CN102002478A - Adipose-derived stem cell separation culture method - Google Patents

Abstract

The invention discloses a method for separating and culturing fat stem cells. The method of the present invention comprises the steps of isolating, obtaining and culturing SVF cells from adipose tissue, and is characterized in that it also includes the following steps: removing Lin ⁺ cells in SVF cells by immunomagnetic bead sorting to obtain ^Lin- cell groups; The sorting method was used to enrich CD271 ⁺ Sca-1 ⁺ cells from the obtained Lin ^- cell population to obtain adipose stem cells; the obtained adipose stem cells were cultured with medium containing LIF and FGF2. The present invention uses the combination of FACS and MACS to efficiently and quickly obtain high-purity adipose stem cells. This group of cells has strong clone formation ability, self-renewal ability and multi-directional differentiation potential. The P16 generation adipose stem cells obtained by the method of the present invention are still It has strong adipogenic ability and osteogenic ability, and provides a new method for obtaining a large number of stem seed cells in vitro for tissue regeneration.

Description

A method for isolating and culturing fat stem cells

technical field

The invention relates to a method for separating and culturing stem cells, in particular to a method for separating and culturing high-purity fat stem cells by combining fluorescence-activated flow cytometry and magnetic-activated cell sorting.

Background technique

Adipose-derived Stem Cells (ASCs) are a type of adult stem cells widely used in the research fields of tissue engineering and regenerative medicine (Cowan CM,. Nat Biotechnol, 2004. 22:560-567). Adipose stem cells, like bone marrow mesenchymal stem cells, have the same multidirectional differentiation potential, and can differentiate into adipocytes, osteoblasts, chondrocytes, myoblasts, endothelial cells, and neuroblasts under specific conditions. In addition, adipose stem cells also have many advantages that other types of adult stem cells do not have, such as sufficient sources of adipose tissue, convenient material collection, slight damage during the acquisition process, and no ethical disputes. On average, about 100 mL of adipose tissue can be obtained. For 1×10 ⁶ stem cells, about 2% of cells derived from adipose tissue have stem cell characteristics, much higher than bone marrow mesenchymal stem cells (BMSCs) (about 0.02% of cells have stem cell characteristics). And adipose stem cells have a stable population doubling rate, self-renewal potential and good immune compatibility (Strem BM,. Trends Biotechnol, 2005.24:1246-1253), its gene transfection efficiency is high, and it can stably express foreign genes. It is widely used in the research of regenerative medicine (Gimble JM,. Circ Res, 2007: 1249-1260). Moreover, ASCs differentiate into adipocytes more efficiently than BMSCs. Therefore, adipose stem cells are one of the ideal seed cells for tissue engineering research, and are the most promising seed cells for adipose tissue engineering.

At present, the commonly used method of isolation and culture of adipose stem cells is to obtain cells from adipose tissue, and after mechanical or enzymatic treatment to remove mature cells such as red blood cells, the culture medium containing 10% fetal bovine serum is used for subculture. Although the medium containing 10% fetal bovine serum can promote the proliferation of adipose stem cells, it is difficult to maintain their undifferentiated state, making the subcultured adipose stem cells prone to aging. Mechanical or enzymatic treatment can only remove mature cells such as erythrocytes. After removing erythrocytes, the essence of these cells is stromal-vascular fraction cells (SVF cells), which still contain a large number of mature cells. Therefore, now The purity of the obtained fat stem cells is not high. Because the growth of the cells is related to the signal traffic between cells, the stem cells with low purity are more likely to age. Often, the fat stem cells will age after 5-6 subcultures in vitro. Stem cell aging (or senescence) refers to the decline in the proliferation ability of stem cells, and the reduction or disappearance of multidirectional differentiation potential (or stemness). Aging means the decrease in the number and function of stem cells, or the inability to regenerate. The aging of adipose-derived stem cells limits the in-depth study of its application in adipose tissue engineering and other tissue engineering.

At present, methods for separating and purifying stem cells include flow cytometry and immunomagnetic bead sorting. AFS cells (amniotic fluid stem cells, AFS) were isolated from amniotic fluid cells by the bead method. However, most of these in vitro separation techniques for stem cells are based on the recognition of cell surface markers. For example, the AFS cells isolated by Coppi et al. are c-Kit or CD117 (stem cell factor receptor) positive cells, and each Each type of adult stem cell has its own unique markers. Rodeheffer MS et al. used cell flow sorting (Fluorescent-activated cell sorting, FACS) to separate the Lin ^- CD29 ⁺ CD34 ⁺ Sca-1 ⁺ CD24 ⁺ cell population from adipose tissue, but this group of cells can only differentiate into adipocytes, And the resulting numbers are low (0.08% ± 0.015)). So far, no specific markers of adipose-derived stem cells have been found, and no stem cell populations in adipose-derived stromal cells (ADSCs) have been found. Research at home and abroad cannot obtain high-purity adipose-derived stem cells ( High-purity adipose stem cells refer to a group of adipose tissue-derived stem cells with the same cell surface antigen markers, homogeneity, self-renewal and multidirectional differentiation capabilities).

The existing isolation of adipose stem cells is not high in purity, and the in vitro culture of adipose stem cells is difficult to maintain self-renewal and multi-directional differentiation potential, which greatly limits the comparison and repetition of research results at home and abroad.

Contents of the invention

The purpose of the present invention is to overcome the deficiency in the prior art that it is difficult for adipose stem cells to maintain self-renewal and multidirectional differentiation potential under in vitro culture conditions, and to provide a method for isolating and culturing adipose stem cells and an effective new medium thereof. After isolation and culture of adipose stem cells according to this method, the adipose stem cells can still maintain their self-renewal and multidirectional differentiation potential after more than 10 generations of subculture.

The technical scheme that the present invention realizes above-mentioned purpose of the invention is:

A method for isolating and culturing adipose stem cells, comprising isolating and obtaining SVF cells from adipose tissue, culturing SVF cells, and removing Lin ⁺ cells in SVF cells by immunomagnetic bead sorting to obtain ^Lin- cell groups; The selection method enriches CD271 ⁺ Sca-1 ⁺ cells from the obtained Lin ^- cell population to obtain adipose stem cells; the obtained adipose stem cells are cultured with a medium containing LIF and FGF2.

