Method for isolated culture and identification of porcine muscle-derived mesenchymal stem cells and fat progenitor cells

Abstract

The invention discloses a method for separating, culturing and identifying porcine muscle-derived mesenchymal stem cells and fat progenitor cells. The method mainly comprises the following steps: dissociating pig muscle tissues through a tissue processor, obtaining mesenchymal stem cells and adipose progenitor cells by utilizing a flow cytometry sorting technology, and completing in-vitro culture and identification of the cells. According to the technical scheme provided by the invention, the technical problems of primary culture, separation and identification of the mesenchymal stem cells and the adipose progenitor cells of the porcine muscle tissue are effectively solved, and a reliable method is provided for further application of a flow cytometry sorting technology and a myogenic mesenchymal stem cell and adipose progenitor cell primary model in animal nutrition research.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a method for isolating, culturing, and identifying porcine myogenic mesenchymal stem cells and adipose-derived progenitor cells. Background Technology

[0002] Existing methods for isolating mesenchymal stem cells (MSCs) mainly focus on tissues such as bone marrow, adipose tissue, umbilical cord, and placenta. In contrast, myogenic MSCs and adipocyte progenitor cells account for a relatively low proportion in skeletal muscle tissue, and related research is limited. Currently, there are many methods for isolating and culturing MSCs and adipocyte progenitor cells, which can be broadly classified into four types based on their technical characteristics: density gradient centrifugation, adherent culture screening, immunomagnetic bead sorting, and flow cytometry sorting. Density gradient centrifugation typically involves slowly adding cell suspension to the upper layer of a separation medium such as Percoll, relying on density differences to achieve cell separation. Adherent culture screening relies on differences in cell adhesion ability and time for preliminary screening. Immunomagnetic bead sorting and flow cytometry sorting achieve separation by recognizing specific antigens on the cell surface, resulting in high cell purity, but significantly affecting cell viability, limiting yield, and requiring specialized equipment; therefore, their application in pig-related research is not yet widespread. Among these methods, adherent culture screening is commonly used. It leverages the fact that mesenchymal stem cells and adipose-derived progenitor cells adhere more readily to the culture medium than mature muscle fiber debris, lymphocytes, erythrocytes, and other impurities, effectively removing impurities and enriching the target population. However, the purity of the resulting cells is limited, and subsequent experiments are easily affected by contaminating cells. Flow cytometry sorting separates target cells based on differences in cell size or surface markers. Existing technologies have combined adherent culture with flow cytometry sorting for isolating muscle stem cells; however, a systematic method for isolating and culturing porcine myogenic mesenchymal stem cells and adipose-derived progenitor cells has not yet been established.

[0003] Pigs and humans share many anatomical and physiological similarities, which provides them with significant advantages as a model organism for human medical research. This is crucial for elucidating disease mechanisms, identifying drug targets, and evaluating drug efficacy. However, as a non-model organism, pigs face challenges in cell experiments, including a lack of species-specific antibodies, species-specific reagents, and insufficient control data. Current research often relies on antibodies from humans or mice, but due to species differences, some antibodies cannot effectively bind to porcine antigens. Therefore, it is necessary to design and validate multiple control experiments to confirm the effectiveness of antibody use. Summary of the Invention

[0004] This invention aims to overcome the shortcomings of the prior art and provide a method for isolating, culturing and identifying porcine myogenic mesenchymal stem cells and adipose-derived progenitor cells.

[0005] To achieve the above objectives, the technical solution provided by this invention is as follows: The method for isolating, culturing, and identifying porcine mesenchymal stem cells and adipocyte progenitor cells includes the following steps: (1) Using porcine muscle tissue dissociation solution and a tissue processor, porcine muscle tissue was dissociated to obtain P0 generation primary muscle cells containing a mixture of mesenchymal stem cells and adipocyte progenitor cells; (2) Based on the specific antibody combination and using flow cytometry, the P0 generation primary muscle cells obtained in step (1) are sorted and purified to obtain sorted and purified mesenchymal stem cells and adipose progenitor cells; the specific antibody combination is CD45, CD31, CD29, CD140a, CD90, CD105 and CD73; (3) Identification of the sorted and purified mesenchymal stem cells and adipose progenitor cells after culturing.

