A method for in vitro expansion of adipose-derived stem cells and SVF

Abstract

The present invention relates to stem cell technology, and specifically, to a method for in vitro amplification of adipose stem cells and SVF, wherein the amplification of the cells includes the steps of cell separation and cell culture, wherein the cell culture includes basic fibroblast growth factor, reduced glutathione and long-chain recombinant human insulin-like growth factor; the concentrations of the basic fibroblast growth factor, reduced glutathione and long-chain recombinant human insulin-like growth factor are 0.3ng / mL, 0.25mg / mL and 50ng / mL, respectively. Compared with the prior art, the present invention has at least the following beneficial effects: 1) The present invention uses ultrasound to more fully separate adipose stem cells. 2) The present invention uses an improved culture medium, and the obtained adipose stem cells and SVF have good cell proliferation ability and effect.

Description

Technical Field

[0001] The present invention relates to stem cell technology, and in particular to a method for in vitro adipose stem cells and SVF expansion. Background Art

[0002] Adipose-derived mesenchymal stem cells are a type of adult mesenchymal stem cells that are rich in human adipose tissue. These stem cells have multidirectional differentiation potential and can differentiate into fat, bone, cartilage, muscle and nerve cells in vitro. In addition, human adipose-derived mesenchymal stem cells can secrete a variety of bioactive substances, including proteins, peptides and cytokines, such as basic fibroblast growth factor (SCGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF) and transforming growth factor, etc. These cell growth factors can control or maintain injured tissue cells and guide their self-repair. Compared with embryonic stem cells, human adipose-derived mesenchymal stem cells as adult stem cells can avoid related ethical issues. Compared with bone marrow stem cells, human adipose-derived mesenchymal stem cells are obtained in a minimally invasive manner. Combining the above advantages, human adipose-derived mesenchymal stem cells have broad application prospects in cell therapy and regenerative medicine.

[0003] Adipose tissue has been considered an endocrine organ due to its role in energy regulation, inflammatory response and immune response. In addition, it is one of the sources of multifunctional cells with multidirectional differentiation potential, and these multifunctional cells exist in the vascular stromal fraction of adipose tissue. The vascular stromal fraction (SVF) in adipose tissue is obtained by filtering and centrifuging adipose tissue to remove mature adipocytes. Because SVF is easy to extract from adipose tissue and contains rich and highly plastic adipose tissue-derived mesenchymal stem cells (ASC), it is of great significance to reveal the cellular composition of SVF and clarify its corresponding biological functions.

[0004] SVF cells represent a heterogeneous group of cells that surround adipocytes in adipose tissue. SVF obtained from human breast and visceral adipose tissue has been reported to contain endothelial cells, uncharacterized stromal cells, blood cells, and tissue macrophages. In addition, SVF is also one of the sources of hematopoietic progenitor cells and mesodermal stem cells (which can differentiate into osteogenic, chondrogenic, and myogenic and neurogenic cell lines).

[0005] Currently, there are two main directions for optimizing stem cell separation methods: one is to optimize enzymatic hydrolysis parameters, and the other is to optimize culture medium.

[0006] Prior art CN108728411A discloses a method for separating and culturing adipose stem cells. The separation and culturing method comprises: standing adipose tissue containing tissue fluid, retaining the upper layer of adipose tissue after stratification, washing the adipose tissue with a PBS solution; digesting the washed adipose tissue with a solution containing type I collagenase and type III collagenase; after digestion, adding FBS, shaking, centrifuging, discarding the supernatant, and obtaining adipose stem cells; inoculating adipose stem cells into a high-glucose DMEM medium containing FBS for cell culture; after culturing for 40 to 50 hours, replacing the DMEM medium containing EGF, bFGF, and FBS for continued culture.

[0007] The prior art CN111690597B discloses an extraction reagent and an extraction, amplification and culture method for providing human autologous adipose vascular matrix component SVF. The extraction reagent includes 0.3-0.6w / w% mixed enzyme solution, 2-5w / w% trehalose solution, 0.05-0.15w / w% gelatin solution, 0.1-0.5w / w% polyglutamic acid solution and 0.03-0.07w / w% chitosan solution. The extraction of SVF cells from adipose tissue using the extraction reagent can significantly improve the extraction amount and extraction efficiency of SVF cells, with high cell viability and simple operation, thereby achieving simpler, faster, high cell yield, and high cell viability adipose SVF cell extraction and amplification culture. The specific cell population amplified and cultured to the P3 generation can show good proliferation ability, stable cell viability and high survival rate.

[0008] Therefore, this application will also focus on optimizing the culture medium to meet the needs of more efficient in vitro expansion of adipose stem cells and SVF. Summary of the invention

[0009] The present invention first provides a method for in vitro adipose stem cell expansion, the method comprising the following steps: (1) separation and extraction of adipose stem cells; and (2) cultivation of adipose stem cells.