In the above method for isolating and culturing adipose stem cells, the markers used in the immunomagnetic bead sorting method are CD5, CD45R, CD11b, Anti-Gr-1 and Ter-119.

In the above method for isolating and culturing adipose stem cells, the markers used in the flow cytometry method are Lin, CD271 and Sca-1.

In the above method for isolating and culturing adipose stem cells, the culture medium used for culturing the obtained adipose stem cells is an α-MEM medium containing LIF and FGF2.

In the above method for isolating and culturing adipose stem cells, the medium used for culturing the obtained adipose stem cells is α-MEM culture medium containing 10% by volume of fetal bovine serum, 10 ³ -10 ⁵ U/mL LIF, and 10-100 ng/mL FGF2 base.

In the above method for isolating and culturing adipose stem cells, the medium used for culturing the obtained adipose stem cells is α-MEM medium containing 10% fetal bovine serum, 10 ³ U/mL LIF, and 20 ng/mL FGF2.

The method for isolating and culturing the adipose stem cells above also includes subculture the obtained adipose stem cells.

In the above method for isolating and culturing adipose stem cells, the subculture is adherent culture.

In the method for isolating and culturing adipose stem cells above, when the growth density of the obtained adipose stem cells reaches 70-90% at the bottom of the culture dish, subculture is carried out.

Preferably, in the above method for isolating and culturing adipose stem cells, subculture is performed when the growth density of the obtained adipose stem cells at the bottom of the culture dish reaches 80%.

In the above method for isolating and culturing adipose stem cells, the process of isolating and obtaining SVF cells from adipose tissue is as follows: cut out the mouse inguinal fat pad under conventional aseptic conditions, rinse, shred, remove blood, etc. Add 0.1% type Ⅰ collagenase equal to the volume of fat, and the cells obtained after digestion, filtration, red cracking and centrifugation are SVF cells.

In the above-mentioned isolation and culture method of adipose stem cells, SVF cells were cultured using α-MEM medium containing 10% fetal bovine serum by volume, and the culture conditions were 37°C and 5% (v/v) CO ₂ .

The adipose stem cells isolated and cultured according to any one of the aforementioned methods for isolating and culturing adipose stem cells are used for the purification or cloning of stem cells, the production of transgenic stem cell lines, or the production of adipogenic/osteogenic cells.

As mentioned above, the method of adipose-derived stem cells used to produce adipogenic/osteogenic cells is:

(1) ASCs were isolated and obtained by combining fluorescence-activated flow cytometry and magnetic-activated cell sorting;

(2) ASCs were respectively compounded with fibrin and BCP scaffold materials, and inoculated on the back of nude mice or the femoral defect of mice.

After 6-12 weeks, the grafts were taken out for HE section staining and immunohistochemical detection.

In the prior art, the subculture of adipose stem cells is prone to aging. In view of this problem, the inventor has conducted extensive research and found that the SVF cells isolated from adipose tissue were sorted by immunomagnetic beads and flow cytometry. The combination of sorting methods can remove mature cells in SVF cells as much as possible, so that high-purity adipose stem cells with the same antigenic markers, homogeneity, self-renewal and multidirectional differentiation capabilities can be obtained. The inventors compared the ability of colony formation and multidirectional differentiation between the addition of the factor LIF that inhibits stem cell differentiation and the factor FGF2 that promotes the proliferation of stem cells in the culture medium, and the ability of multi-lineage differentiation, and found that adding the factor LIF that inhibits cell differentiation to the medium And the growth factor FGF2 that promotes the proliferation of stem cells can maintain the stemness of the obtained high-purity stem cells.

Therefore, in the present invention, SVF cells are isolated and cultured from adipose tissue, and then hematopoietic and mature cells such as CD5 ⁺ , CD45R ⁺ , CD11b ⁺ and Ter ^- 119 ⁺ are removed from SVF cells by MACS method, and Lin ^- cells are enriched; Then FACS was used to sort CD271 and Sca-1 positive cells from the Lin ^- cell population to enrich CD271 ⁺ Sca-1 ⁺ cells (that is, adipose stem cell ASCs); The differentiation factor LIF and the growth factor FGF2 that promotes stem cell proliferation maintain the stemness of stem cells. The present invention compares the clonogenic ability and multidirectional differentiation ability of adding the factor LIF which inhibits the differentiation of stem cells and the factor FGF2 which promotes the proliferation of stem cells in the culture medium and conventional culture conditions, and proves that adding LIF and FGF2 in the culture medium is helpful to maintain the stemness of adipose-derived stem cells. The present invention measures the culture and clone formation ability of ASCs, and confirms that the obtained ASCs have functions such as multi-directional differentiation potential in vivo and in vitro from aspects such as morphology, immunocytochemistry, mRNA and protein levels.

The present invention uses the combination of FACS and MACS to efficiently and quickly obtain high-purity adipose stem cells. Compared with the prior art, the present invention has the following advantages and positive effects:

1. The present invention adopts the method of combining fluorescence-activated flow cytometry (FACS) and magnetic-activated cell sorting (MACS) to enrich high-purity adipose-derived stem cells by using Lin ^- :CD271 ⁺ :Sca-1 ⁺ for the first time at home and abroad Subpopulation, this group of cells has strong clone formation ability, self-renewal ability and multi-lineage differentiation potential, while other cells do not have this ability.

2. The present invention uses the conditioned medium containing LIF and FGF2 to culture cells, and the P16 adipose stem cells still have a strong adipogenic ability, and the P16 adipose stem cells still have the osteogenic ability.

3. The present invention uses fibrin and biphasic calcium phosphate ceramics (BCP) as carriers of adipose stem cells, and uses ASCs of green fluorescent protein transgenic mice as tracers to prove that ASCs can transform in the adipogenic/osteogenic microenvironment in vivo for adipocytes and osteoblasts.

