CN106754685A - A kind of construction method of Human fat mesenchymal stem cell bank - Google Patents
A kind of construction method of Human fat mesenchymal stem cell bank Download PDFInfo
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Abstract
The invention provides a kind of construction method of Human fat mesenchymal stem cell bank, comprise the following steps：(1) adipose tissue is gathered, physiological saline centrifugation is added；(2) oil reservoir is removed, adipose tissue is cleaned；(3) adipose tissue after cleaning is inoculated in complete medium, is placed in 37 DEG C, 5%CO2Under the conditions of cultivated；(4) subculture was changed every 58 days, when cell fusion degree is 70 80%, uses TrypLETMSelect vitellophags；(5) Secondary Culture；(6) quality testing；(7) freeze；(8) storehouse is built.The present invention removes grease and red blood cell using centrifugal process, does not use collagenase digesting, can shorten the operating time, makes process is simple feasible；Also a large amount of red blood cells will not be produced, it is not necessary to using erythrocyte cracked liquid or Low-molecular-weight Heparins Calcium removal red blood cell, potential risk will not be caused to human adipose mesenchymal stem cells, while also a saving cost.
Description
Technical field
The invention belongs to the preparing technical field of mescenchymal stem cell, and in particular to a kind of Human fat mesenchymal stem cell bank
Construction method.
Background technology
Adipose tissue is largely present in human body, is a kind of good tissue engineering material being readily available, and soft tissue is lacked
Damage filling, chest enlarge, smoothing wrinkle have preferable aesthetic medicine therapeutic effect, but the survival rate of adipose tissue transplantation is relatively low, easily quilt
The shortcomings of absorption, liquefaction of fat and necrosis, significantly limit the application of adipose tissue.Research shows, fat mesenchymal stem cell
The survival rate of adipose tissue transplantation can be remarkably promoted.
Fat mesenchymal stem cell (Adipose-derived mesenchymal stem cells, AD-MSCs) is one
Class is present in adipose tissue and the adult stem cell with self-renewing and multi-lineage potential.Fat mesenchymal stem cell conduct
A kind of preferable seed cell has the advantage that：1) substantial amounts of mescenchymal stem cell, and fatty group are contained in adipose tissue
Easily, abundance, the pain brought to patient is smaller for the acquisition knitted；2) fat mesenchymal stem cell has stronger immune tune
Energy-conservation power, even if carrying out heteroplastic transplantation will not also cause strong suppression versus-host disease；3) fat mesenchymal stem cell is increasing
Growing the aspects such as activity, cytokine secretion ability, ability of going back to the nest, genetic background stabilization all has stronger biological characteristics, this
A little characteristics help lend some impetus to regeneration and scar reparation, are all had broad application prospects in medical cosmetology.
At present, there are shortcomings, such as Chinese patent in the extraction of fat mesenchymal stem cell and banking process
CN103074298A discloses a kind of Human fat mesenchymal stem cell bank and its construction method, and the method is comprised the following steps：1)
Aseptic collection body fat；2) grease removal；3) collagenase digesting；4) cracking of red blood cell；5) Amplification Culture；6) freeze.In
State patent CN104357387A discloses a kind of method that human adipose-derived stem cell is separated from human fat tissue, first with containing low point
The physiological saline of sub- calciparine rinses adipose tissue repeatedly, then between NTx enzyme solutions digestion adipose tissue extraction fat
Mesenchymal stem cells.
Above two method all employs collagenase digestion and extracts mescenchymal stem cell so that mescenchymal stem cell is extracted
Operating time is more long, technique complicates；There is substantial amounts of red blood cell in the cell suspension that collagenase digesting is obtained, it is above-mentioned the first
Method removes red blood cell using erythrocyte cracked liquid, and second method removes red blood cell, these methods using Low-molecular-weight Heparins Calcium
Potential danger can be not only caused to the quality of mescenchymal stem cell, and also add production cost, be also unsuitable for clinic
The application of level fat mesenchymal stem cell.
In sum, human adipose mesenchymal stem cells are extracted in the prior art to have the following disadvantages：Cell purity is not high, behaviour
Make it is cumbersome, take time and effort, production cost is higher etc..
The content of the invention
For above shortcomings in the prior art, the present invention provides a kind of structure of Human fat mesenchymal stem cell bank
Method, can effectively solve the complexity of operating process in the prior art, and cell purity is low and red blood cell removes cumbersome problem.