[0006] Preferably, the porcine muscle tissue dissociation solution in step (1) is prepared by adding type I collagenase and anhydrous calcium chloride to DMEM F12 medium; the concentration of type I collagenase in DMEM F12 medium is 2.8±0.5 mg / mL, and the concentration of anhydrous calcium chloride in DMEM F12 medium is 5±0.5 mM; the dissociation program conditions during the processing by the automated tissue processor are: temperature 37℃, time 50-70 min, and rotation speed 1800-2200 rpm.

[0007] More preferably, the concentration of type I collagenase in DMEM F12 medium is 2.8 mg / mL, and the concentration of anhydrous calcium chloride in DMEM F12 medium is 5 mM; the dissociation program conditions for the automated tissue processor are: temperature 37°C, time 60 min, and rotation speed 2000 rpm.

[0008] Preferably, the specific antibody combination in step (2) includes an antibody combination targeting mesenchymal stem cells and an antibody combination targeting adipose progenitor cells; the antibody combination for mesenchymal stem cells is CD45, CD31, CD90, CD105 and CD73, and the antibody combination for adipose progenitor cells is CD45, CD31, CD29 and CD140a.

[0009] More preferably, the target mesenchymal stem cell phenotype of the antibody combination for the mesenchymal stem cells is CD45. - CD31 - CD90 + CD105 + CD73 + The target adipocyte phenotype of the antibody combination for the adipocyte progenitor cells is CD45. - CD31 - CD29 + CD140a+ .

[0010] Preferably, the culture in step (3) includes adipogenic differentiation culture and osteogenic differentiation culture; the culture medium for adipogenic differentiation culture includes complete adipocyte progenitor cell culture medium, adipogenic differentiation culture medium, and insulin-inducing culture medium; the culture medium for osteogenic differentiation culture includes complete mesenchymal stem cell culture medium and osteogenic differentiation culture medium; the adipogenic differentiation culture involves seeding mesenchymal stem cells and adipocyte progenitor cells into complete adipocyte progenitor cell culture medium, then culturing them in adipogenic differentiation culture medium, then culturing them in insulin-inducing culture medium, and finally maintaining them in complete adipocyte progenitor cell culture medium before identification; the osteogenic differentiation culture involves seeding mesenchymal stem cells into complete mesenchymal stem cell culture medium, then culturing them in osteogenic differentiation culture medium before identification.

[0011] More preferably, the identification of adipocyte differentiation after adipogenic induction culture is performed by Oil Red O staining to identify the adipocyte differentiation results, and positive staining indicates the presence of fat droplets in the cells; the identification of osteogenic differentiation after osteogenic induction culture is performed by Alizarin Red staining to identify the osteogenic differentiation results, and positive staining indicates the presence of calcium nodules in the extracellular matrix.

[0012] More preferably, the complete culture medium for adipocyte progenitor cells is DMEM F12 medium supplemented with 20% fetal bovine serum, 1% Glutamax, 1% NEAA, 1% sodium pyruvate, 2 ng / mL bFGF, and 1% penicillin-streptomycin antibiotics; the adipogenic differentiation induction medium is DMEM F12 medium supplemented with 10% fetal bovine serum, 1 μg / μL insulin, 2.5 mM dexamethasone, and 0.5 M IBMX, or MEMα medium supplemented with 10% fetal bovine serum, 1 μg / μL insulin, 2.5 mM dexamethasone, and 0.5 M IBMX; the insulin induction medium is DMEM supplemented with 10% fetal bovine serum and 1 μg / μL insulin. F12 medium or MEMα medium supplemented with 10% fetal bovine serum and 1 μg / μL insulin; the complete mesenchymal stem cell medium is MEMα medium supplemented with 20% fetal bovine serum, 1% Glutamax, and 1% penicillin-streptomycin antibiotics; the osteogenic induction differentiation medium is MEMα medium supplemented with 20% fetal bovine serum, 10 μL / mL β-glycerophosphate sodium, 2 μL / mL ascorbic acid, and 0.1 μL / mL dexamethasone.