4. The present invention has found a feasible and efficient method for obtaining fat stem cells, which provides a new method for obtaining a large number of stem cells in vitro for tissue regeneration.

Description of drawings

Figure 1 shows the Lin ^- cell population obtained by MACS, and then co-stained with two antibodies CD271 and Sca-1 and then flow cytometrically sorted to obtain Lin ^- :CD271 ⁺ :Sca-1 ⁺ cell population (ie adipose stem cells).

Figure 2 shows the clonal culture of ASCs using the limiting dilution method: where A is the Lin ^- :CD271 ⁺ :Sca-1 ⁺ cell population formed a clone (day 3), Scale bars= 100 μm; B is Lin ^- :CD271 ⁺ : Colony formed by Sca-1 ⁺ cell population (day 5), Scale bars = 100 μm.

Figure 3 A and B are light micrographs of the growth state of adipose-derived stem cells. The cells are fibroblast-like spindle-shaped, with typical swirl-shaped growth. The Scale bars in A are 40 μm, and the Scale bars in B are 100 μm.

Figure 4 is the multidirectional differentiation diagram of ASCs in vitro, in which A: After adipogenic induction, Oil Red O stains lipids in orange red, Scale bars= 200μm; B: After osteogenic differentiation, Alizarin red staining shows calcium nodules Scale bars= 100μm; C: After chondrogenic differentiation, toluidine blue staining showed that the cartilage matrix was blue, with cartilage lacuna formation, Scale bars= 400μm; D: After myogenic differentiation, α-SMA immunocytochemical staining was positive, DAPI stained cell nuclei in blue, Scale bars= 400 μm.

Figure 5 is the RT-PCR detection of ASCs after adipogenic (PPARγ2, C/EBP-α, LPL), osteogenic (Runx2, Opn), chondrogenic (Acan, Sox9) and myogenic (Myog, Myod1) induction, the correlation Gene expression, the internal reference was GAPDH. The results showed that after multidirectional induction, the expression levels of PPARγ2, C/EBP-α, LPL, Acan, Sox9, Myog, and Myod1 genes increased, which fully demonstrated from the mRNA level that our cultured adipose stem cells can promote fat, bone , cartilage, and muscle cell differentiation.

Figure 6 Effects of FGF2 and LIF on the proliferation ability of ASCs. The medium added with FGF2 and LIF maintained the rapid proliferation ability of ASCs and stabilized the doubling time of the cell population; in the contorl group without FGF2 and LIF medium, the doubling time of the cell population prolongs as the number of passages increases.

Figure 7 shows the effects of FGF2 and LIF on the CFU-F formation ability of ASCs. The formation ability of CFU-F in the FGF2 and LIF medium groups did not decrease significantly; while the formation of CFU-F in the contorl group without FGF2 and LIF medium decreased significantly.

Figure 8 shows the effects of FGF2 and LIF on maintaining the adipogenic differentiation potential of ASCs. A is the oil red O staining result of P16 ASCs in FGF2 and LIF medium after 10 days of adipogenic induction, B is the oil red O staining result of P16 ASCs cultured without FGF2 and LIF after 10 days of adipogenic induction, the adipogenic ability of ASCs is obvious Lower, Scale bars = 200μm.

Figure 9 Quantitative determination of glycerol-3-phosphate dehydrogenase activity. From the P6 generation, there was a significant difference in the glycerol-3-phosphate dehydrogenase activity between the two groups of ASCs cultured with FGF2, LIF and without FGF2, LIF after adipogenic induction, and the data were expressed as mean±SE (n=3), ( P﹤0.01).

Fig. 10 A: Under the culture condition of FGF2 and LIF, after osteogenic induction of P16 ASCs, the formation of mineralized nodules can be seen by alizarin red staining; B: Under the culture condition of no FGF2 and LIF, the formation of mineralized nodules of P16 ASCs after osteogenic induction See mineralized nodule formation. Scale bars = 100μm.

Figure 11 The GFP adipose stem cells of C57 mice are fibroblast-like spindle-shaped, without lipid droplets in the cells, and all express green fluorescent protein. Figure 11A is an image of an ordinary optical microscope, and Figure 11B is an image observed by an inverted fluorescence microscope. Scale bars = 40μm.

Fig. 12 Adipogenesis of the cell-fibrin complex 6 weeks after implantation in vivo. A: HE staining shows that it is round or polygonal, and contains vacuoles in the cytoplasm. The vacuoles are the parts where the lipid droplets are dissolved during the sectioning process. The nucleus is squeezed to one side by the lipid droplets, and it is oblate. bars=200μm. B: Oil red O staining shows that the lipid droplets are orange-red, and the nuclei are squeezed aside and appear blue, Scale bars=200μm. C: Anti-GFP immunohistochemistry of newborn adipocytes is positive, Scale bars=100μm.

Figure 13 Osteogenesis of the cell-BCP scaffold material composite implanted in vivo. A and B are HE and Masson staining. The new bone is mainly formed in the pores of the BCP scaffold material, and it is distributed like a bone island. The new bone is surrounded by osteoblasts. HE staining shows pale pink bone trabeculae and mineralizing bone Collagen fibers, the formation of bone lacuna can be seen; Masson staining shows the newly formed bone trabeculae and collagen fibers that are being mineralized in blue. Scale bars=200μm. C is anti-GFP immunohistochemical detection of new bone, in which light brown cells are positive cells, and osteoblasts are positive for anti-GFP (indicated by the red arrow). Scale bars=200μm.

Detailed ways

The present invention will be further described in detail below in conjunction with test examples and specific embodiments. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

The adipose tissue mentioned in the embodiments of the present invention is derived from mice, and the main materials, reagents and equipment used in the embodiments of the present invention are as follows:

C57 mice (provided by the West China Medical Experimental Animal Center of Sichuan University);

Green fluorescent protein transgenic C57 mice (gifted by the Stem Cell Biology Laboratory of the State Key Laboratory of Biotherapy);

Nude mice (purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences);

Fibrin glue scaffold (Hangzhou Puji, China);

Porous biphasic calcium phosphate ceramic (BCP) scaffold material (provided by the National Engineering Research Center for Biomedical Materials, Sichuan University).

