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CN105886462A - Composition ADSCs for ADSCs culture and ADSCs culture method - Google Patents

  • ️Wed Aug 24 2016

CN105886462A - Composition ADSCs for ADSCs culture and ADSCs culture method - Google Patents

Composition ADSCs for ADSCs culture and ADSCs culture method Download PDF

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Publication number
CN105886462A
CN105886462A CN201410442734.5A CN201410442734A CN105886462A CN 105886462 A CN105886462 A CN 105886462A CN 201410442734 A CN201410442734 A CN 201410442734A CN 105886462 A CN105886462 A CN 105886462A Authority
CN
China
Prior art keywords
compositions
culture
adipose
stem cell
mesenchymal stem
Prior art date
2014-08-27
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201410442734.5A
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Chinese (zh)
Inventor
杨钟华
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
RUIAN PULUO BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
Original Assignee
RUIAN PULUO BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
2014-08-27
Filing date
2014-08-27
Publication date
2016-08-24
2014-08-27 Application filed by RUIAN PULUO BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd filed Critical RUIAN PULUO BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
2014-08-27 Priority to CN201410442734.5A priority Critical patent/CN105886462A/en
2016-08-24 Publication of CN105886462A publication Critical patent/CN105886462A/en
Status Pending legal-status Critical Current

Links

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Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a composition for culture of adipose-derived mesenchymal stem cells. The composition comprises human serum albumin, vitamin C, resveratrol, hydrocortisone, basic fibroblast growth factor (bFGF), progesterone and glutathione. The invention also discloses a culture method of adipose-derived mesenchymal stem cells. The above composition is added in a basic medium, and then adipose-derived mesenchymal stem cells are cultured. By the method for culture of adipose-derived stem cells, growth and proliferation speed of adipose-derived stem cells can be promoted, premature differentiation and maturation and premature senility can be inhibited, and differentiation potential of adipose-derived stem cells is maintained. The culture method is very suitable for culture of adipose-derived stem cells.

Description

The compositions cultivated for ADSCs and ADSCs cultural method

Technical field

The present invention relates to biomedicine field, particularly relate to a kind of fat mesenchymal stem cell culture fluid and cultivation side Method.

Background technology

Fat mesenchymal stem cell (adipose-derived stem cells, ADSCs), is in fatty tissue one Class multipotent stem cells.Due to adipose tissue-derived extensively, draw materials easily, be not related to ethical issues, be easy to autologous The misery transplant, brought to patient is less, is therefore increasingly subject to the attention of researcher.At present, research worker becomes Merit ground is in vivo, under conditions in vitro, induced organization types such as being differentiated to form fat, bone, cartilage, muscle thin Born of the same parents.

Fat mesenchymal stem cell in source, obtain and the advantage of the aspect such as differentiation and proliferation potentiality own, imply that it As tissue engineering seed cell, there is good application prospect.The most adipose-derived mescenchymal stem cell draws Play scientific circles' extensive concern, carried out substantial amounts of Basic Experiment Study, clinical treatment also has certain applications.Bone, The tissue defects such as cartilage, nerve, muscle, an always one insoluble difficult problem, but based on stem cell The treatment appearing as these diseases of organizational project brings new hope.Adipose-derived stem cell in organizational project and Application clinically has following several respects: 1. cell therapy: directly entered by adipose-derived mesenchymal stem cell transplantation Internal, repair deficiency tissue, organ, or In vitro culture regenerating tissues and organ, this will be another in medical history Secondary revolution.2. as gene therapy vector: Polymorphism rotaring dyeing technology, target gene or growth factor gene are carried, Treatment hereditary.3. the optimum selection of seed cell, with its multinomial unique advantage, becomes the kind of organizational project Word bank is originated, simultaneously with biodegradable bracket material Combined culture, the lived implantation body of external structure, repair tissue Defect or the partial function of substitute organ, treat refractory disease.4. stem cell bank can be established future, for immunity Defective disease, or autologous disease damage tissue and the reparation of organ in the future, it is possible to after distribution type success, for allogeneic Transplantation treatment.

At present, conventional ADSCs isolated culture method is from fatty tissue separation cell mostly, at machinery, enzyme Reason removes the mature cells such as erythrocyte, then utilizes feeder layer cells to train with the culture medium containing 10% hyclone Support.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 the highest, continues Generation propagation is slow, premature cell senescence.Therefore those skilled in the art be devoted to develop one can improve ADSCs The technology of subculture multiplication efficiency.

Summary of the invention

It is an object of the invention to provide a kind of technology improving ADSCs subculture multiplication efficiency.

The invention provides a kind of compositions cultivated for fat mesenchymal stem cell, including: human albumin, Vitamin C, resveratrol, hydrocortisone, basic fibroblast growth factor (bFGF), progesterone and paddy The sweet peptide of Guang.

