CN102757936B - Proliferation accelerator for human adipose-derived stem cells and application method thereof - Google Patents
Proliferation accelerator for human adipose-derived stem cells and application method thereof Download PDFInfo
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Abstract
The invention discloses a proliferation accelerator for human adipose-derived stem cells and an application method thereof. The proliferation accelerator for the human adipose-derived stem cells comprises insulin, hydrocortisone, parathyroid hormone, estradiol, testosterone, a platelet-derived growth factor-AB, an epidermal growth factor, a basic fibroblast growth factor, L-glutamine, 2-mercaptoethanol, a leukemia inhibitory factor, L-ascorbic acid, biotin, dexamethasone, an IGF-I (Insulin-like Growth Factor-I) and a SCF (Stem Cell Factor). The proliferation accelerator for the human adipose-derived stem cells, disclosed by the invention, is applied to proliferation and subculture of the adipose-derived stem cells, can be used to not only specifically promote the growth sped and the proliferation quantity of the adipose-derived stem cells to increase in the form of geometric progression, but also suppress early differentiation and maturity of the adipose-derived stem cells so as to prevent apoptosis or premature senility of the stem cells during culturing and keep the vigorous and active differentiation potentials of the adipose-derived stem cells. The proliferation accelerator for the human adipose-derived stem cells, disclosed by the invention, has good application prospect in the field of culture of the adipose-derived stem cells.
Description
Technical field
The present invention relates to biotechnology and cell engineering field, particularly a kind of human adipose-derived stem cell enhancer of proliferation.
Background technology
Human adipose-derived stem cell is body fat mescenchymal stem cell (adipose-derived stem cells, ADSCs) be a kind of adult stem cell that is widely used at present organizational project and regenerative medicine field, the same with mesenchymal stem cells MSCs have a multi-lineage potential.ADSCs is the growth of inoblast sample, and endochylema and kernel are abundant, is parallel or the arrangement of whirlpool sample.Cell cycle analysis shows that the cell of G0/G1 phase accounts for 69%, the S phase and accounts for 24%, the G2/M phase and account for 8%.Under the existence condition of foetal calf serum, go down to posterity and cultivate 1 times of cell proliferation in 2-3 days.Repeatedly go down to posterity after (10-20 generation), cell proliferation rate is without obviously slowing down, and senile cell occurs in the cell colony after 6 times going down to posterity, and senile cell accounts for 15%[3 in the 15th generation cell colony].ADSCs has and the essentially identical Immunophenotyping of mesenchymal stem cells MSCs, through flow cytometry analysis CD9, CD10, CD13, CD29, CD44, CD49d, CD49e, CD54, CD55, CD59, CD90, CD105, CD117, CD146, CD166, STRO-1 are all positive, and CD31, CD34, CD45 are all negative.Maximum difference is that ADSCs expresses CD49d, does not express CD106; And just in time opposite, BMSCs expresses CD106, does not express CD49d.
At present ADSCs isolation cultivation method commonly used is to separate from fatty tissue to obtain cell, remove the mature cell such as red corpuscle through machinery and method of enzymatically treating after, utilize feeder layer cells and the substratum that employing contains 10% foetal calf serum to cultivate.The shortcoming of this culturing stem cells method is that method is complicated, and the stem cell population of extraction is few, and purity is not high, and shoot proliferation is slow.
Summary of the invention
The purpose of this invention is to provide a kind of human adipose-derived stem cell enhancer of proliferation, be used for improving shoot proliferation efficient.
The shortcoming of culturing stem cells method commonly used is that method is complicated, and the stem cell population of extraction is few, and purity is not high, and shoot proliferation is slow.