α-MEM medium (Hyclone, USA);

Fetal bovine serum (Hyclone, USA);

Trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco, USA);

FGF2 (Peprotech Inc, USA);

LIF, Anti-GFP antibody (Millipore, USA);

Type I collagenase (COLLAGENASE TYPE I), dimethyl sulfoxide (DMSO), dexamethasone (Dex), L-ascorbic acid (L-ascorbic acid), 1, 25-dihydroxyvitamin D3 (1, 25-( OH)2VitD3), 3-isobutyl-1-methylxanthine (IBMX), indomethacin, oil red O, all purchased from Sigma, USA;

RNA extraction kit (Shanghai Huashun, China);

TaKaRa one step RNA PCR Kit (AMV) and DL2000 were purchased from Japan TaKaRa Company.

Constant temperature water bath (Heto-Hoten, Denmark); micropipette (Gilson Company, France); culture bottle (Corning, USA), electronic balance (Strtorius, USA), 0.22pm sterile syringe filter (Millipore, USA) ), ultrapure water machine UNIQUE-R30 (Millipore, USA); CO2 incubator (Thermo, Germany), tabletop centrifuge SORVALLR LEGEND RM (Thermo, Germany), temperature-controlled tabletop centrifuge SORVALLR LEGEND T (Thermo, Germany), Thermo KS12 ultra-clean bench (Thermo, Germany), Thermo -86℃ HERA freeze, (Thermo, Germany); OLYMPUS IX71 inverted phase-contrast fluorescence microscope (Olympus, Japan), OLYMPUS CKX41 inverted microscope (Olympus, Japan), OLYMPUS CX41 upright Microscope (Olympus, Japan), -152°C ultra-low temperature refrigerator (SANYO, Japan), scanning electron microscope (JSM-5900LV) (JEOL Ltd.); Tanon-2500 gel imager (Shanghai Tianneng, China); PCR instrument (BIO-RAD, USA), gel imaging analysis software Quantity One 4.6.2 (BIO-RAD, USA).

The method for isolating and culturing adipose stem cells described in the examples cited in the present invention includes isolating, obtaining and culturing SVF cells from mouse adipose tissue, and also includes removing Lin ⁺ cells in SVF cells by immunomagnetic bead sorting to obtain Lin ^- cell population; CD271 ⁺ Sca-1 ⁺ cells were enriched from the obtained Lin ^- cell population by flow cytometry to obtain adipose stem cells; the obtained adipose stem cells were cultured and subcultured with a medium containing LIF and FGF2 .

In the above method for isolating and culturing adipose stem cells, isolating, obtaining and culturing SVF cells from mouse adipose tissue includes cutting out the inguinal fat pad of the mouse under conventional aseptic conditions, rinsing, shredding, and removing blood, and dissolving the SVF cells in the adipose tissue. Add 0.1% type Ⅰ collagenase equal to the volume of fat, and the cells obtained after digestion, filtration, red cracking and centrifugation are SVF cells.

SVF cells were cultured in α-MEM medium containing 10% fetal bovine serum, and the culture conditions were 37°C and 5% (v/v) CO ₂ .

The markers used in the immunomagnetic bead sorting method were CD5, CD45R, CD11b, Anti-Gr-1 and Ter-119. Markers used in flow cytometry were Lin, CD271 and Sca-1.

The medium used for culturing the obtained adipose stem cells was α-MEM medium containing 10% by volume of fetal bovine serum, 10 ³ U/mLLIF, and 20ng/mLFGF2.

Carry out adherent subculture, and when the growth density of the obtained adipose stem cells reaches 80% at the bottom of the culture dish, subculture is carried out, and the adipose stem cells at P16 are used to produce adipogenic/osteogenic cells.

1. Acquisition and culture of stromal vascular fragment (SVF) cells:

1) 4-week-old female C57 mice were anesthetized by intraperitoneal injection of pentobarbital sodium 0.1mg/100g. After the anesthesia was effective, the inguinal fat pad of the rat was taken under aseptic conditions, and the tissue block was washed with sterile PBS solution;

2) Cut the adipose tissue pieces into pieces in an ultra-clean workbench, and digest them with an equal volume of 0.1% type I collagenase on a constant temperature shaker at 37°C for 45-60 min, shaking properly during the period to fully digest;

3) Add medium containing 10% fetal bovine serum by volume to neutralize type Ⅰ collagenase, use a 70 μm cell sieve to filter out undigested large tissue pieces and residues left after digestion, and centrifuge at low speed (1200g, 5 min) , the bottom of the centrifuge tube is the cell mass;

4) Aspirate the adipose tissue and mature adipocytes floating on the surface, resuspend the cell mass with freshly prepared erythrocyte lysate and let it stand for 10 min, then centrifuge (300g, 5 min);

5) Collect the cell clusters (interstitial vascular fragment cells, SVF cells) at the bottom of the centrifuge tube, wash the medium three times, inoculate in a 10cm-diameter petri dish at a cell density of 1×105/mL, and store in saturated humidity at 37°C , 5% CO ₂ incubator for cultivation, replace the medium every 3 days, the medium is 10% volume ratio FBS+α-MEM medium containing 100u/mL penicillin and 100 μg/mL streptomycin;

6) When the cells grow in a monolayer at the bottom of the culture dish and reach about 80% confluence, subculture.

2. Enrichment of adipose stem cells from SVF cells

2.1 Obtain SVF and use Lineage Cell Depletion Kit to negatively select Lin ^- cells:

1) Count the obtained SVF cells, wash with PBS solution, centrifuge (300×g, 10 min), and remove the supernatant completely.

2) Resuspend 10 ⁷ cells in 40 μL buffer.

3) Add 10 μL Biotin-Antibody Cocktail to 10 ⁷ cells.

4) Mix well and incubate at 4°C for 10 min.

5) Add 30 μL buffer to 10 ⁷ cells.

6) Add 20μL Anti-Biotin Microbeads.

7) Mix well and incubate at 4°C for 15 min.

8) Wash the cells with 1-2 mL buffer, centrifuge (300×g, 10 min), and aspirate the supernatant.

