mescenchymal stem cell surface marker, differentiation detect, freezen protective.
Stem cell use between step 6, clinical use: get frozen cell, with brine twice after recovery, statistics cell count and Cell viability, mix stand-by by clinical needs by cell and compound electrolyte solution according to count results; And keep sample and carry out intracellular toxin and Bacteria Detection.
performance test
Carry out clinical criteria detection to mescenchymal stem cell prepared by embodiment 1 ~ 3, detected result is as described below:
| ? | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| Human immunodeficiency virus | Negative | Negative | Negative |
| HIV-RNA fluorescence is qualitative | Negative | Negative | Negative |
| HTLV-I/II type antibody | Negative | Negative | Negative |
| Hepatitis B surface antigen is qualitative | Negative | Negative | Negative |
| Hepatitis B surface antibody is qualitative | Negative | Negative | Negative |
| Hepatitis B virus e antigen is qualitative | Negative | Negative | Negative |
| Hepatitis B e antibody is qualitative | Negative | Negative | Negative |
| Hepatitis B core antibody is qualitative | Negative | Negative | Negative |
| Anti-HCV | Negative | Negative | Negative |
| HCVRNA is qualitative | Negative | Negative | Negative |
| CMV-IgM antibody | Negative | Negative | Negative |
| Treponema pallidum specific antibody | Negative | Negative | Negative |
| Bacterium, fungi hemoculture | Asepsis growth | Asepsis growth | Asepsis growth |
| Culture Mycoplasma | Negative | Negative | Negative |
| Intracellular toxin detects | Negative | Negative | Negative |
| CD73 | 99.97％ | 99.98％ | 99.99％ |
| CD90 | 99.17％ | 100％ | 100％ |
| CD105 | 99.99％ | 99.99％ | 100％ |
| CD14 | 1.00％ | 0.92％ | 0.95％ |
| CD34 | 1.08％ | 1.03％ | 0.69％ |
| CD45 | 0.78％ | 0.78％ | 0.88％ |
| CD79a | 0.91％ | 0.49％ | 0.71％ |
| HLA-DR | 1.08％ | 1.12％ | 0.96％ |
| Cell count | 2.34×10 7 | 2.54×10 7 | 2.35×10 7 |
| Cell viability | 98％ | 96％ | 97％ |
| Cell becomes fat to break up qualification | Normally | Normally | Normally |
| Cell becomes cartilage differentiation to identify | Normally | Normally | Normally |
| Cell Osteoblast Differentiation is identified | Normally | Normally | Normally |
Detected result shows, and all standard that preparation method of the present invention prepares the mescenchymal stem cell of gained all meets clinical application requirement.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (7)
1. a preparation method for clinical mescenchymal stem cell, is characterized in that, described preparation method comprises the steps:
The screening of step one, umbilical cord and preservation: screen, gather health full term puerpera volunteer umbilical cord, be placed in umbilical cord preserving fluid 4 DEG C refrigeration, and carried out separating treatment in 24 hours;
Step 2, umbilical cord are separated: transferred to by umbilical cord in culture dish, and physiological saline cleans; Remove ligation position, two ends, divest umbilical vein and two Umbilical artery; Flatten, magnificent Tong Shi glue of tearing, remove amnion; The magnificent Tong Shi glue of tearing is placed in centrifuge tube shred to 1 ~ 4mm
3fritter;
Step 3, mesenchymal cell original cuiture: the magnificent Tong Shi glue shredded is gone to and is equipped with in the ventilating cover culturing bottle of perfect medium, at 37 DEG C, 5%CO
2, cultivate in saturated humidity environment; Cell density reaches about 1g/T75 bottle, within the 5th day, changes liquid first, within after this every 3 days, changes liquid once, to about the 14th day; When cytogamy degree reaches more than 80%, go down to posterity with tryptic digestion, stop by former culture supernatant;
Step 4, Secondary Culture: when going down to posterity, passage cell inoculum density is about 6000/cm
2; After 3 days, cell density reaches more than 80%, again goes down to posterity; Reach the third generation, cell count is about 3 ~ 5 × 10
9, collecting cell also counts and motility rate, by 2 × 10
7/ ml/ pipe carries out freezen protective; Cell conditioned medium is carried out Sterility testing, detection of mycoplasma, gets 1 × 10
7cell does cell surface Mark Detection;
Step 5, mescenchymal stem cell surface marker, differentiation detect: according to ISCT mescenchymal stem cell standard detection positive indication and negative indication; Analytical Chemical Experiment is carried out to third generation cell and detects differentiation of stem cells ability, respectively to one-tenth fat, skeletonization, three the direction differentiation of one-tenth cartilage, carry out oil red O stain, Alizarin red staining and alcian blue dyeing qualification; After mescenchymal stem cell surface marker, differentiation detect, freezen protective;
Stem cell use between step 6, clinical use: get frozen cell, with brine twice after recovery, statistics cell count and Cell viability, mix stand-by by clinical needs by cell and compound electrolyte solution according to count results; And keep sample and carry out intracellular toxin and Bacteria Detection.