To achieve the above object, the technical solution adopted for the present invention to solve the technical problems is：
A kind of construction method of Human fat mesenchymal stem cell bank, comprises the following steps：
(1) adipose tissue of donor is gathered, 1-5mm is cut into3Size, loaded in centrifuge tube (Corning), Ran Houjia
Enter 3 times of SPSSs of volume (Cologne medicine company), 10-20min is centrifuged under the conditions of 20 DEG C, 2000-5000r/min；Its
In, the donor age is 18-30 Sui, and adipose tissue collection position is belly, buttocks, thigh both sides；
(2) after centrifugation is finished, the oil reservoir on upper strata is removed, the adipose tissue in intermediate layer is then transferred to new centrifugation
Pipe, adds physiological saline to clean 1-2 times；
(3) adipose tissue after cleaning is inoculated in complete medium, is placed in 37 DEG C, 5%CO2Under the conditions of trained
Support；Wherein complete medium composition is：Serum free medium (Cellgenix), 30ng/mL bFGF (Cellgenix), 10ng/
ML insulin (Gibco) and the GlutaMAX (Gibco) that volumetric concentration is 2%；
(4) subculture was changed every 5-8 days, when cell fusion degree is 70-80%, uses TrypLETM Select
(Gibco) vitellophag；
(5) Secondary Culture：Carry out centrifugal treating after digestion, 2000r/min centrifugation 3-5min collect cell, according to 1 ×
104-2×104Individual cell/cm2Density (density is that the ratio of number of cells and blake bottle bottom surface area calculates herein) be inoculated with
In T175 blake bottles, 20mL complete medium Secondary Cultures are then added, obtain fat mesenchymal stem cell；
(6) for the fat mesenchymal stem cell obtained by step (5), detection following items at least one：Cell is lived
Property, the detection of Sterility testing, cell surface marker, caryogram detection, HLA-ABC/DR distribution type, hereditary disease detection, detection of mycoplasma,
Endotoxin is detected；
(7) freeze：The fat mesenchymal stem cell of Testing index will be met after Secondary Culture according to 4 × 106-8×106It is individual
Cell/mL density is frozen in 1mL frozen stock solutions, and freeze-stored cell is preserved to being transferred in liquid nitrogen container after -80 DEG C for a long time through programmed cooling；
(8) database of the fat mesenchymal stem cell comprising information above is set up, and makes the database with step (7)
Freeze-stored cell is associated.
Further, the treatment in step (1) to adipose tissue needs to be carried out in 48h after acquisition.
Further, adipose tissue block and the w/v of complete medium are (1-2 in step (3):10-15)g/
mL。
Further, cell density is 4 × 10 when being frozen in step (7)6Individual cell/mL.
Further, frozen stock solution is composed of the following components and mass fraction of each component is in step (7)：80% serum-free
Culture medium (Cellgenix), 10% human serum albumin (Sigma) and 10% dimethyl sulfone (Origen).
A kind of construction method of Human fat mesenchymal stem cell bank that the present invention is provided, has the advantages that：
(1) extracting method that the present invention is used, step is simple, and adipose tissue does not use collagenase digesting, only needs direct
Adhere-wall culture is that can obtain purity fat mesenchymal stem cell high, not only simplify technological process, and avoids introducing more
Foreign aid's disturbing factor so that technology stability is relatively easy to control.
(2) present invention removes grease and red blood cell using centrifugal process, will not produce a large amount of red blood cells, it is not necessary to using red thin
Human adipose mesenchymal stem cells will not be caused potential risk, while also by cellular lysate liquid or Low-molecular-weight Heparins Calcium removal red blood cell
Cost is saved.
(3) the inventive method uses complete free serum culture technology, it is to avoid the interference of animal sources serum compositions, security
Height, reaches clinical rank and builds storehouse technique, is conducive to the clinical practice in later stage.
(4) storehouse is normal adults depot fat mescenchymal stem cell, is that he or she and kinsfolk perhaps can the 3
Side is when needing to be treated various diseases or carried out medical and beauty treatment using adipose tissue-derived mesenchymal stem cell transplantation, there is provided the applicable fat of clinic
Fat mescenchymal stem cell and stem cell medicine.