[0010] In certain embodiments, the (2) adipose stem cell culture is performed using a culture medium comprising basic fibroblast growth factor, reduced glutathione and long-chain recombinant human insulin-like growth factor.

[0011] In certain embodiments, the long-chain recombinant human insulin-like growth factor is LR3-I GF-I.

[0012] In certain embodiments, in the culture medium, the concentrations of basic fibroblast growth factor, reduced glutathione and long-chain recombinant human insulin-like growth factor are 0.1-0.5 ng / mL, 0.1-0.5 mg / mL and 20-80 ng / mL, respectively.

[0013] In certain embodiments, in the culture medium, the concentrations of basic fibroblast growth factor, reduced glutathione and long-chain recombinant human insulin-like growth factor are 0.3 ng / mL, 0.25 mg / mL and 50 ng / mL, respectively.

[0014] The present invention also provides a method for in vitro amplification of SVF, the method comprising the following steps: (1) SVF separation and extraction; (2) SVF culture.

[0015] In certain embodiments, the (2) SVF culture is performed using a culture medium comprising basic fibroblast growth factor, reduced glutathione and long-chain recombinant human insulin-like growth factor.

[0016] In certain embodiments, the long-chain recombinant human insulin-like growth factor is LR3-I GF-I.

[0017] In certain embodiments, in the culture medium, the concentrations of basic fibroblast growth factor, reduced glutathione and long-chain recombinant human insulin-like growth factor are 0.1-0.5 ng / mL, 0.1-0.5 mg / mL and 20-80 ng / mL, respectively.

[0018] In certain embodiments, in the culture medium, the concentrations of basic fibroblast growth factor, reduced glutathione and long-chain recombinant human insulin-like growth factor are 0.3 ng / mL, 0.25 mg / mL and 50 ng / mL, respectively.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] 1) The present invention uses adipose stem cells to be more fully isolated.

[0021] 2) The present invention uses an improved culture medium, and the obtained adipose stem cells and SVF have good cell proliferation ability and effect. DETAILED DESCRIPTION

[0022] In order to make the technical problems to be solved, technical solutions and advantages of the present invention more clear, they are described in detail below in conjunction with specific embodiments.

[0023] Low-glucose DMEM containing 10% FBS (fetal bovine serum) and bFGF were purchased from Gibco;

[0024] Reduced glutathione was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0025] LR3-IGF-1 was purchased from Shanghai Puxin Biotechnology Co., Ltd.;

[0026] Other conventional reagents were also purchased from commercial companies.

[0027] Example 1 Culture Configuration

[0028] Medium 1: 10% FBS (fetal bovine serum) low-glucose DMEM; 0.3 ng / mL basic fibroblast growth factor (bFGF), 0.25 mg / mL reduced glutathione and 50 ng / mL long-chain recombinant human insulin-like growth factor (LR 3 -IGF-Ⅰ).

[0029] Culture medium 2 (Comparative Example 1): low-glucose DMEM containing 10% FBS (fetal bovine serum); 0.3 ng / mL basic fibroblast growth factor (bFGF), and 0.25 mg / mL reduced glutathione.

[0030] Medium 3 (Comparative Example 2): 10% FBS (fetal bovine serum) low-glucose DMEM; 0.3 ng / mL basic fibroblast growth factor (bFGF) and 50 ng / mL long-chain recombinant human insulin-like growth factor (LR 3 -I GF-Ⅰ).

[0031] Medium 4 (Comparative Example 3): 10% FBS (fetal bovine serum) low-glucose DMEM; 0.25 mg / mL reduced glutathione and 50 ng / mL long-chain recombinant human insulin-like growth factor (LR 3 -IGF-Ⅰ).

[0032] Culture medium 5 (Comparative Example 4): low-glucose DMEM containing 10% FBS (fetal bovine serum); 0.3 ng / mL basic fibroblast growth factor (bFGF).

[0033] Culture medium 6 (Comparative Example 5): low-glucose DMEM containing 10% FBS (fetal bovine serum); 0.25 mg / mL reduced glutathione.

[0034] Medium 7 (Comparative Example 6): 10% FBS (fetal bovine serum) low-glucose DMEM; 50 ng / mL long-chain recombinant human insulin-like growth factor (LR 3 -I GF-Ⅰ).

[0035] Culture medium 8 (Comparative Example 7): low-glucose DMEM containing 10% FBS (fetal bovine serum).

[0036] Example 2 Expansion of adipose stem cells

[0037] 1) Under sterile conditions, obtain 10 g of human adipose tissue obtained by liposuction. Rinse the adipose tissue with physiological saline and dispense into 50 mL centrifuge tubes at a rate of 5 mL / tube.