[0013] The present invention will be further described below: This invention combines differential adhesion and flow cytometry cell sorting. Differential adhesion is used for initial cell separation, followed by further purification using flow cytometry. Compared to separating tissue suspensions, the flow cytometry sorting of adherent cells using this invention is more efficient. Traditional physical cell fragmentation methods are time-consuming, difficult to standardize, and struggle to guarantee cell viability. This invention employs a fully automated tissue processor to dissociate tissue, enabling faster, gentler, standardized, and automated preparation of single-cell suspensions, with innovation particularly in the use of specific antibody combinations. The biological significance and sorting value of the antibodies described in this invention are as follows: 1. FVS780 Live cell-specific fluorescent dyes distinguish live cells from dead cells by assessing cell membrane integrity, ensuring the viability and purity of cells in subsequent sorting. 2.CD45 CD45 (leukocyte common antigen, LCA) is a universal marker of hematopoietic cells, expressed in all white blood cells (lymphocytes, monocytes, granulocytes, etc.). It is not expressed in non-hematopoietic cells (such as mesenchymal stem cells, adipocyte progenitor cells, and endothelial cells). Since mesenchymal stem cells and adipocyte progenitor cells originate from the mesenchyme (not the hematopoietic system), CD45 is not used as a marker. - Excluding hematopoietic cell contamination is a fundamental step in sorting mesenchymal-derived cells; 3.CD31 CD31 (PECAM-1, platelet endothelial cell adhesion molecule) is a specific marker of endothelial cells, expressed in vascular endothelial cells and platelets, but not in mesenchymal stem cells and adipocyte progenitor cells. Mesenchymal tissues (such as bone marrow, fat, and muscle) contain a large number of endothelial cells, and CD31 is used to identify these markers. - Exclude endothelial cells and further purify mesenchymal-derived cells; 4.CD29 CD29 (integrin β1) is a transmembrane glycoprotein that forms heterodimers with integrin α subunits (such as α1 and α2) to mediate cell adhesion to the extracellular matrix (ECM) (such as fibronectin and collagen). Mesenchymal stem cells and adipocyte progenitor cells highly express CD29. CD29⁺ is used to enrich mesenchymal-derived cells and reduce interference from non-mesenchymal cells (such as epithelial cells). 5.CD104a CD140a (platelet-derived growth factor receptor α, PDGFRα) is a tyrosine kinase receptor that is highly expressed in adipocytes, but not expressed or poorly expressed in mesenchymal stem cells. CD140a is a key biomarker distinguishing adipocytes from mesenchymal stem cells; adipocytes express CD140a. + Mesenchymal stem cells are CD140a - Therefore, CD140a is used. + Sorting adipocyte progenitor cells; 6.CD90 CD90 (Thy1) is a glycosylphosphatidylinositol (GPI) anchoring protein that is expressed in fibroblasts, mesenchymal stem cells, and adipocyte progenitor cells, but not in endothelial cells or epithelial cells. CD90 is a common positive marker for mesenchymal-derived cells. Further enrichment of mesenchymal-derived cells and exclusion of cells that do not express CD90, such as endothelial cells, are necessary. 7.CD105 CD105 (endoglin) is a helper protein of the transforming growth factor β (TGF-β) receptor, which is highly expressed in mesenchymal stem cells, but not expressed or poorly expressed in adipocyte progenitor cells; CD105 is a specific marker of mesenchymal stem cells. 8.CD73 CD73 (5'-nucleotidase) is an exonuclease that catalyzes the conversion of AMP to adenosine. It is highly expressed in mesenchymal stem cells, but not expressed or expressed at low levels in adipocyte progenitor cells. CD73 is a classic specific marker of mesenchymal stem cells, and together with the other two core markers of mesenchymal stem cells, CD90 and CD105, it constitutes a "triple positive" result. The sorting effect of adipocyte progenitor cells was calculated as follows: percentage of target cells (78.8%) × percentage of de-adhesive cells (98.3%) × percentage of viable cells (50.9%) × CD45 - / CD31 - Cell percentage (97.2%) × CD29 + Cell percentage (97.1%) × CD140a + The cell percentage was 1.26%, and more than 4,000 cells were sorted.