According to an aspect of the present invention, a kind of human adipose-derived stem cell enhancer of proliferation is provided, it is characterized in that: formed by Regular Insulin, hydrocortisone, Rat parathyroid hormone 1-34, estradiol, testosterone, PDGF-AB, Urogastron, Prostatropin, L-glutaminate, 2 mercapto ethanol, leukaemia inhibitory factor, L-AA, vitamin H, dexamethasone, insulin-like growth factor I GF-I and human stem cell factor SCF.After adding the human adipose-derived stem cell substratum in the human adipose-derived stem cell enhancer of proliferation each composition activity to be followed successively by insulin concentration be that 0.1 ~ 10 μ g/mL, hydrocortisone concentration are 10
-10~ 5 * 10
-9Mol/L, Rat parathyroid hormone 1-34 concentration are 2 * 10
-10~ 7 * 10
-10Mol/L, estradiol concentration 0.2 ~ 3.5ng/mL, testosterone concentration is 0.8 ~ 15ng/mL, PDGF-AB concentration is 1 ~ 8ng/mL, Urogastron concentration is 2 ~ 16ng/mL, Prostatropin concentration is 2 ~ 20ng/mL, the L-glutaminate preferred concentration is 0.4 ~ 4mmol/L, 2 mercapto ethanol concentration is 0.07 ~ 0.15mmol/L, leukaemia inhibitory factor concentration is 1 ~ 7ng/mL, L-AA concentration is 10 ~ 80 μ g/mL, biotin concentration is 10 ~ 45 μ g/mL, dexamethasone concentration is 10
-8~ 5 * 10
-8Mol/L, IGF-I concentration are that 1 ~ 10ng/mL and SCF concentration are 0.8 ~ 15ng/mL.Wherein the Regular Insulin most preferable concentrations is 6.25 μ g/mL, and the hydrocortisone most preferable concentrations is 1 * 10
-9Mol/L, the Rat parathyroid hormone 1-34 most preferable concentrations is 5 * 10
-10Mol/L, the estradiol most preferable concentrations is 1ng/mL, the testosterone most preferable concentrations is 4ng/mL, the PDGF-AB most preferable concentrations is 4ng/mL, the Urogastron most preferable concentrations is 10ng/mL, the Prostatropin most preferable concentrations is 10ng/mL, the L-glutaminate most preferable concentrations is 2mmol/L, the 2 mercapto ethanol most preferable concentrations is 0.1mmol/L, the leukaemia inhibitory factor most preferable concentrations is 5ng/mL, the L-AA most preferable concentrations is 50 μ g/mL, and the vitamin H most preferable concentrations is 33 μ g/mL, and the dexamethasone most preferable concentrations is 10
-8Mol/L, the IGF-I most preferable concentrations is 5ng/mL, it is 5ng/mL that SCF forms most preferable concentrations.
The invention provides hormone and the various somatomedin effect of keeping the cell index growth as follows:
Insulin Regular Insulin: can promote cell to utilize glucose and amino acid
Hydrocortisone hydrocortisone: stimulate the growth of stem cell, guarantee cell function
Parathyroid hormone Rat parathyroid hormone 1-34: the growth that stimulates stem cell
Estradiol estradiol: stimulate the growth of stem cell, guarantee cell function
Testosterone testosterone: stimulate the growth of stem cell, guarantee cell function
The PDGF-AB PDGF-AB: mitogen can stimulate inoblast and other various kinds of cell division growths.
EGF Urogastron: the Various Tissues cell is had strong short splitting action
The bFGF Prostatropin: can stimulate cellular proliferation, move, induce plasminogen activator and collagenase activities, be the cell mitogen that high-affinity is arranged with heparin.
L-Glutamine L-glutaminate: increase the volume of cell, promote cell enlargement
The 2-Mercaptoethanol 2 mercapto ethanol: can the Cell protection endoenzyme and protein in sulfydryl not oxidized, promote the adherent of cell and propagation.
The LIF leukaemia inhibitory factor: have the functions such as the differentiation of stem cells of inhibition, the stem cell that is used for maintain is in undifferentiated state.
L-ascorbic acid L-AA: antioxidant all can promote the growth of stem cell, increases surviving rate.
The biotin vitamin H: vitamin H is known as again vitamin H, belongs to water-soluble vitamin B group family, participates in helping normally synthetic and metabolism of material in the adipocyte.Promote stem cell growth.
Dexamethasone dexamethasone: belong to adrenal cortex hormones drug, in cell cultivation process, play the effect of somatomedin, promote the proliferation and differentiation of cell.
IGF-I rhIGF-1: be the multi-functional regulation of cell proliferation factor of a class.In the differentiation of cell, propagation, individual growing, has important promoter action.
SCF human stem cell factor: be a kind of I type transmembrane glycoprotein of wide expression, can promote stem cell to survive, accelerate propagation.
According to an aspect of the present invention, a kind of human adipose-derived stem cell enhancer of proliferation that provides, comprise keeping hormone and the various somatomedin of cell index growth, the propagation that the is used for fat stem cell cultivation of going down to posterity, not only can specificity promote the fat stem cell speed of growth and amplification quantity to increase by geometric progression, suppress again its too early differentiation and maturation, avoid stem cell apoptosis or early ageing in cultivation, keep the potential of the vigorous active differentiation of fat stem cell, promote the stem cell that extracts to survive.