2. the preparation method of clinical mescenchymal stem cell according to claim 1, is characterized in that, in step one, described umbilical cord preserving fluid is serum free medium, not containing microbiotic.
3. the preparation method of clinical mescenchymal stem cell according to claim 1, is characterized in that, in step one, the test item of described screening comprises and carries virus or hereditary family history.
4. the preparation method of clinical mescenchymal stem cell according to claim 1, is characterized in that, in step 3, described tryptic concentration is 0.05%, dilutes 5 times by 0.25% finished product pancreatin.
5. the preparation method of clinical mescenchymal stem cell according to claim 1, is characterized in that, in step 3, described perfect medium is formed by basic medium and serum substitute and glutamine proportioning, and serum substitute ratio is 10%; Glutamine ratio 0.02mmol/ml.
6. the preparation method of clinical mescenchymal stem cell according to claim 1, is characterized in that, in step 4, described Sterility testing comprises and carries out bacterium and fungal detection with hemoculture instrument; Described detection of mycoplasma comprises carries out detection of mycoplasma by PCR method.
7. the preparation method of clinical mescenchymal stem cell according to claim 1, is characterized in that, in step 4, described freezen protective cell freezing used protection liquid by perfect medium and dimethyl sulfoxide (DMSO) formulated, wherein dimethyl sulfoxide (DMSO) final concentration 10%.
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| CN104770363A (en) * | 2015-04-21 | 2015-07-15 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation solution and cryopreservation method for mesenchymal stem cells |
| CN104862274A (en) * | 2015-05-27 | 2015-08-26 | 贵州北科泛特尔生物科技有限公司 | Method for efficiently separating umbilical cord mesenchymal stem cells |
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| CN105920042A (en) * | 2016-04-13 | 2016-09-07 | 上海华颜医药科技有限公司 | Umbilical cord mesenchymal stem cell injection with anti-aging function and preparation method thereof |
| CN108285888A (en) * | 2017-01-10 | 2018-07-17 | 刘金宏 | The preparation method of fresh mescenchymal stem cell |
| CN109468273A (en) * | 2018-12-29 | 2019-03-15 | 厦门博瑞思生物科技有限公司 | A method of obtaining mescenchymal stem cell from umbilical cord |
| CN112646773A (en) * | 2021-01-21 | 2021-04-13 | 华夏源细胞工程集团股份有限公司 | Method for inducing differentiation of human mesenchymal stem cells into lipid with high efficiency |
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| CN104726402A (en) * | 2015-03-03 | 2015-06-24 | 吉林省拓华生物科技有限公司 | Method for culturing mesenchymal stem cell from umbilical cord and application thereof |
| CN104770363A (en) * | 2015-04-21 | 2015-07-15 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation solution and cryopreservation method for mesenchymal stem cells |
| CN104770363B (en) * | 2015-04-21 | 2017-03-15 | 广州赛莱拉干细胞科技股份有限公司 | A kind of frozen stock solution of mescenchymal stem cell and cryopreservation methods |
| CN104862274A (en) * | 2015-05-27 | 2015-08-26 | 贵州北科泛特尔生物科技有限公司 | Method for efficiently separating umbilical cord mesenchymal stem cells |
| CN105132520A (en) * | 2015-07-29 | 2015-12-09 | 赫柏慧康生物科技无锡有限公司 | Tissue-engineered epidermis quality detection method |
| CN105238748A (en) * | 2015-10-27 | 2016-01-13 | 上海百众源生物科技有限公司 | Preparation method of placenta-source decidua parietalis mesenchymal stem cells by separation and refrigeration |
| CN105462919A (en) * | 2015-12-11 | 2016-04-06 | 郭镭 | Method for separating and extracting hUC-MSC (human Umbilical Cord mesenchymal stem cells) from wharton jelly tissue of umbilical cord |
| CN105420183A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord Wharton jelly tissue |
| CN105920042A (en) * | 2016-04-13 | 2016-09-07 | 上海华颜医药科技有限公司 | Umbilical cord mesenchymal stem cell injection with anti-aging function and preparation method thereof |
| CN108285888A (en) * | 2017-01-10 | 2018-07-17 | 刘金宏 | The preparation method of fresh mescenchymal stem cell |
| CN109468273A (en) * | 2018-12-29 | 2019-03-15 | 厦门博瑞思生物科技有限公司 | A method of obtaining mescenchymal stem cell from umbilical cord |
| CN112646773A (en) * | 2021-01-21 | 2021-04-13 | 华夏源细胞工程集团股份有限公司 | Method for inducing differentiation of human mesenchymal stem cells into lipid with high efficiency |
| CN112646773B (en) * | 2021-01-21 | 2021-12-21 | 华夏源(上海)生物科技有限公司 | Method for inducing differentiation of human mesenchymal stem cells into lipid with high efficiency |
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