[0014] The sorting effect of mesenchymal stem cells is calculated as follows: percentage of target cells (79.9%) × percentage of de-adhesive cells (94.5%) × percentage of viable cells (76.7%) × CD45 - / CD31 - Cell percentage (96.4%) × CD90 + / CD105 + Cell percentage (98.1%) × CD73 + Cell percentage (1.33%), a total of more than 10,000 cells were sorted; Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Improved cell separation efficiency and purity: By optimizing the tissue dissociation system and employing a fully automated tissue processor, rapid, gentle, and standardized preparation of single-cell suspensions from porcine muscle tissue was achieved, significantly improving cell viability and yield. Combined with flow cytometry sorting technology and a validated antibody labeling strategy, high-purity myogenic mesenchymal stem cells and adipocyte progenitor cells can be accurately sorted, effectively avoiding interference from impurity cells in traditional methods.

[0015] 2. This invention addresses a key technical bottleneck in porcine cell research: Due to the lack of specific reagents and antibody resources for pigs as non-model organisms, existing methods struggle to achieve precise isolation of their myogenic stem cells. This invention establishes a reliable cell sorting and identification system by rationally selecting and validating antibody combinations suitable for porcine cells, filling a technological gap in this field.

[0016] 3. Advances in animal nutrition research and medical models: The successful establishment of primary models of porcine myogenic mesenchymal stem cells and adipocyte progenitor cells provides an important tool for research on animal nutritional metabolism, muscle development, and fat deposition mechanisms. Furthermore, the high degree of physiological similarity between pigs and humans makes this method promising for applications in researching related human diseases and drug screening.

[0017] This invention provides a multi-parameter flow cytometry antibody combination for the precise sorting of porcine mesenchymal stem cells and adipocyte progenitor cells. It can simultaneously but independently sort porcine mesenchymal stem cells (CD90) + CD105 + CD73 + ) and adipocyte progenitor cells (CD29) + CD140a + This invention clarifies the different surface marker profiles of the two cell types. It provides a complete "isolation-sorting-identification" technique for porcine mesenchymal stem cells and adipose-derived progenitor cells, achieving for the first time reproducible, high-purity acquisition and functional verification of muscle tissue mesenchymal stem cells and adipose-derived progenitor cells in a porcine model.

[0018] In summary, this invention, based on the method for isolating, culturing, and identifying porcine myogenic mesenchymal stem cells and adipose-derived progenitor cells, solves the technical challenges of primary culture, isolation, and identification of porcine muscle tissue mesenchymal stem cells and adipose-derived progenitor cells, and promotes the application of flow cytometry cell sorting technology and primary cell models of myogenic mesenchymal stem cells and adipose-derived progenitor cells in animal nutrition research. Attached Figure Description