Description of drawings
Fig. 1 is the basic medium and the base culture base human adipose-derived stem cell growth curve chart that does not add the human adipose-derived stem cell enhancer of proliferation that adds the human adipose-derived stem cell enhancer of proliferation among the present invention;
Fig. 2 is the form of cell when using common basic medium group cultivator fat stem cell to cultivate 6 days among the present invention;
Fig. 3 is the form of cell when end user's fat stem cell enhancer of proliferation cultivator fat stem cell is cultivated 6 days among the present invention;
Fig. 4 is end user's fat stem cell enhancer of proliferation cultivator fat stem cell human adipose-derived stem cell telomerase activation detected result among the present invention.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.Testing used utensil instrument reagent all can obtain by commercial sources.
Embodiment 1: the preparation of the general population's fat stem cell basic medium:
Get DMEM/F12 cell culture fluid (buying from the TAKARA BIO INC.) added with antibiotic of 900mL: penicillin 90000U, Streptomycin sulphate 90000U.
Adjust pH: use massfraction 5%NaHCO
3Regulate pH to 7.2.
Filtration sterilization: adopt each one of aperture 0.45 μ m and 0.22 μ m filter membrane, the upper strata is 0.45 μ m, and lower floor is 0.22um, to guarantee filter effect.
Add calf serum: will add before use calf serum and be settled to 1000mL(and buy from TAKARA BIO INC.).
Embodiment 2: prepare a kind of proliferated culture medium A that contains the human adipose-derived stem cell enhancer of proliferation
Predetermined amount is the DMEM/F12 cell culture fluid (TAKARA BIO INC.) that 1000mL then gets 900mL, added with antibiotic: penicillin 90000U, Streptomycin sulphate 90000U.
Adjust pH: use massfraction 5%NaHCO
3Regulate pH to 7.2.
Add enhancer of proliferation: composition shown in the according to the form below 1 also joins in the above-mentioned DMEM/F12 cell culture fluid for preparing successively with the amount weighing, and vibration or ultrasonic dissolution assisting do not heat hydrotropy.
Table 1
Then each composition concentration in proliferated culture medium A is followed successively by Regular Insulin 0.1 μ g/mL, hydrocortisone 10 shown in the upper table
-10Mol/L, Rat parathyroid hormone 1-34 2 * 10
-10Mol/L, estradiol 0.2ng/mL, testosterone 0.8ng/mL, PDGF-AB 1ng/mL, Urogastron 2ng/mL, HBGH-2 ng/mL, L-glutaminate 0.4mmol/L, 2 mercapto ethanol 0.07mmol/L, leukaemia inhibitory factor 1ng/mL, L-AA 10 μ g/mL, vitamin H 10 μ g/mL, dexamethasone 10
-8Mol/L, IGF-I 1ng/mL and SCF 0.8ng/mL
Filtration sterilization: adopt each one of aperture 0.45um and 0.22um filter membrane, the upper strata is 0.45um, and lower floor is 0.22um, to guarantee filter effect.
Add calf serum: will add before use calf serum and be settled to 1000mL(and buy from TAKARA BIO INC.).
Embodiment 3: prepare a kind of proliferated culture medium B that contains the human adipose-derived stem cell enhancer of proliferation
Preparation method and predetermined amount such as embodiment 2, but during weighing with each composition in the table 1 by making its concentration in proliferated culture medium B be followed successively by Regular Insulin 10 μ g/mL, hydrocortisone 5 * 10
-9Mol/L, Rat parathyroid hormone 1-34 7 * 10
-10Mol/L, estradiol 3.5ng/mL, testosterone 15ng/mL, PDGF-AB 8ng/mL, Urogastron 16ng/mL, HBGH-2 0ng/mL, L-glutaminate 4mmol/L, 2 mercapto ethanol 0.15mmol/L, leukaemia inhibitory factor 7ng/mL, L-AA 80 μ g/mL, vitamin H 45 μ g/mL, dexamethasone 5 * 10
-8The amount weighing of mol/L, IGF-I10ng/mL and SCF 15ng/mL.