Brief description of the drawings
Fig. 1 freezes 2 months, 6 months, the cell growth status of culture of recovering after 12 months for adipose tissue；Wherein Fig. 1-1
The P0 without the adipose tissue culture for freezing is respectively for cell growth status to 1-4, and adipose tissue is recovered after freezing 2 months and trained
Foster P0 for cell growth status, adipose tissue recovered after freezing 6 months culture P0 for cell growth status, adipose tissue freezes
Recovered after depositing 12 months culture P0 for cell growth status；Fig. 1-5 to 1-8 is respectively without the adipose tissue culture for freezing
P3 for cell growth status, adipose tissue recovered after freezing 2 months culture P3 for cell growth status, adipose tissue freezes 6
Recovered after individual month culture P3 for cell growth status, adipose tissue recovered after freezing 12 months culture P3 for cell growth feelings
Condition.
Fig. 2 is the multidirectional induction differentiation figure of fat mesenchymal stem cell；Wherein Fig. 2-1 is compares figure；Wherein Fig. 2-2 is into fat
Induction differentiation；Fig. 2-3 is into chondrocyte induction differentiation；Fig. 2-4 is Osteoinductive differentiation.
Fig. 3 is fat mesenchymal stem cell negative cells surface marker testing result figure；Wherein Fig. 3-1 to 3-5 is not for
The P3 of the adipose tissue culture through freezing is for cell surface marker testing result；Fig. 3-6 to 3-10 freezes 2 for adipose tissue
Month after recover culture P3 for cell surface marker testing result；Fig. 3-11 to 3-15 is multiple after being frozen 6 months for adipose tissue
The P3 for cultivating revive for cell surface marker testing result；Fig. 3-16 to 3-20 recovers after being frozen 12 months for adipose tissue and cultivates
P3 for cell surface marker testing result.
Fig. 4 is fat mesenchymal stem cell positive cell surface marker testing result figure；Wherein Fig. 4-1 to 4-3 is not for
The P3 of the adipose tissue culture through freezing is for cell surface marker testing result；Fig. 4-4 to 4-6 freezes 2 for adipose tissue
Month after recover culture P3 for cell surface marker testing result；Fig. 4-7 to 4-9 recovers after being frozen 6 months for adipose tissue
The P3 of culture is for cell surface marker testing result；Fig. 4-10 to 4-12 recovers what is cultivated after being frozen 12 months for adipose tissue
P3 is for cell surface marker testing result.
Fig. 5 is fat mesenchymal stem cell karyotyping figure；Wherein Fig. 5-1 is the P3 without the adipose tissue culture for freezing
For cell karyotyping；Fig. 5-2 recovered after being frozen 2 months for adipose tissue culture P3 for cell karyotyping；Fig. 5-3 is fat
Fat tissue freezing recovered after 6 months culture P3 for cell karyotyping；Fig. 5-4 recovers after being frozen 12 months for adipose tissue and trains
Foster P3 is for cell karyotyping.
Fig. 6 is the cytokines measurement result figure of fat mesenchymal stem cell secretion.
Specific embodiment
The separation of the fat mesenchymal stem cell of embodiment 1
(1) Informed Consent Form is signed with donor, gathers 30mL women (25 years old) abdominal adipose tissue, while gather 10mL supplying
Person's blood sample and detect donor blood group, HLA-ABC/DR distribution type, infectious disease (Hepatitis B virus, hepatitis, cytomegalovirus, Epstein-Barr virus,
Syphilis, AIDS virus), it is ensured that its indices is normal, and the adipose tissue of donor then is cut into 1-5mm3Size and by its
3 parts are divided into, respectively loaded in 50mL centrifuge tubes, 30mL SPSSs are added, under the conditions of 20 DEG C, 4000r/min
Centrifugation 15min, it is therefore an objective to carry out the red blood cell in adipose tissue de-oiling and removal adipose tissue；
(2) after centrifugation is finished, the oil reservoir on upper strata is removed, the adipose tissue in intermediate layer is then transferred to new centrifugation
Pipe, adds physiological saline to clean 1-2 times；
(3) the adipose tissue block in collection step (2), weighs 2g and is inoculated in T175 blake bottles (Corning), adds
12mL complete mediums, are subsequently placed in 37 DEG C, 5%CO2Cultivated in humidified incubator；Wherein complete medium composition is：Without blood
Clear culture medium (Cellgenix), 30ng/mL bFGF (Cellgenix), 10ng/mL insulin (Gibco) and volumetric concentration are
2% GlutaMAX (Gibco)；Cell growth status are shown in Fig. 1-1；
(4) the adipose tissue block in collection step (2), weighs 6g and freezes in 6mL frozen stock solutions, and frozen stock solution is by following components
Constitute and the mass fraction of each component is：80% serum free medium (Cellgenix), 10% human serum albumin (Sigma) and
10% dimethyl sulfone (Origen), the adipose tissue for freezing, to being transferred in liquid nitrogen container after -80 DEG C, freezes the time point through programmed cooling
Wei 2 months, 6 months, 12 months.