[0019] Figure 1 A schematic diagram of the antibodies and channels used in flow cytometry sorting; Figure 2 Morphological image of primary muscle cells at passage PO, adherent for 72 hours (40×10 magnification). Figure 3 Image showing the results of flow cytometry sorting of adipocyte progenitor cells from primary muscle cells that adhered for 72 hours (P0 generation). Figure 4 Image showing the results of flow cytometry sorting of mesenchymal stem cells from primary muscle cells that have adhered for 72 hours (P0 passage). Figure 5 Morphological image of adipocyte progenitor cells sorted by flow cytometry (40×10 magnification). Figure 6 Morphological image of adipogenic progenitor cells on day 8 of adipogenic differentiation induced by flow cytometry (40×20 magnification). Figure 7 Oil Red O staining image of adipogenic progenitor cells on day 8 of flow cytometry sorting for adipogenic differentiation (40×20 magnification). Figure 8 Morphological images of mesenchymal stem cells sorted by flow cytometry (40×10 magnification). Figure 9 Mesenchymal stem cell morphology on day 8 after adipogenic induction differentiation by flow cytometry (40×20 magnification). Figure 10 Oil Red O staining image of mesenchymal stem cells on day 8 of adipogenic differentiation sorted by flow cytometry (40×20 magnification). Figure 11 Mesenchymal stem cell morphology on day 8 of osteogenic induction differentiation by flow cytometry (40×20 magnification). Figure 12 Alizarin red staining image of mesenchymal stem cells on day 8 of osteogenic induction differentiation by flow cytometry (40×20 magnification). Detailed Implementation

[0020] The following is in conjunction with the appendix Figures 1 to 12 The present invention will be further illustrated by the examples.

[0021] The method for isolating, culturing, and identifying porcine mesenchymal stem cells and adipose-derived progenitor cells includes the following three parts: A, B, and C. A. A mixture of mesenchymal stem cells and adipocyte progenitor cells was obtained by dissociating porcine muscle tissue using a tissue processor: (1) Prepare porcine muscle tissue dissociation solution, which is prepared by adding type I collagenase and anhydrous calcium chloride to DMEM F12 medium; the concentration of type I collagenase in DMEM F12 medium is 2.8 mg / mL, and the concentration of anhydrous calcium chloride in DMEM F12 medium is 5 ± 0.5 mM; preheat the porcine muscle tissue dissociation solution to 15-25℃ before use; rinse the sterile enzyme-free centrifuge tubes with DMEM F12 medium and then discard all liquid; tighten the caps of the centrifuge tubes and place them on ice for later use; the sterile enzyme-free centrifuge tubes are 15 mL and 50 mL in size; prepare PBS and 75 vol% ethanol containing 2 vol% penicillin-streptomycin double antibody solution, wherein the penicillin content in the penicillin-streptomycin double antibody solution is 10000 U / mL and the streptomycin content is 10 mg / mL; (2) The piglets were euthanized by bleeding from the neck, washed with running water, and the pigs’ bodies, eyes, ears, noses and anus were cleaned 3 times with benzalkonium chloride disinfectant, and then cleaned 3 times with 75 vol% ethanol. The piglets were then transferred to a clean bench, the longissimus dorsi muscle was separated and placed in a 50 mL centrifuge tube containing 20 mL of PBS containing 2 vol% penicillin-streptomycin antibiotic solution. The tube was then cleaned 3 times with 75 vol% ethanol and 3 times with PBS containing 2 vol% penicillin-streptomycin antibiotic solution. Finally, the tube was placed in PBS containing 2 vol% penicillin-streptomycin antibiotic solution. (3) Use the fully automated tissue processor to dissociate the tissue; divide the muscle tissue into 8 portions and mince them into 2-4mm pieces in tube C. Add 5mL of type I collagenase to each tube. Use the tissue processor's built-in program for dissociation. The program is set to 37℃, 60min, 2000rpm; (4) The liquid obtained from the dissociation in step (3) is filtered through a 100μm cell sieve into a 50mL sterile, enzyme-free centrifuge tube that has been treated in step (1) and placed on ice to obtain the filtrate after the first step of filtration. (5) Filter the filtrate obtained in step (4) again through a 70μm bidirectional cell sieve, and dispense the filtrate into two 15mL sterile enzyme-free centrifuge tubes that have been treated in step (1). Centrifuge at 2000rpm and 25℃ for 12min and remove the supernatant. (6) After resuspending the precipitate in PBS with 2 vol% penicillin-streptomycin solution, centrifuge at 1500 rpm and 25°C for 10 min to remove the supernatant; and repeat the centrifugation operation in step (6) once. (7) Resuspend the cells in complete adipose progenitor cell culture medium, plate them in T25 culture flasks, and culture for 48 hours. Discard the supernatant and change the medium. These are P0 generation primary muscle cells. The morphology of P0 generation primary muscle cells is shown in the figure below. Figure 2 As shown.