Embodiment 4: the proliferated culture medium C of preparation human adipose-derived stem cell enhancer of proliferation
Preparation method and predetermined amount such as embodiment 2, but during weighing with each composition in the table 1 by making its concentration in proliferated culture medium C be followed successively by Regular Insulin 6.25 μ g/mL, hydrocortisone 1 * 10
-9Mol/L, Rat parathyroid hormone 1-34 5 * 10
-10Mol/L, estradiol 1ng/mL, testosterone 4ng/mL, PDGF-AB 4ng/mL, Urogastron 10ng/mL, Prostatropin 10ng/mL, L-glutaminate 2mmol/L, 2 mercapto ethanol 0.1mmol/L, leukaemia inhibitory factor 5ng/mL, L-AA 50 μ g/mL, vitamin H 33 μ g/mL, dexamethasone 10
-8Mol/L, IGF-I5ng/mL, the amount weighing of SCF 5ng/mL.
Embodiment 5: utilize multiplication agent culturing cell of the present invention and test
Use above-mentioned 4 kinds of culture medium culturing human adipose-derived stem cells
1. with 1 * 10
6Individual fat stem cell is inoculated into respectively in four groups of diameter 100mm culture dish that fill different substratum and mixing, and every group of 10 culture dish place under the inverted microscope and observe;
2. adding substratum:
10 culture dish of control group add ordinary culture medium 5mL among the embodiment 1;
A organizes 10 culture dish, adds proliferated culture medium A5mL;
B organizes 10 culture dish, adds proliferated culture medium B5mL;
C organizes 10 culture dish, adds proliferated culture medium C5mL.
3. cell is placed at 37 ℃ 5%CO
2Incubator in cultivate; Inverted microscope is observed, and sucks the upper strata substratum every 3 days with transfer pipet, adds new substratum, continues to cultivate.
4. cell counting: get a culture dish and put into clean bench every day in each group, then inhales with transfer pipet and abandon substratum.PBS damping fluid washing 2 times adds 1mL trypsinase complex body Trypsin-EDTA, and after the discovery attached cell separated under the inverted microscope, the basic medium that adding 4mL contains 10% bovine serum FBS stopped the pancreatin effect.
Use trypan blue (Typan Blue) dyeing blood cell counting count board living cell counting number.
5. experimental result uses proliferated culture medium C can very effectively accelerate the breeding of fat stem cell as shown in Figure 1.Use the approximate serpentine of cell growth curve of common basic medium and proliferated culture medium A, be in residence time at the front 3d cell of inoculating, then enter logarithmic phase, reached the climax about the 6th day.Afterwards cell enlargement deceleration enters platform area, and to the cytosis of the 9th day apoptosis, cell dishful rate reaches more than 90%, but uses proliferated culture medium A still to be better than common basic medium.The human adipose-derived stem cell of use high dosage (B group) and most preferred dose (C group) cell proliferating agent can be very fast from surviving and accelerate propagation, show that residence time is very short, only just begin rapid division growth less than time of two days, enter in advance increased logarithmic phase than ordinary culture medium group cell, the growth number approximately is 5-6 times of ordinary culture medium group.The cell enlargement rate of high dose group and most preferred dose group that rose by the 5th day is distinguished, 7 days values of peaking of the cell sustainable growth to the of most preferred dose group, and cell quantity reaches 7x10
7, after this enter platform area, 9 days minority apoptotic cells that begin to see.And the cell of high dose group is at most only long to 5x10
7, after this apoptotic cell constantly increases, cell quantity decrescence, later this group cell quantity and low dose group and common basic medium did not have significant difference by the 8th day.So the high dosage multiplication agent can not obtain the effect of best human adipose-derived stem cell propagation, C group dosage promotes the stem cell breeding to increase and did not occur a large amount of apoptosis in 10 days in vitro culture.
The security test of cell proliferating agent cultivator fat stem cell
1 cellular form:
Beginning is adherent behind the cell inoculation 4h that just separates, and the cellular form of which group cell in lag phase all is small circular, and nucleus is placed in the middle, double-core or multinuclear can occur.Cell occurs stretching growth after entering the rise period, and cell enlargement is obviously accelerated, and is the changes such as fusiform, polygon, and fusiformis and fibrocyte shape form appear in visible attached cell when then entering the peak period.Four groups of cells show cell quantity notable difference (Fig. 2, Fig. 3) identical only on sometime on the form, still highlight and use the fat stem cell Growth of Cells of C dosage of cells multiplication agent intensive and active.