The recovery of the adipose tissue of embodiment 2, Secondary Culture and freeze
(1) by the time of freezing reach 2 months, 6 months, the adipose tissue of 12 months recovers through 37 DEG C of water-baths respectively；
(2) take out adipose tissue to be added in 50mL centrifuge tubes, 30mL SPSSs are added, in 4 DEG C, 1200r/
3min is centrifuged under the conditions of min；
(3) the adipose tissue block in collection step (2), weighs 2g and is inoculated in T175 blake bottles (Corning), adds
12mL complete mediums, are subsequently placed in 37 DEG C, 5%CO2Cultivated in humidified incubator, change a subculture within every 7 days；Freeze 2
Individual month, 6 months, the adipose tissue of 12 months see Fig. 1-2,1-3,1-4 for cell growth status through the P0 of recovery culture；
(4) when cell fusion degree is 70%, TrypLE is usedTMSelect (Gibco) vitellophag, carried out after digestion from
Heart treatment, 1200r/min centrifugation 3min, collects cell, according to 1 × 104Individual cell/cm2Density be inoculated in T175 blake bottles
In, 20mL complete medium Secondary Cultures are then added, culture to cell fusion degree reaches 70%~90%.Without freezing, freeze
Deposit 2 months, freeze 6 months, freeze 12 months adipose tissue culture P3 for cell growth status see Fig. 1-5,1-6,1-7,
1-8；
(5) P3 is pressed into density 4 × 10 for cell6Individual cell/mL is resuspended in frozen stock solution (composition and mass fraction：80% nothing
Blood serum medium (Cellgenix), 10% human serum albumin (Sigma), 10% dimethyl sulfone (Origen)) in, freeze volume
It is 1mL, is then preserved for a long time to being positioned in liquid nitrogen container after -80 DEG C through programmed cooling.
Can be obtained by Fig. 1, the P0 generations without the adipose tissue culture for freezing, freezing 2 months, freeze 6 months, freeze 12 months
Containing fatty oil droplet in cell culture, rounded shinny spot, when passage culture to P3 for when, in cell culture
Fatty oil droplet disappear.
The fat mesenchymal stem cell Identification of Biological Characteristics of embodiment 3
1st, the identification of multidirectional induction differentiation potential
P3 fat subsitutes mescenchymal stem cells are inoculated in six orifice plates, the inoculation 1 × 10 per hole5Individual cell, 2mL is added per hole
Complete medium, each sample sets 3 repetitions, changes a subculture within every two days, distinguishes when cell fusion degree is 70%
Add skeletonization, into fat, into chondrocyte induction culture medium (Cyagen) each 2mL, to add complete medium as blank, per 2-
3 days change an inducing culture, persistently cultivate 2-3 week after, remove inducing culture and with PBS once, addition 2mL it is many
Polyformaldehyde solution (4%, v/v) fix 30min, then remove paraformaldehyde solution and with PBS twice, using dyeing liquor contaminate
Color, osteogenic induction, into chondrocyte induction dyeing time be 2-3min；Adipogenic induction dyeing time is 30min, then removes dyeing liquor
And add 1mL PBS solutions to clean once, and observation, shooting photo under inverted microscope are finally placed in, as a result see Fig. 2.
As shown in Figure 2, by the Fiber differentiation of 2-3 weeks, fat mesenchymal stem cell can carry out into fat, skeletonization, into soft
Self-bone grafting breaks up.