[0022] B. Flow cytometry separation and purification of mesenchymal stem cells and adipocyte progenitor cells: (8) P0 generation primary muscle cells were digested with trypsin, centrifuged at 1000 rpm and 4℃ for 5 min, the supernatant was removed, and the cells were resuspended in FACS buffer and diluted for counting. (9) Prepare blank control, single-stain control and mixed-stain tubes. The appropriate cell concentration for the single-stain control tube is 5×10^5 cells / mL; add 1μL Fixable Viability Stain 780 to the single-stain control and mixed-stain tubes in 1mL LFACS buffer system, place on ice, incubate in the dark for 10min, centrifuge at 1000rpm and 4℃ for 10min, remove the supernatant; resuspend in 800μL LFACS buffer, and repeat the centrifugation operation in step (9) once; (10) Prepare single-staining tubes for CD45, CD31, CD29, CD140a, CD90, CD105, and CD73 respectively; add 1 TEST antibody to each 5×10^5 cells / mL in a 100μL LFACS buffer system for single-staining tubes. The specific amount of each antibody used in single-staining tubes is shown in Table 1; prepare mixed-staining tubes for mesenchymal stem cells (CD45, CD31, CD90, CD105, CD73) and mixed-staining tubes for adipocyte progenitor cells (CD45, CD31, CD29, CD140a); the amount of antibody in the mixed-staining tubes can be increased proportionally according to the number of target cells; place on ice and incubate in the dark for 30 min, add 800μL LFACS buffer to resuspend, centrifuge at 1000rpm and 4℃ for 10 min, and remove the supernatant; add 800μL LFACS buffer to resuspend, and repeat the centrifugation operation in step (10) once; Table 1

[0023] (11) Based on the cell concentration, add 200 μL LFACS buffer to 5 × 10^5 cells / mL, place on ice, and store in the dark until ready for flow cytometry to separate mesenchymal stem cells and adipocyte progenitor cells. The flow cytometry results of mesenchymal stem cells and adipocyte progenitor cells are shown in the figure below. Figure 3 and Figure 4 As shown in the figure, the morphological images of sorted mesenchymal stem cells and adipocyte progenitor cells after adherent culture are as follows. Figure 5 and Figure 8 As shown.