9) Resuspend cells in 500 μL buffer.

10) Put the magnetic column in a suitable position on the magnetic rack, and wash the column once with 500 μL buffer.

11) Add 500 μL of cell suspension to the magnetic column, collect the liquid that flows out, which contains Lin ^- cells

12) Wash the magnetic column 3 times with 500 μL buffer solution, and mix the collected effluent with the effluent collected in the previous step.

13) Remove the magnetic column, place it on the top of a new collection tube, add 1 mL of buffer solution to the magnetic column, and quickly push out the magnetically labeled cells. The cells obtained in this section are Lin ⁺ cells (positive for CD5, CD45R, CD11b, Anti-Gr-1 or Ter-119).

2.2 FACS enrichment of CD271 ⁺ Sca-1 ⁺ cells from Lin ^- cell population

1) Count the Lin ^- cells collected in the previous step and place them in a 1.5mL EP tube.

2) Wash once with PBS, and resuspend the cells with serum-free α-MEM medium.

3) Add purified CD271 primary antibody (rabbit anti-mouse) and incubate for 30 min in the dark.

4) Wash with PBS, resuspend cells in serum-free α-MEM medium, add fluorescent secondary antibody (monkey anti-rabbit), and incubate for 30 min.

5) Wash with PBS, resuspend the cells in serum-free α-MEM medium, add Sca-1 and 7AAD or DAPI, and incubate for 30 min.

6) Wash with PBS, resuspend the cells with serum-free α-MEM medium, and sort them aseptically on a flow cytometer to obtain a group of Lin ^- :CD271 ⁺ :Sca-1 ⁺ cells and Lin ^- :CD271 ^- and Lin ^- :CD271 ⁺ :Sca-1 ^- cell population.

7) The obtained Lin ^- :CD271 ⁺ :Sca-1 ⁺ cell population was cultured. At the same time, the collected Lin ⁺ cell population, Lin ⁻ :CD271 ⁻ cell population and Lin ⁻ :CD271 ⁺ :Sca-1 ⁻ cell population were also cultured.

3. Determination of ASCs clone forming ability (Clony-forming unit-fibroblast, CFU-F)

The freshly isolated cell population was cultured by limiting dilution method, and a single cell suspension was made with 20% FBS+α-MEM medium, and a single cell was sucked into each well of a 48-well plate with a micropipette. Add 20% FBS+α-MEM medium.

After 14 days, the colony formation was detected. The colony formation rate=(cloning formation/number of inoculated cells)×100%. It was determined that the colony formation rate was 15.7%±0.58. As shown in Figure 2, the cells formed good clones.

4. Detection of multi-directional differentiation ability of ASCs in vitro, this step is to verify the multi-directional differentiation ability of the obtained adipose stem cells.

4.1 Adipogenic induction

The P1-generation ASCs were inoculated in six-well plates, and cultured in adipogenic induction medium after 24 h. Oil red O staining, DAPI staining nuclei. As shown in Figure 4A, the round lipid droplets are red and distributed around the cells, indicating that the cells can differentiate towards fat.

Table 1 Formulas of various induction solutions

RT-PCR detection of adipogenesis-related genes peroxisome proliferator-activated receptor-γ2 (PPARγ2) and CCAAT enhancer-binding protein α (CCAAT/enhancer-binding α, C/EBP-α) and lipoproteinase (lipoprotein lipase, Lpl) expression. The total cellular RNA was extracted by referring to the RNA extraction kit of Shanghai Huashun Biotechnology Co., Ltd., and then cDNA was synthesized according to the operation instructions of TaKaRa One Step RNA PCR Kit (AMV), and the obtained cDNA template was stored at -20°C for later use. According to the gene sequence to be detected, primers were designed using Primer 5.0 primer design software, and the housekeeping gene GAPDH was used as an internal reference. The designed forward primer (Forward primer), reverse primer (Reverse primer), annealing temperature (annealing temperature, Tm) and expected product size are shown in Table 2. As shown in Figure 5, after the cells were induced to adipocytes, the expression of adipocyte-specific genes PPARγ2, C/EBP-α, and Lpl was significantly enhanced, which further indicated successful adipogenic differentiation from the gene level.

4.2 Osteogenic induction

The P1-generation ASCs were inoculated in six-well plates, and replaced with osteogenic induction medium for culture after 24 hours. The composition of the induction medium is shown in Table 1, and the medium was changed every 3 days. After 14 days of osteogenic induction culture, the cell slides were stained with alizarin red to observe the formation of mineralized nodules. As shown in Figure 4B, after osteotropic induction, adipose-derived stem cells differentiated into osteoblasts, with typical red calcium nodules formed.

RT-PCR detection of osteogenesis-related genes Runt-related transcription factor 2/core binding factor a1 (runt related transcription factor 2/core binding factor a1, Runx2/Cbfal) and osteopontin (osteopontin, OPN) expression during osteogenic culture . As shown in Figure 5, the expressions of Runx2 and OPN were significantly increased, further indicating successful osteogenic differentiation at the gene level.

4.3 Chondrogenic induction

P1 generation ASCs were resuspended in 1.2% low-viscosity alginate gel at a cell concentration of 1×107/mL, cross-linked with 100 mM CaCl2 solution, and cultured in chondrogenic induction medium. The composition of the induction medium is shown in Table 1. Change the medium every 3 days. After 3 weeks of chondrogenic induction, the specimens were fixed, sliced and stained with toluidine blue to observe the formation of cartilage matrix. As shown in FIG. 4C , the toluidine blue staining of the adipose-derived stem cells was positive after chondrogenic induction, indicating that the adipose-derived stem cells could differentiate toward cartilage.

RT-PCR detected the expression of cartilage-related gene SRY box containing gene 9 (SRY-box containing gene 9, Sox9) and aggrecan (aggrecan, Acan). As shown in Figure 5, the expression of these two cartilage-specific genes was enhanced, further indicating the success of chondrogenic differentiation at the gene level.