2nd, surface marker analyte detection
By the P3 fat subsitutes mescenchymal stem cells TrypLE that degrees of fusion is 90%TMAfter Select digestion process, count simultaneously
Regulation density is 1 × 106The cell suspending liquid of individual cell/mL, after 100 μm of cell screen clothes filterings, is separately added into 10 μ L fluorescence marks
The cell surface marker antibody of note is placed in 4 DEG C, is incubated 20min under the conditions of lucifuge in 1mL cell suspensions, and not carry out
The equal cell suspension of antibody labeling is used as blank；The antibody for being used has：CD11b-FITC、CD19-ECD、CD34-
PE、CD45-PC7、CD73-PE、CD90-FITC、CD105-PE、HLA-DR-PC7；After the completion of incubation, with cold physiological saline (4
DEG C) clean once, 1200r/min centrifugation 5min abandon supernatant, add 250 μ L physiological saline to mix, and add more than the 1% of 250 μ L
Polyformaldehyde is fixed, and the cell after mark is tested and analyzed using the stream type cell analyzer of Beckman companies, the cell of detection
Divide 4 kinds：P3 without the adipose tissue culture for freezing is for cell；In the P3 generations of the adipose tissue culture recovered after freezing 2 months, are thin
Born of the same parents；The P3 of the adipose tissue culture recovered after freezing 6 months is for cell；The P3 of the adipose tissue culture recovered after freezing 12 months
For cell；Result is shown in Fig. 3 and 4.
From Fig. 3 and 4, cell rate of the surface containing mark is respectively：
Do not freeze：CD11b：0.03%；CD19：0.90%；CD34：1.98%；CD45：0.42%；HLA-DR：
0.16%；CD73：99.39%；CD90：99.98%；CD105：83.63%；
Freeze 2 months：CD11b：1.29%；CD19：0.98%；CD34：0.40%；CD45：0.87%；HLA-DR：
1.06%；CD73：97.51%；CD90：99.99%；CD105：99.91%；
Freeze 6 months：CD11b：0.41%；CD19：1.15%；CD34：1.38%；CD45：0.41%；HLA-DR：
0.59%；CD73：97.44%；CD90：99.91%；CD105：99.92%；
Freeze 12 months：CD11b：0.44%；CD19：1.91%；CD34：1.40%；CD45：0.38%；HLA-DR：
0.54%；CD73：99.39%；CD90：99.89%；CD105：98.78%.
Through flow detection and analysis find fat mesenchymal stem cell hardly express hematopoietic cell surface marker such as CD11b,
CD19、CD34、CD45、HLA-DR；Expression mescenchymal stem cell positive indication thing such as CD73, CD90, CD105.
3rd, fat mesenchymal stem cell karyotyping
By 1 × 106Individual P3 is inoculated in T75 blake bottles respectively for cell, is subsequently adding 10mL complete mediums, is positioned over
37 DEG C, 5%CO2In incubator, culture is harvested after 48-72 hours, and the colchicine of 0.2% concentration is added before harvesting, and 37 DEG C, is incubated
Harvested after educating 4 hours, by being centrifuged, abandoning supernatant, hypotonic, fixed, drop piece after results, finally dye aobvious band；In addition by results
Cell cryopreservation 2 months, 6 months, recover respectively after 12 months, carry out karyotyping, analysis result is shown in Fig. 5.
Through karyotyping, fat mesenchymal stem cell does not occur obvious chromosome abnormality after passage.
4th, the cytokines measurement of fat mesenchymal stem cell secretion
2 × 10 are inoculated with by each T175 blake bottle6Individual P3 fat subsitutes mescenchymal stem cell, adds 20mL complete mediums,
It is placed in 37 DEG C, 5%CO2Cultivated in incubator, culture medium is removed when cell fusion degree reaches 80%, add 10mL DMEM
(Hyclone) and it is positioned over 37 DEG C, 5%CO224h is cultivated in incubator, nutrient solution, 20 DEG C, 4000r/min centrifugations is collected
20min, Aspirate supernatant, with reference to ELISA detection kit specification (R＆D) detect each cell factor (IGF-1, PDGF,
HGF, EGF, FGF, VEGF) expression quantity, as a result see Fig. 6.
From result, fat mesenchymal stem cell secretes VEGF (VEGF) higher, (liver is thin for HGF
Intracellular cytokine), FGF (fibroblast growth factor), (Insulin-Like is given birth to secrete a small amount of PDGF (PDGF), IGF-1
The factor long) and EGF (EGF).
The foundation of the fat mesenchymal stem cell database of embodiment 4
1st, the detection of cytoactive
The number for freezing front and rear living cells is counted using trypan blue staining.
2nd, the detection of cell infection
Using a small amount of cell culture, whether detection cell is polluted by fungi and bacterium.Using aetology method, detection
Whether cell is subject to Hepatitis B virus (HbsAg, HbsAb, HBcAb, HbeAg, HbeAb), hepatitis (HCVAb), AIDS (HIV-1/
2Ab), cytomegalovirus (CMV-IgA), Epstein-Barr virus and syphilis.