[0024] C. Culture and identification of mesenchymal stem cells and adipose-derived progenitor cells: (12) Prepare complete culture medium for adipocyte progenitor cells. The complete culture medium for adipocyte progenitor cells is prepared by adding 20% ​​fetal bovine serum, 1% Glutamax, 1% NEAA, 1% sodium pyruvate, 2 ng / mL bFGF, and 1% penicillin-streptomycin antibiotics to DMEM F12 medium. (13) Prepare a complete culture medium for mesenchymal stem cells, wherein the complete culture medium for mesenchymal stem cells is prepared by adding 20% ​​fetal bovine serum, 1% Glutamax and 1% penicillin-streptomycin antibiotics to MEMα culture medium; (14) Prepare an adipogenic differentiation medium, wherein the adipogenic differentiation medium is prepared by adding 10% fetal bovine serum, 1 μg / μL insulin, 2.5 mM dexamethasone and 0.5 M IBMX to DMEM F12 / MEMα medium; (15) Insulin induction medium, wherein the insulin induction medium is prepared by adding 10% fetal bovine serum and 1 μg / μL insulin to DMEM F12 / MEMα medium; (16) After cell seeding, culture for 24 hours until the cells reach 100% confluence. Change the medium and continue culturing for 2 days to induce contact inhibition. Remove the old culture medium and add adipogenic differentiation-inducing medium for 2 days. Remove the old culture medium and add insulin-inducing medium for 2 days of induction. At this time, cell adhesion is poor and cells are prone to detachment, so handle them gently. Remove the old culture medium and add complete medium for maintenance culture. Change the medium every 2 days. After about 6 days of induction, round and bright lipid droplets appear. By day 8, large, round, spherical lipid droplets appear in adipocyte progenitor cells and mesenchyme. Figure 6 and Figure 9 As shown; (17) Identify fat droplets using Oil Red O staining; remove the old culture medium from step (15); wash cells three times with PBS; add 4% paraformaldehyde; fix at room temperature for 1 hour; remove the fixative; add Oil Red O staining solution and stain at room temperature for 10-15 minutes; remove the staining agent; wash twice with sterile double-distilled water to remove unstained dye; observe under a microscope and take pictures. Fat droplets turn red after being stained with Oil Red O. Oil Red O staining images of adipogenic progenitor cells and mesenchymal stem cells on day 8 of adipogenic differentiation are shown below. Figure 7 and Figure 10 As shown; (18) Prepare osteogenic differentiation induction medium, wherein the osteogenic differentiation induction medium is prepared by adding 20% ​​fetal bovine serum, 10 μL / mL sodium β-glycerophosphate, 2 μL / mL ascorbic acid and 0.1 μL / mL dexamethasone to MEMα medium; (19) Press 2-3×10 4 cells / cm 2Cells were seeded at a density suitable for osteogenic differentiation in six-well plates, with 2 mL of complete mesenchymal stem cell culture medium added to each well. When cell confluence reached 80-95%, the old culture medium was removed, and 2 mL of complete human bone marrow mesenchymal stem cell osteogenic differentiation induction culture medium was added to each well. The culture medium was replaced with preheated fresh osteogenic differentiation induction medium every 48-72 hours. After 2-4 weeks of induction, cell morphology and growth were assessed. The cell morphology on day 8 of osteogenic differentiation induction is shown in the image below. Figure 11 As shown; (20) Identify calcium nodules using alizarin red staining; remove the old culture medium from step (19); wash cells three times with PBS; add 4% paraformaldehyde; fix at room temperature for 1 hour; remove the fixative; add alizarin red staining solution and stain at room temperature for 5-10 minutes; remove the staining agent; wash cells three times with PBS to remove unstained dye; observe under a microscope and take pictures; on the 8th day of osteogenic induction differentiation of mesenchymal stem cells, calcium nodules appear red, reddish-yellow, or reddish-purple after being stained with alizarin red. Figure 12 As shown.

Claims

1. A method for isolating, culturing, and identifying porcine mesenchymal stem cells and adipocyte progenitor cells, characterized in that, The method includes the following steps: (1) Pig muscle tissue was dissected using a porcine muscle tissue dissociation solution and a tissue processor to obtain P0 generation primary muscle cells containing a mixture of mesenchymal stem cells and adipocyte progenitor cells. (2) Based on the specific antibody combination and using flow cytometry, the P0 generation primary muscle cells obtained in step (1) are sorted and purified to obtain sorted and purified mesenchymal stem cells and adipose progenitor cells; the specific antibody combination is CD45, CD31, CD29, CD140a, CD90, CD105 and CD73; (3) Identification of the sorted and purified mesenchymal stem cells and adipose progenitor cells after culturing.