4.4 Myogenic induction

The P1-generation ASCs were inoculated in a six-well plate, and replaced with a myogenic induction medium for culture after 24 hours. ). After 3 weeks, the cells were fixed with 4% paraformaldehyde and immunofluorescence stained with α-SMA antibody. As shown in Figure 4D, immunocytochemical fluorescent staining for the smooth muscle cell-specific protein α-SMA was positive (green).

The expression of myoblast-related genes Myog and Myod1 was detected by RT-PCR. As shown in Figure 5, the expression levels of Myog and Myod1 were significantly increased, which further confirmed that ASCs differentiated into myogenic direction after myogenic induction from the mRNA level.

The primer sequences of the genes to be detected are shown in Table 2:

Table 2 Primers and expected products of the genes to be tested

 

Note: PPAR-γ2: peroxisome proliferator-activated receptor γ2; C/EBP-α: CCAAT enhancer-binding protein α; Lpl: lipoproteinase; Runx2: Runt-related transcription factor 2; Opn: osteopontin ; Sox9: SRY box containing gene 9; Acan: aggrecan; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

5. The effect of adding FGF2 and LIF on the proliferation and multilineage differentiation ability of ASCs

5.1 The effect of FGF2 and LIF on the proliferation ability of ASCs

1) The ASCs were divided into groups without FGF2 and LIF and groups with FGF2 and LIF. In the FGF2 and LIF groups, 20 ng/mL FGF2 and 1000 units/mL were added to 10% FBS(v/v)+α-MEM medium. The LIF. The ASCs of the 1st, 6th, 11th and 16th passages in the two groups were respectively taken, and the cells were planted into a 12-well plate at 5×103/well, and the ASCs with and without FGF2, For LIF cells, the cell number was detected by MTT method, the growth curve was drawn, and the population doubling time of the cells was calculated.

2) Cells were inoculated at 10 cells/cm ² , and 14 days later, the colony formation in the groups with and without FGF2 and LIF were detected, and the colony formation rate was calculated.

5.2 The role of FGF2 and LIF in maintaining the multilineage differentiation potential of ASCs

5.2.1 Effect on adipogenic differentiation potential

The ASCs of the 1st, 6th, 11th and 16th passages in the two groups were taken respectively, and the cells were inoculated at the same density. After 24 hours, adipogenic induction medium was added for culture, and oil red O staining was performed 10 days after adipogenic induction. Glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate dehydrogenase, GPDH) activity was used to determine the amount of adipocyte formation. The method of GPDH activity was determined by referring to the method reported by Hauner et al.: wash the target cells with PBS, pre-cooled containing 1 mM EDTA Cells were collected with 25 mM Tris-HCl buffer solution (pH 7.5), the supernatant was obtained after the cells were lysed by ultrasonic, and the activity of GPDH was detected by ultraviolet spectrophotometer. Reaction system: 100 mM triethanolamine HCl buffer solution (pH 7.5), 2.5 mM EDTA, 0.12 mM NADH, 0.1 mM mercaptoethanol, and 0.2 mM dihydroxyacetone phosphate.

5.2.2 Effects on osteogenic differentiation potential

1) Take the ASCs of the 1st, 3rd, 6th, 11th and 16th passages in the two groups, and inoculate the cells at the same density. After 24 hours, osteogenic induction medium was added for culture, and Alizarin red staining was performed 14 days later to observe the formation of mineralized nodules.

2) A group of P1 generation ASCs was subjected to conventional osteogenic induction, and 20 ng/mL of FGF2 and 1000 units/mL of LIF were added to the osteogenic induction solution of the other group. Total RNA was extracted on days 4, 7, 14, and 21, respectively, and osteogenesis-related genes Runx2 and Ocn were detected according to the aforementioned method.

3) According to the same grouping as in 1), the total RNA of ASCs at passages 1, 3, 6, 11, and 16 cultured with or without FGF2 and LIF were extracted, and the mRNA expression of the early osteogenesis-related gene Runx2 was detected by RT-PCR , to determine whether it has osteogenic potential.

The results showed that under the culture conditions without FGF-2 and LIF, ASCs cultured to P16 generation, no mineralized nodules were formed after osteogenic induction, while under the culture conditions of FGF-2 and LIF, ASCs cultured to P16 generation still remained Formation of mineralized nodules can be seen. In P1 generation ASCs in the osteogenic induction medium without FGF-2 and LIF, the expression level of the early osteogenic gene Runx2 was high at 7-14 days, the expression disappeared at 21 days, and the late osteogenic marker gene Ocn appeared at the 7th day Expression lasted up to 21 days. In the osteogenic induction medium containing FGF-2 and LIF, the early osteogenic gene Runx2 in ASCs was still expressed at 21 days, while the late osteogenic gene Ocn was not expressed during the induction process. In the basal medium with FGF-2 and LIF, the expression of the early osteogenesis gene Runx2 could still be detected in ASCs at passage P16; while in the basal medium without FGF-2 and LIF, ASCs could not detect the expression of the early osteogenic gene Runx2 at passage P16. Expression of Runx2 was detected.

6. Acquisition and culture of GFP adipose stem cells (same as C57 mice), this experiment is to obtain adipose stem cells labeled with GFP, and further conduct in vivo test verification.

1) The Lin ^- :CD271 ⁺ :Sca-1 ⁺ GFP adipose stem cell population was enriched from the adipose tissue of GFP transgenic C57 mice using the same method as above;

2) Inoculate the obtained cells in a 10cm-diameter petri dish at a cell density of 1×10 ⁵ /mL for culture, 10% FBS (v/v)+α-MEM medium (containing FGF-2 and LIF), Culture in an incubator with saturated humidity at 37°C and 5% _CO2 , and replace the medium every 3 days.

3) In vitro adipogenic differentiation of Lin ^- :CD271 ⁺ :Sca-1 ⁺ GFP adipose stem cell population

The obtained P3 generation GFP adipose stem cells were inoculated in a culture dish with a diameter of 3.5 cm as 1×10 ⁵ cells, and adipogenic induction began after 24 h. The adipogenic induction medium contained α-MEM basal medium, 10% FBS, 1 μM Dexamethasone, 10 μM insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 0.2 mM indomethacin, 100 u/mL penicillin, 100 μg/mL streptomycin. The medium containing α-MEM basal medium and 10% FBS (v/v) was replaced every 3 days in half.