2 surface antigen analyses:
Get the stem cell of the lipfanogen culture of cultivation, remove nutrient solution, 2.5% trypsin solution and the mixture slaking of 0.02%EDTA solution with volume ratio 1: 1 contain 1 * 10 with making every 100ul after the PBS washing that contains 1% bovine serum albumin (BSA)
6The single cell suspension of individual cell, be divided into 7 parts, add respectively in 7 Eppendorf pipes, pipe add FITC Mouse IgG1, the APC-CY7Mouse IgG2b of totally 20 μ L and dyeing damping fluid for detection of since antibody non-specific binding reasons for its use in contrast, other test tubes add respectively CD29, CD34, CD44, CD45, CD105, each 20 μ L of HLA.DR monoclonal antibody, and every pipe adds respectively cell suspension 100 μ L and (contains 1 * 10
6Individual cell), incubated at room 25min, after the PBS washing that contains 1%BSA, flow cytometer detects.
Flow cytometry analysis human adipose-derived stem cell phenotype of former generation: CD29+ (77.5%), CD34-(3.3%), CD44+ (26.8%), CD45-(3.8%), CD105+ (30.1%), HLA.DR-(2.0%) shows that this cell has the stem cell characteristic.HLA.DR expresses not obvious, and getting rid of such cell is inoblast.
The mensuration of 3 propagation Telomerase Activity:
Detect the human adipose-derived stem cell of vitro culture more than 6 days with TeloTAGGG Telomerase PCR ELISA test kit (Chemocon Inc.) by specification (TRAP method), simultaneously with the positive contrast of HEKC 293 cell strain cells (293T), the negative contrast of Interferon, rabbit (providing in the test kit).Detection method is as follows, gets 2 * 10
6Individual cell, lysing cell is got the PCR that centrifugal supernatant 2 μ L carry out the reaction of telomerase catalytic specific substrate, and the specific probe with the DIG mark after the sex change of PCR product is hybridized, then with the capable ELISA reaction of anti-DIG antibody of coupling peroxidase, measure ELISA result in microplate reader.Measuring wavelength is 450nm and 690nm absorbancy (A) value, absorbance difference △ A=A450-A690, and △ A represents telomerase activation.Judging criterion as a result: positive control △ A〉1.5, negative control △ A＜0.25, testing sample △ A value deducts negative control △ A value, △ A〉0.2 positive.Adopt the SAS9.0 software package to carry out interpretation of result, measured value represents with means standard deviation.
The human adipose-derived stem cell sample measured value of using optimum dosage of cells multiplication agent to cultivate is that the 6th day human adipose-derived stem cell is (0.050 ± 0.002), human adipose-derived stem cell was (0.055 ± 0.012) in the 10th day, positive control 293 cells are 2.1, and the negative control Interferon, rabbit is 0.048(Fig. 4).
Telomere is the special construction that end of chromosome is comprised of TTAGGG tumor-necrosis factor glycoproteins and Binding proteins matter, and is closely related with the multiple fission ability of cell.Proved that approximately 80% malignant tumour and immortalized cell line cell (such as positive control 293 cells of this experiment) are expressed high-caliber telomerase activation, and normal somatic cell Telomerase Expression level is very low.No matter what therefore, proliferated culture medium was cultivated is that Cell Telomerase Activity remained normal level, illustrates that cultured cells is normal, does not have canceration the most vigorous the 6th day that grows and lasting the 10th day of breeding.
The chromosome karyotype analysis of 4 human adipose-derived stem cells:
Respectively get the 6th day, the 10th day the fat stem cell that multiplication agent is cultivated, add the colchicine (whole mass concentration is 0.04mg/L) of 5g/L, then place 37 ℃, 5% (volume fraction) CO
2Cultivate 5h in the incubator, digestion, centrifugal adds 0.075%kcl, leaves standstill 20min, adds a little stationary liquid (10% formalin) and pre-fixes rear centrifugally, and then fixing 30min is centrifugal, drips sheet, dry after Giemsa dyeing, oven dry, microscopically is observed.Above each method is with different sample cell repeated experiments 3 times.The result shows: use this patent cell proliferating agent under the condition of cultured and amplified in vitro, the ANOMALOUS VARIATIONS such as the karyotype of human adipose-derived stem cell transposition, inversion does not occur, lack or copies, fusion.The generation close relation of the abnormal change of karyotype and cancer.Present research has confirmed that the generation of tumour and cell chromosome reciprocity, disappearance, insertion and inversion cause the oncogene activation expression of chromosome specific breaking point zone or cancer suppressor gene disappearance, cause existing inevitable contact between the change of correlative coding abnormal protein and cytobiology proterties.Therefore, after separation and Culture, amplification number generation, whether abnormal changes its karyotype human adipose-derived stem cell under conditions in vitro, this and its as the security of tissue engineering seed cell important relation is arranged.This patent experimental result shows: cultivated in many days through cell proliferating agent, the ANOMALOUS VARIATIONS such as the human adipose-derived stem cell caryogram transposition, inversion does not all occur, lack or copies, fusion are normal cell caryogram.