Preferably, human albumin 10-30mg/ml, vitamin C 10-50 μ g/ml, white Herba chenopodii in described compositions Reed alcohol 10-20 μ g/ml, hydrocortisone 3-6ng/ml, basic fibroblast growth factor (bFGF) 0.1-0.5 Ng/ml, progesterone 3-6ng/ml and glutathion 30-50 μ g/ml.

It is highly preferred that human albumin 20mg/ml, vitamin C 40 μ g/ml, resveratrol in described compositions 15 μ g/ml, hydrocortisone 4ng/ml, basic fibroblast growth factor (bFGF) 0.3ng/ml, Progesterone 4ng/ml and glutathion 40 μ g/ml.

Preferably, also including vitamin E in described compositions, preferably content of vitamin E is 15-20 μ g/ml.

Preferably, also including antibiotic in described compositions, described antibiotic can be penicillin and/or streptomycin.

Preferably, in described compositions, the content of penicillin is that the content of 20-250 unit/ml and streptomycin is 20-250 unit/ml.

The content of above-mentioned each component is each component final content in the medium.

A second aspect of the present invention provides a kind of fat mesenchymal stem cell cultural method, wherein, cultivates on basis Base adds the compositions described in first aspect present invention, then fat mesenchymal stem cell is cultivated.

Preferably, described basal medium is DMEM/F12 culture medium.

The invention has the beneficial effects as follows:

1, the culture fluid compositions of the present invention, formula components determines have higher safety, is suitable for clinical treatment Application；

2, the method using the present invention cultivates fat stem cell, not only can promote the growing multiplication speed of fat stem cell Degree, can suppress again its premature differentiation ripe and senilism, keep the differentiation potential of fat stem cell, be very suitable for fat The cultivation of stem cell.

Accompanying drawing explanation

Fig. 1 shows living cells technical result in the embodiment of the present invention 2.

Detailed description of the invention

Embodiment 1

Culture medium based on DMEM/F12 culture medium (purchased from TAKARA BIO INC.), adds penicillin 200 Unit/ml, adds streptomycin 200 units/ml.Preparing according to following table, each constituent content is that each component is in culture medium In final content.

After above-mentioned each group of culture medium has been prepared, pH7.2 is standby in regulation, adds the little of volume fraction 10% before use Ox blood serum.

Based on matched group, culture medium adds calf serum (basal medium: calf serum is 9: 1, volume ratio).

Embodiment 2

With above-mentioned each group of culture medium culturing fat mesenchymal stem cell, detect culture effect.

In equipped with embodiment 1 in the culture medium ware of each group culture medium, inoculation basic crack is 1 × 10 respectively6Individual Fat stem cell, is placed in 37 DEG C, 5%CO2Incubator in cultivate；Sucked with pipet every 3 days Layer culture medium, adds new culture medium, and inverted microscope is observed.

In culture dish after sampling, PBS washs 2 times, adds trypsin complex Trypsin-EDTA 1mL, jog to attached cell separates, and adds the basal medium termination pancreatin that 4mL contains 10% Ox blood serum FBS Effect.Use blood cell counting plate statistics viable count after Trypan Blue.Cultivate to the 8th day, the work of matched group Cell number is about 60 × 106, experimental group viable count result as it is shown in figure 1, as can be seen from the figure A group training The cell proliferation effect supporting base is best；Being not added with resveratrol in B group culture medium, cell proliferation effect is worst, with Matched group is suitable, makes veratryl alcohol clear and can be obviously promoted the propagation of fat mesenchymal stem cell；Although in C group With the addition of resveratrol, but do not add vitamin C, although cell proliferation effect is better than B group, but still It is not reaching to the level of A group, illustrates that vitamin C and resveratrol have collaborative promotion fat mesenchymal stem cell The effect of propagation.The counting situation of D group is it can be seen that the propagation of fat mesenchymal stem cell is just had by glutathion The impact in face still affects less significant.

In an experiment, inventor finds, using the fat stem cell of above-mentioned culture medium culturing, shuttle occurs in cellular morphology Shape and fibrocyte shape form, cellular morphology concordance is poor, through repeatedly verifying, the inventors discovered that in culture medium The vitamin E of middle interpolation 15-20 μ g/ml can significantly improve the homogeneity of cellular morphology.

Embodiment 3 surface antigen analysis

Take the fat mesenchymal stem cell cultivated 8 days, abandon the culture fluid, (trypsin of 2.5% after Digestive system digestion Solution and 0.02%EDTA solution are mixed into Digestive system, volume ratio 1: 1), with containing 1% bovine serum albumin (BSA) PBS washing after make every 100ul and contain 1 × 106The single cell suspension of individual cell, is separately added into 7 Ep pipes In, often pipe add cell suspension 100 μ L, control tube add totally 20 μ L FITC Mouse IgGl, APC-CY7Mouse IgG2b and dye solution are used for detecting due to antibody non-specific binding reasons for its use, Other test tubes are separately added into CD29, CD34, CD44, CD45, CD105, HLA.DR monoclonal antibody, and (monoclonal antibody is purchased From TAKARA BIO INC.) each 20 μ L, incubated at room 25min, after washing with the PBS containing 1%BSA, stream Formula cell instrument detects.