RT-PCR was used to detect the mRNA expression of adipogenic related genes PPARγ2 and C/EBP-α during adipogenic induction. Oil red O staining was used to detect the formation of lipid droplets in GFP adipose-derived stem cells 10 days after lipid orientation induction.

4) In vitro osteogenic differentiation of Lin ^- :CD271 ⁺ :Sca-1 ⁺ GFP adipose stem cell population

P3 generation GFP adipose stem cells were seeded in six-well plates, and cultured in osteogenic induction medium after 24 h. The osteogenic induction medium contained α-MEM basal medium, 10% FBS, 10 mM β-sodium glycerophosphate, 10 ^-8 mol/L dexamethasone, 50 μM ascorbic acid and 0.01 μM 1, 25-dihydroxyvitamin D3, 100 u/mL penicillin, and 100 μg/mL streptomycin.

RT-PCR was used to detect the expression of osteogenesis-related genes Runx2 and Ocn during osteogenesis culture. Alizarin red staining was performed to observe the formation of mineralized nodules on the slices of cells cultured for 21 days after osteogenic induction.

The results showed that GFP adipose stem cells exhibited a spindle-shaped fibroblast-like morphology, 80%-90% cell confluence was achieved in about 6 days, and the expression of green fluorescent protein in all cells was positive. After adipogenic induction of GFP adipose stem cells, the lipids formed in the cytoplasm were identified by Oil Red O staining, and the lipids were stained orange-red, while the staining of the control group was negative; by RT-PCR detection, during the adipogenic induction The mRNA expression of adipogenic specific genes PPARγ2 and C/EBP-α was significantly enhanced; it confirmed that GFP adipose stem cells successfully differentiated into adipocytes after adipogenic induction. Alizarin red staining was performed on the cell slides of GFP adipose-derived stem cells 21 days after osteogenic induction, and the formation of red mineralized substances could be observed; RT-PCR detected the expression of osteogenic specific genes Runx2 and Ocn during osteogenic culture; confirmed GFP After osteotropic induction, adipose-derived stem cells differentiate into osteoblasts.

7. Construction of cell-fibrin complex and implantation in vivo. The purpose of this experiment is to verify the adipogenic differentiation of adipose stem cells in vivo.

1) Use P3 GFP ⁺ Lin ^- :CD271 ⁺ :Sca-1 ⁺ adipose stem cells and mix evenly with 100 μl fibrinogen solution containing 1 μg FGF2 at a density of 2 × 10 ⁷ cells/mL. Lin ^- :CD271 ^- and Lin ^- :CD271 ⁺ :Sca-1 ^- cells were used as controls.

2) Under sterile conditions, nude mice were anesthetized with 3.5% chloral hydrate.

3) After the anesthesia became effective, the fibrinogen solution containing cells in 1) and 100 μL thrombin solution were simultaneously injected subcutaneously on the back of the nude mice.

8. The construction and in vivo implantation of the cell-BCP scaffold material composite. The purpose of this experiment is to verify the osteogenic differentiation of adipose-derived stem cells in vivo.

8.1 Characteristics of BCP scaffold material and construction of cell-BCP scaffold material composite

The ratio of hydroxyapatite (HA) to β-tricalcium phosphate (β-TCP) in the porous biphasic calcium phosphate ceramics (Biphasic calcium phosphate ceramics, BCP) scaffold material is 30:70, with a length of 5 mm and a diameter of 1.5 mm. The pores are made by H ₂ O ₂ microwave foaming method. The size of the macropores is _200-500 μm, and the pores are well connected. There are a large number of interconnected micropores in the wall of the macropores. After ultrasonic cleaning and high temperature steam sterilization, it is ready for use.

GFP adipose stem cells were seeded on the surface of the BCP scaffold material at a density of 5×10 ⁶ cells/mL. After 5 days, the cell-scaffold complex was washed twice with PBS, fixed overnight at 4°C with 2.5% glutaraldehyde; 30%, 50%, 75%, 85%, 95%, 100% volume fraction alcohol gradient dehydration; after dehydration Soak in isoamyl acetate for replacement for 1 h; critical point drying, gold spraying; scanning electron microscope inspection.

8.2 In vivo implantation of cell-BCP scaffold composite

1) Use the same method as above to construct GFP adipose stem cell-BCP scaffold material composites, a total of 4 samples. Lin ^- :CD271 ^- and Lin ^- :CD271 ⁺ :Sca-1 ^- cells were used as controls.

2) Under sterile conditions, 8-week-old male common C57 mice were anesthetized with 3.5% chloral hydrate.

3) After the anesthesia is effective, an incision is made in the femoral area under sterile conditions, and the muscles are bluntly separated to expose the femur. A 5mm long femoral defect was prepared with a low-speed electric drill.

4) The GFP adipose stem cell-BCP scaffold composite was implanted into the bone defect area and fixed with silk thread. Layered registration stitching.

9. Detection of planting cell-scaffold composites.

9.1 Detection of cell-fibrin complexes

1) 6 weeks after the complex was implanted, the experimental animals were sacrificed by overdose anesthesia, and the implanted cell-fibrin complex was taken out, routinely embedded in paraffin, and stained with HE sections.

2) Oil red O staining of frozen sections, the steps are as follows:

A The removed new organisms were fixed overnight with 10% neutral formaldehyde.

B Frozen sections in a constant temperature box at -25°C, with a thickness of 10 μm.

Wash in 60% isopropanol for a while.

D Infect with oil red O staining solution for 10 min.

E Rinse with running water for 5 min.

F Soak with hematoxylin solution for 2 min.

G Rinse with running water for 5 min, and differentiate with 1% (v/v) hydrochloric acid alcohol.

Wash with H water for 10 min until the nuclei turn blue.

I Absorb the excess water around, and seal the slide with glycerin gelatin.