[0038] 2) Add equal volumes of type I collagenase and type III collagenase mixture (mass percentage concentrations of 0.05% and 0.05% respectively) into the centrifuge tube containing fat, seal it and shake it upside down to mix it thoroughly. Transfer it to a 37°C constant temperature air bath shaker, digest it at 200R for 50 minutes, and then inactivate the enzyme to obtain the adipose tissue enzymatic solution.

[0039] 3) First, ultrasonically treat the tube at 100 W for 90 seconds, then place the centrifuge tube in a 37°C constant temperature shaker and shake at 120 rpm for 20 minutes until the tube is digested into a uniform suspension.

[0040] 4) The obtained suspension was centrifuged at 1500 rpm for 10 minutes, the upper layer of fat cells and the middle liquid were discarded, the cell precipitate was collected, and the fat stem cell clusters at the bottom layer were retained.

[0041] 5) Add 10 mL of phosphate buffered saline solution to the centrifuge tube containing the adipose stem cell mass, repeatedly blow and resuspend the cells to make a uniform single-cell suspension, wash thoroughly, let the cells pass through a filter with a diameter of 70 microns, collect single cells, and then centrifuge at 1500 rpm for 5 minutes, discard the upper layer of liquid, and retain the adipose stem cell mass at the bottom; repeat this washing step twice.

[0042] 6) After thorough washing, add an appropriate amount of physiological saline, repeatedly blow and beat the cells to make a single cell suspension of adipose stem cells, add them to a culture bottle containing DMEM complete medium, and place them in a 5% CO2 concentration 37°C incubator for culture;

[0043] 7) When the cells are in culture on the 3rd and 6th days, the medium should be changed in time; on the 7th to 10th day, when 3-5 cell colonies reach more than 80%, the cells can be subcultured.

[0044] 8) Remove the culture supernatant; add buffer to rinse the culture flask, remove the supernatant and add trypsin to digest for 3-5 minutes. After digestion, observe the cells under a microscope and tap the culture flask with your palm to ensure that the cells are completely detached from the bottom wall of the cell flask;

[0045] 9) Add culture medium to stop digestion and blow down the cells, transfer to a new centrifuge tube, add buffer to rinse the culture bottle, collect the remaining cells and centrifuge at 400g×3min. After centrifugation, discard the supernatant, add culture medium to resuspend and blow the cells evenly, take 100μL of cell suspension for cell counting and viability detection, and culture the cells at a ratio of 1:3. Take the third generation of adipose stem cells for the experiment. The cultured third generation of adipose stem cells were tested for surface antigens, and the positive surface markers CD29, CD49, CD73, CD90 and CD105 were detected. The cell surface antigen identification results proved that the cells were adipose stem cells.

[0046] 10) Following the steps in step (9), the adipose-derived stem cells were cultured to the third generation, and the supernatant was discarded; 10 mL of new culture medium 1 was added, and the culture was continued at 5% CO2 concentration and 37°C. When the cell confluence reached 80%, the cell supernatant was collected. The harvested supernatant was then filtered with a 0.22 μm filter.

[0047] Comparative Example 1

[0048] Except that medium 2 was used to replace medium 1, other conditions were the same as those in Example 2.

[0049] Comparative Example 2

[0050] Except that medium 3 was used to replace medium 1, other conditions were the same as those in Example 2.

[0051] Comparative Example 3

[0052] Except that medium 4 was used to replace medium 1, other conditions were the same as those in Example 2.

[0053] Comparative Example 4

[0054] Except that medium 5 was used to replace medium 1, other conditions were the same as those in Example 2.

[0055] Comparative Example 5

[0056] Except that medium 6 was used to replace medium 1, other conditions were the same as those in Example 2.

[0057] Comparative Example 6

[0058] Except that medium 7 was used to replace medium 1, other conditions were the same as those in Example 2.

[0059] Comparative Example 7

[0060] Except that medium 8 was used to replace medium 1, other conditions were the same as those in Example 2.

[0061] Example 3 Viability of Adipose Stem Cells

[0062] Adipose stem cells were cultured using the culture methods of Example 2 and Comparative Examples 1-7. After the digestion step, after the primary culture and after the subculture, the cells were counted using the trypan blue classic staining method, observed and counted under a microscope, and the cell viability was calculated based on the staining. Based on the cell number of Comparative Example 7, the relative cell number of each group was calculated, and the results are shown in Table 1.

[0063] Table 1 Relative cell number of adipose stem cells cultured and expanded in vitro

[0064] Group P10 Example 2 20 Comparative Example 1 13 Comparative Example 2 11 Comparative Example 3 9 Comparative Example 4 8 Comparative Example 5 8 Comparative Example 6 7

[0065] The cell counting results are shown in Table 1. The culture medium 1 of Example 2 can increase the number of cells by 20 times, and compared with the control (Comparative Example 7), the effect is significantly different (p<0.001), indicating that the culture medium 1 can significantly promote the proliferation of adipose stem cells.