Flow cytometry analysis human adipose-derived stem cell phenotype, experimental result is shown in Table 1.

Table 1 human adipose-derived stem cell phenotype analytical

As can be seen from the above table, the cell cultivated in experimental group A, B, C, E, F and matched group is respectively provided with dry thin Born of the same parents' characteristic.But, the HLA.DR expression in D group is higher, illustrates to be not added with the culture fluid of glutathion, though So also can promote to effectively facilitate cell proliferation, but the cell of propagation has the tendency of fibroblastization, this experiment Illustrate, the cell culture fluid of the present invention adds glutathion and can effectively prevent the one-tenth of fat mesenchymal stem cell Fibrocyte.

The preferred embodiment of the present invention described in detail above.Should be appreciated that those of ordinary skill in the art Just many modifications and variations can be made according to the design of the present invention without creative work.Therefore, all technology neck In territory, technical staff is the most on the basis of existing technology by logical analysis, reasoning or limited Test available technical scheme, all should be in the protection domain being defined in the patent claims.

Claims (8)

1. the compositions cultivated for fat mesenchymal stem cell, it is characterised in that described compositions includes: people Blood albumin, vitamin C, resveratrol, hydrocortisone, basic fibroblast growth factor (bFGF), Progesterone and glutathion.

2. compositions as claimed in claim 1, wherein, human albumin 10-30mg/ml, dimension in described compositions Raw element C 10-50 μ g/ml, resveratrol 10-20 μ g/ml, hydrocortisone 3-6ng/ml, alkalescence fibroblast Dimension cell growth factor (bFGF) 0.1-0.5ng/ml, progesterone 3-6ng/ml and glutathion 30-50 μ g/ml.

3. compositions as claimed in claim 1, wherein, human albumin 20mg/ml, dimension life in described compositions Element C 40 μ g/ml, resveratrol 15 μ g/ml, hydrocortisone 4ng/ml, basic fibroblast growth The factor (bFGF) 0.3ng/ml, progesterone 4ng/ml and glutathion 40 μ g/ml.

4. compositions as claimed in claim 1, wherein, also includes vitamin E, preferably ties up in described compositions Raw element E content is 15-20 μ g/ml.

5. compositions as claimed in claim 1, wherein, also includes antibiotic, described antibiotic in described compositions Can be penicillin and/or streptomycin.

6. compositions as claimed in claim 1, wherein, in described compositions, the content of penicillin is that 20-250 is mono- The content of position/ml and streptomycin is 20-250 unit/ml.

7. a fat mesenchymal stem cell cultural method, it is characterised in that add present invention power in basal medium Profit requires the compositions described in 1, then cultivates fat mesenchymal stem cell.

8. method as claimed in claim 7, wherein it is preferred to described basal medium is DMEM/F12 culture medium.

CN201410442734.5A 2014-08-27 2014-08-27 Composition ADSCs for ADSCs culture and ADSCs culture method Pending CN105886462A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106754683A (en) * 2017-01-03 2017-05-31 黄兵 A kind of people's umbilical cord/fat mesenchymal stem cell without differentiation amplification anti-aging culture medium
CN108753712A (en) * 2018-07-04 2018-11-06 成都清科生物科技有限公司 A kind of fat stem cell extracting method
CN109825471A (en) * 2019-03-19 2019-05-31 杨姣姣 A kind of external efficient amplification agent of fat mesenchymal stem cell, amplification method
CN112553154A (en) * 2020-12-24 2021-03-26 南京农业大学 Improved proliferation culture medium for maintaining functions of adipose-derived mesenchymal stem cells

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106754683A (en) * 2017-01-03 2017-05-31 黄兵 A kind of people's umbilical cord/fat mesenchymal stem cell without differentiation amplification anti-aging culture medium
CN106754683B (en) * 2017-01-03 2020-03-24 北京海康殷氏生物科技有限责任公司 Differentiation-free amplification anti-aging culture medium for human umbilical cord/adipose-derived mesenchymal stem cells
CN108753712A (en) * 2018-07-04 2018-11-06 成都清科生物科技有限公司 A kind of fat stem cell extracting method
CN108753712B (en) * 2018-07-04 2022-11-01 成都清科生物科技有限公司 Method for extracting adipose-derived stem cells
CN109825471A (en) * 2019-03-19 2019-05-31 杨姣姣 A kind of external efficient amplification agent of fat mesenchymal stem cell, amplification method
CN109825471B (en) * 2019-03-19 2022-08-19 上海揽微赛尔生物科技有限公司 Adipose-derived mesenchymal stem cell in-vitro efficient amplification agent and amplification method
CN112553154A (en) * 2020-12-24 2021-03-26 南京农业大学 Improved proliferation culture medium for maintaining functions of adipose-derived mesenchymal stem cells

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