3) Immunohistochemical detection of anti-GFP, the steps are as follows:

A Make paraffin sections with a thickness of 5 μm, and place them in a 60°C oven for 24 h.

B xylene for 10 min, then soak for another 10 min after replacing the xylene.

C Absolute ethanol for 5 minutes, 95%, 85%, and 75% (v/v) ethanol for 5 minutes each.

D Rinse with running water for 5 min.

E 3% (v/v) H ₂ O ₂ was incubated at room temperature for 15 min to block the activity of endogenous peroxidase.

Soak in F ddH ₂ O for 3 min x 3 times, and shake on a low-speed shaker during the soaking process.

G Add antigen retrieval solution and bathe in water at 95°C for 45 min.

Cool at room temperature for 30 min. Pour off the antigen retrieval solution and soak in ddH2O for 1 min.

I 1% (v/v) hydrochloric acid alcohol differentiation 3 sec.

J 10% (v/v) normal goat serum was blocked and incubated at room temperature for 10 min. Pour off the serum, do not wash, add 1:500 mouse anti-GFP primary antibody dropwise, overnight at 4°C.

Incubate at K 37°C for 45 min, wash with PBS, 5 min×3 times.

L drop 1:100 rabbit anti-mouse secondary antibody, incubate at 37°C for 30 min, wash with PBS, 5 min×3 times

M Add 1:100 horseradish peroxidase complex dropwise, incubate at 37°C for 30 min, wash with PBS, 5 min×3 times.

Develop color with NDAB, grasp the degree of staining under a microscope, and rinse with running water for 10 min.

Counterstain with O hematoxylin for 2 minutes, differentiate with 1% (v/v) hydrochloric acid alcohol, and rinse with running water for 10 minutes.

P Dehydrated, transparent, sealed with neutral gum, observed under a microscope.

9.2 Detection of cell-BCP scaffold composites

1) The composite was implanted for 6w, and the experimental animals were killed by overdose anesthesia, and the implanted GFP adipose stem cell-BCP scaffold material composite was taken out, and decalcified with 12% EDTA in a 37°C water bath for 2w. Paraffin sections with a thickness of 5 μm were made and routinely stained with HE and Masson.

2) Make paraffin sections with a thickness of 5 μm, and perform immunohistochemical detection of anti-GFP.

The results showed that after 6 weeks of implantation in nude mice, the new organisms taken out were yellowish-white, and their volume was significantly smaller than that of implantation. After HE and Oil Red O staining, it was confirmed that the neoplasm contained adipose tissue. The amount of adipocytes in the lipotropic induced group was significantly more than that in the uninduced group. Newly formed adipocytes were positive for anti-GFP immunohistochemistry, indicating transformation from engrafted GFP adipose-derived stem cells.

The GFP adipose stem cell-BCP scaffold material composite was implanted into the femoral defect area of C57 mice for 6 weeks, and the new organisms taken out were stained by HE and Masson, and new bone formation was confirmed. Anti-GFP immunohistochemical detection of tissue sections showed that some of the osteoblasts were positive, while the other osteoblasts were negative, indicating that the implanted GFP adipose stem cells were partially converted into osteoblasts and participated in the formation of new bone.

In the present invention, it has been proved that the obtained ASCs have self-renewal ability and multi-directional differentiation potential from the aspects of morphology, immunocytochemistry, mRNA and protein levels, etc., and the clone formation ability is strong, and it has been confirmed that it has the ability of adipogenic/osteogenic It can be transformed into adipocytes/osteoblasts under the microenvironment. This method establishes a new method for obtaining high-purity ASCs efficiently and quickly, and provides a new way for obtaining a large number of stem seed cells in vitro and applying them to tissue regeneration.

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Claims (10)

1. A method for isolating and culturing adipose stem cells, comprising isolating and obtaining SVF cells from adipose tissue, and cultivating SVF cells, characterized in that it also includes the following steps: removing Lin ⁺ cells in SVF cells by immunomagnetic bead sorting to obtain ^Lin- Cell population; CD271 ⁺ Sca-1 ⁺ cells were enriched from the obtained Lin ^- cell population by flow cytometry to obtain adipose stem cells; the obtained adipose stem cells were cultured with medium containing LIF and FGF2. 2. The method for isolating and culturing adipose-derived stem cells according to claim 1, wherein the markers used in the immunomagnetic bead sorting method are CD5, CD45R, CD11b, Anti-Gr-1 and Ter-119. 3. The method for isolating and culturing adipose stem cells according to claim 1, wherein the markers used in the flow cytometry method are Lin, CD271 and Sca-1. 4. The method for isolating and culturing adipose stem cells according to claim 1, characterized in that: the medium used for culturing the obtained adipose stem cells contains 10% by volume of fetal bovine serum, 10 ³ -10 ⁵ U/mL LIF, 10 -100 ng/mL FGF2 in α-MEM medium. 5. The method for isolating and culturing adipose stem cells according to claim 4, characterized in that: the medium used for culturing the obtained adipose stem cells contains 10% by volume of fetal bovine serum, 10 ³ U/mL LIF, 20 ng/mL α-MEM medium for FGF2. 6. The method for isolating and culturing adipose stem cells according to claim 1, further comprising subculturing the obtained adipose stem cells. 7. The method for isolating and culturing adipose stem cells according to claim 6, characterized in that: said subculture is adherent culture. 8. The method for isolating and culturing adipose stem cells according to claim 7, characterized in that: subculture is carried out when the growth density of the obtained adipose stem cells at the bottom of the culture dish reaches 70-90%. 9. The application of the adipose stem cells isolated and cultured according to any one of the adipose stem cell separation and culture methods of claims 1-8, is characterized in that it is applied to the production of purified or cloned stem cells, transgenic stem cell lines or for the production of bone cells. 10. the application of adipose stem cell according to claim 9 is characterized in that it is applied to the method for producing adipogenic/osteoblast: (1) ASCs were isolated and obtained by combining fluorescence-activated flow cytometry and magnetic-activated cell sorting; (2) ASCs were respectively compounded with fibrin and BCP scaffold materials, and inoculated on the back of nude mice or the femoral defect of mice.

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