3rd, the detection of hereditary disease
Using the method for molecular genetics, detection freeze-stored cell whether there is hereditary disease.
4th, HLA-ABC/DR distribution type
Detection cell HLA-ABC/DR phenotypes, and place on record.
5th, the investigation of cell derived
Record donor particulars, and place on record.
6th, the foundation of fat mesenchymal stem cell database
After normal fat mesenchymal stem cell is preserved, the database of fat mesenchymal stem cell is set up, including
First five items data, and set up and associated with freeze-stored cell.
Claims (6)
1. a kind of construction method of Human fat mesenchymal stem cell bank, it is characterised in that comprise the following steps：
(1) adipose tissue of donor is gathered, 1-5mm is cut into3Size, is subsequently adding 3 times of SPSSs of volume, in
20 DEG C, 10-20min is centrifuged under the conditions of 2000-5000/min；Wherein, adipose tissue collection position is belly, buttocks, thigh two
Side；
(2) after centrifugation is finished, the oil reservoir on upper strata is removed, collects adipose tissue, be subsequently adding physiological saline and clean 1-2 times；
(3) adipose tissue after cleaning is inoculated in complete medium, is placed in 37 DEG C, 5%CO2Under the conditions of cultivated；Wherein
Complete medium composition is：Serum free medium, 30ng/mL bFGF, 10ng/mL insulin and volumetric concentration are 2%
GlutaMAX；
(4) subculture was changed every 5-8 days, when cell fusion degree is 70-80%, uses TrypLETMSelect digestion is thin
Born of the same parents；
(5) Secondary Culture：Digestion is centrifuged 3-5min after 2000r/min, cell is collected, according to 1 × 104-2×104Individual cell/
cm2Density be inoculated in T175 blake bottles, then add 20mL complete medium Secondary Cultures, obtain fat mesenchymal dry thin
Born of the same parents；
(6) for the fat mesenchymal stem cell obtained by step (5), detection following items at least one：Cytoactive, nothing
Bacterium detection, cell surface marker detection, caryogram detection, HLA-ABC/DR distribution type, hereditary disease detection, detection of mycoplasma, endogenous toxic material
Element detection；
(7) freeze：The fat mesenchymal stem cell of Testing index will be met after Secondary Culture according to 4 × 106-8×106Individual cell/
ML density is frozen in 1mL frozen stock solutions, and freeze-stored cell is preserved to being transferred in liquid nitrogen container after -80 DEG C for a long time through programmed cooling；
(8) database of the fat mesenchymal stem cell comprising information above is set up, and makes freezing for the database and step (7)
Cell is associated.
2. the construction method of Human fat mesenchymal stem cell bank according to claim 1, it is characterised in that in step (1)
Treatment to adipose tissue needs to be carried out in 48h after acquisition.
3. the construction method of Human fat mesenchymal stem cell bank according to claim 1, it is characterised in that in step (3)
Adipose tissue block is (1-2 with the w/v of complete medium:10-15)g/mL.
4. the construction method of Human fat mesenchymal stem cell bank according to claim 1, it is characterised in that in step (7)
Cell density is 4 × 10 when freezing6Individual cell/mL.
5. the construction method of Human fat mesenchymal stem cell bank according to claim 1, it is characterised in that in step (7)
Frozen stock solution is composed of the following components and mass fraction of each component is：80% serum free medium, 10% human serum albumin and
10% dimethyl sulfone.
6. such as the human adipose mesenchymal stem cells of claim any one of 1-5 construction method acquisition.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107974430A (en) * | 2018-01-24 | 2018-05-01 | 北京臻溪谷医学研究中心（有限合伙） | A kind of construction method that obtains isolated culture method and stem cell bank of human adipose-derived stem cell |
| CN108546679A (en) * | 2018-04-23 | 2018-09-18 | 北京翊博普惠生物科技发展有限公司 | The method and its application of large amplification human mature high activity Dendritic Cells in vitro |
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| CN112342191A (en) * | 2019-08-07 | 2021-02-09 | 路春光 | hMSC production kit development and quality management standard optimization and system establishment |
| CN110438072A (en) * | 2019-08-26 | 2019-11-12 | 广东唯泰生物科技有限公司 | The preparation method of fat mesenchymal stem cell |
| CN113201489A (en) * | 2021-05-14 | 2021-08-03 | 达瑟儿(上海)生命科技有限公司 | Preparation method of adipose-derived mesenchymal stem cell working cell bank |
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Application publication date: 20170531 |