Therefore, use the cell proliferating agent of this patent invention stimulate human adipose-derived stem cell external breed with fission process in its Telomerase be to be negative or to be lower than the low expression level of TRAP method detection level, and unlike tumour cell, continue the high expression level telomerase activation.In addition, also no abnormality seen change of its karyotype.The human adipose-derived stem cell of this research indication vitro culture may be safe, provides safety indexes and theoretical basis for it further is applied to every research.
Above-described only is some embodiments of the present invention.For the person of ordinary skill of the art, under the prerequisite that does not break away from the invention design, can also make some changes and improvements, these all belong to protection scope of the present invention.
Claims (4)
1. human adipose-derived stem cell enhancer of proliferation, it is characterized in that: by Regular Insulin, hydrocortisone, Rat parathyroid hormone 1-34, estradiol, testosterone, PDGF-AB, Urogastron, Prostatropin, L-glutaminate, 2 mercapto ethanol, leukaemia inhibitory factor, L-AA, vitamin H, dexamethasone, insulin-like growth factor I GF-I and human stem cell factor SCF form, the ratio of each composition for so that after adding the human adipose-derived stem cell substratum in the human adipose-derived stem cell enhancer of proliferation each composition activity to be followed successively by insulin concentration be 0.1～10 μ g/mL, hydrocortisone concentration is 10
-10～5 * 10
-9Mol/L, Rat parathyroid hormone 1-34 concentration are 2 * 10
-10～7 * 10
-10Mol/L, estradiol concentration 0.2～3.5ng/mL, testosterone concentration is 0.8～15ng/mL, PDGF-AB concentration is 1～8ng/mL, Urogastron concentration is 2～16ng/mL, Prostatropin concentration is 2～20ng/mL, L-glutaminate concentration is 0.4～4mmol/L, 2 mercapto ethanol concentration is 0.07～0.15mmol/L, leukaemia inhibitory factor concentration is 1～7ng/mL, L-AA concentration is 10～80 μ g/mL, biotin concentration is 10～45 μ g/mL, dexamethasone concentration is 10
-8～5 * 10
-8Mol/L, IGF-I concentration are that 1～10ng/mL and SCF concentration are 0.8～15ng/mL.
2. human adipose-derived stem cell enhancer of proliferation as claimed in claim 1, it is characterized in that: the ratio of each composition for so that after adding the human adipose-derived stem cell substratum in the human adipose-derived stem cell enhancer of proliferation each composition activity to be followed successively by insulin concentration be 6.25 μ g/mL, hydrocortisone concentration is 1 * 10
-9Mol/L, Rat parathyroid hormone 1-34 concentration is 5 * 10
-10Mol/L, estradiol concentration is 1ng/mL, and testosterone concentration is 4ng/mL, and PDGF-AB concentration is 4ng/mL, Urogastron concentration is 10ng/mL, Prostatropin concentration is 10ng/mL, and L-glutaminate concentration is 2mmol/L, and 2 mercapto ethanol concentration is 0.1mmol/L, leukaemia inhibitory factor concentration is 5ng/mL, L-AA concentration is 50 μ g/mL, and biotin concentration is 33 μ g/mL, and dexamethasone concentration is 10
-8Mol/L, IGF-I concentration is 5ng/mL, SCF concentration is 5ng/mL.
3. the application method of each described human adipose-derived stem cell enhancer of proliferation among the claim 1-2 is characterized in that: each composition of described human adipose-derived stem cell enhancer of proliferation is added in the substratum of human adipose-derived stem cell.
4. the application method of a kind of human adipose-derived stem cell enhancer of proliferation according to claim 3, it is characterized in that: the substratum of described human adipose-derived stem cell is the DMEM/F12 cell culture medium.
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| CN102485885A (en) * | 2011-06-09 | 2012-06-06 | 臻景生物技术（上海）有限公司 | Separating method and application of fat stem cells |
| CN102433298A (en) * | 2011-11-17 | 2012-05-02 | 安徽农业大学 | Mouse fat stem cell culture solution |
| CN102443566A (en) * | 2012-01-20 | 2012-05-09 | 天津和泽干细胞科技有限公司 | Acquisition method of adipose-derived stem cells |
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