[0066] Example 4 Method for amplifying human autologous adipose vascular stromal fraction SVF

[0067] The steps include:

[0068] (1) Adipose tissue was collected and washed four times with phosphate buffer solution, and the washing solution was removed; 0.05 times the volume of a mixed solution of type I collagenase and type III collagenase (mass percentage concentrations of 0.05% and 0.05%, respectively) and 3.5w / w% trehalose solution were added to the washed adipose tissue, and digested at 200R in a 37°C water bath for 50 min, and then centrifuged at 200 rpm for 3 min, the upper layer of digestive solution, lipids and red blood cells were discarded, and the precipitated cells were taken;

[0069] (2) Wash the precipitated cells with 10 volumes of saline, centrifuge at 2800 rpm for 5 min, remove the supernatant, add 10 volumes of saline to the precipitate to resuspend the cells, filter the SVF cells through 120 μm, resuspend the cells with 10 volumes of saline again, filter the cells through a 60 μm filter, and obtain SVF cells;

[0070] (3) Fibroblast stem cells, chondrocytes, mesenchymal stem cells and endothelial cells were separated from the extracted SVF cells by flow cytometry. The fibroblast stem cells, chondrocytes, mesenchymal stem cells and endothelial cells were inoculated into culture bottles for passage and proliferation culture until the P3 generation. The culture medium was replaced every 3 days, and the inoculation ratio was 4000 cells / cm 2 After the cells grow to 90% of the culture flask, they are collected and mixed to obtain a specific cell population for expansion culture.

[0071] Comparative Example 8

[0072] Except that medium 2 was used to replace medium 1, other conditions were the same as those in Example 4.

[0073] Comparative Example 9

[0074] Except that medium 3 was used to replace medium 1, other conditions were the same as those in Example 4.

[0075] Comparative Example 10

[0076] Except that medium 4 was used to replace medium 1, other conditions were the same as those in Example 4.

[0077] Comparative Example 11

[0078] Except that medium 5 was used to replace medium 1, other conditions were the same as those in Example 4.

[0079] Comparative Example 12

[0080] Except that medium 6 was used to replace medium 1, other conditions were the same as those in Example 4.

[0081] Comparative Example 13

[0082] Except that medium 7 was used to replace medium 1, other conditions were the same as those in Example 4.

[0083] Comparative Example 14

[0084] Except that medium 8 was used to replace medium 1, other conditions were the same as those in Example 4.

[0085] Example 5 SVF activity

[0086] The SVF culture was carried out by the culture method of Example 4 and Comparative Examples 8-14. After the digestion step, the primary culture and the subculture were completed, the cells were counted by the trypan blue classic staining method, and the cells were observed and counted under a microscope, and the cell viability was calculated according to the staining. Based on the cell number of Comparative Example 14, the relative cell number of each group was calculated, and the results are shown in Table 2.

[0087] Table 2 Relative cell number of SVF cultured in vitro

[0088]

[0089]

[0090] The cell counting results are shown in Table 2. The culture medium 1 of Example 8 can increase the number of cells by about 11 times, and compared with the control (Comparative Example 14), the effect has a very significant difference (p<0.001), indicating that the above culture medium 1 can significantly promote the proliferation of SVF.

[0091] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for in vitro expansion of adipose-derived stem cells, characterized in that: The method comprises the following steps: (1) isolating and extracting adipose stem cells; (2) culturing adipose stem cells; The (2) adipose stem cell culture is carried out using a culture medium consisting of 10% FBS (fetal bovine serum) low-glucose DMEM, bFGF, reduced glutathione and long-chain recombinant human insulin-like growth factor; In the culture medium, the concentrations of bFGF, reduced glutathione and long-chain recombinant human insulin-like growth factor are 0.3 ng / mL, 0.25 mg / mL and 50 ng / mL respectively; The long-chain recombinant human insulin-like growth factor is LR3-IGF-Ⅰ.

2. A method for in vitro expansion of SVF, characterized in that: The method comprises the following steps: (1) SVF separation and extraction; (2) SVF culture; The (2) SVF culture is carried out using a medium consisting of low-glucose DMEM containing 10% FBS (fetal bovine serum), bFGF, reduced glutathione and long-chain recombinant human insulin-like growth factor; In the culture medium, the concentrations of bFGF, reduced glutathione and long-chain recombinant human insulin-like growth factor are 0.3 ng / mL, 0.25 mg / mL and 50 ng / mL respectively; The long-chain recombinant human insulin-like growth factor is LR3-IGF-Ⅰ.

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