2. The method for isolating, culturing, and identifying porcine mesenchymal stem cells and adipocyte progenitor cells as described in claim 1, characterized in that, The porcine muscle tissue dissociation solution in step (1) was prepared by adding type I collagenase and anhydrous calcium chloride to DMEM F12 medium; the concentration of type I collagenase in DMEM F12 medium was 2.8±0.5 mg / mL, and the concentration of anhydrous calcium chloride in DMEM F12 medium was 5±0.5 mM; the dissociation program conditions during the processing by the automated tissue processor were: temperature 37℃, time 50-70 min, and rotation speed 1800-2200 rpm.

3. The method for isolating, culturing, and identifying porcine mesenchymal stem cells and adipocyte progenitor cells as described in claim 2, characterized in that... The concentration of type I collagenase in DMEM F12 medium is 2.8 mg / mL, and the concentration of anhydrous calcium chloride in DMEM F12 medium is 5 mM; the dissociation program conditions for the automated tissue processor are: temperature 37℃, time 60 min, and rotation speed 2000 rpm.

4. The method for isolating, culturing, and identifying porcine mesenchymal stem cells and adipocyte progenitor cells as described in claim 1, characterized in that... The specific antibody combination mentioned in step (2) includes an antibody combination targeting mesenchymal stem cells and an antibody combination targeting adipose progenitor cells; the antibody combination for mesenchymal stem cells is CD45, CD31, CD90, CD105 and CD73, and the antibody combination for adipose progenitor cells is CD45, CD31, CD29 and CD140a.

5. The method for isolating, culturing, and identifying porcine mesenchymal stem cells and adipocyte progenitor cells as described in claim 4, characterized in that... The target mesenchymal stem cell phenotype of the antibody combination for mesenchymal stem cells is CD45⁻CD31⁻CD90⁺CD105⁺CD73⁺; the target adipocyte progenitor cell phenotype of the antibody combination for adipocytes is CD45⁻CD31⁻CD29⁺CD140a⁺.

6. The method for isolating, culturing, and identifying porcine mesenchymal stem cells and adipocyte progenitor cells as described in claim 1, characterized in that, The culture in step (3) includes adipogenic differentiation culture and osteogenic differentiation culture; the culture medium for adipogenic differentiation culture includes complete adipocyte progenitor cell culture medium, adipogenic differentiation culture medium, and insulin-inducing culture medium; the culture medium for osteogenic differentiation culture includes complete mesenchymal stem cell culture medium and osteogenic differentiation culture medium; the adipogenic differentiation culture involves seeding mesenchymal stem cells and adipocyte progenitor cells into complete adipocyte progenitor cell culture medium, then culturing them in adipogenic differentiation culture medium, then culturing them in insulin-inducing culture medium, and finally maintaining them in complete adipocyte progenitor cell culture medium before identification; the osteogenic differentiation culture involves seeding mesenchymal stem cells into complete mesenchymal stem cell culture medium, then culturing them in osteogenic differentiation culture medium before identification.

7. The method for isolating, culturing, and identifying porcine mesenchymal stem cells and adipocyte progenitor cells as described in claim 6, characterized in that... The identification of adipocytes after adipogenic induction differentiation culture was performed by Oil Red O staining, which showed the presence of fat droplets within the cells. The identification of osteogenic induction differentiation culture was performed by Alizarin Red staining, which showed the presence of calcium nodules in the extracellular matrix.

8. The method for isolating, culturing, and identifying porcine mesenchymal stem cells and adipocyte progenitor cells as described in claim 6, characterized in that, The complete culture medium for adipocyte progenitor cells is DMEM F12 medium supplemented with 20% fetal bovine serum, 1% Glutamax, 1% NEAA, 1% sodium pyruvate, 2 ng / mL bFGF, and 1% penicillin-streptomycin antibiotics; the adipogenic differentiation induction medium is DMEM F12 medium supplemented with 10% fetal bovine serum, 1 μg / μL insulin, 2.5 mM dexamethasone, and 0.5 M IBMX, or MEMα medium supplemented with 10% fetal bovine serum, 1 μg / μL insulin, 2.5 mM dexamethasone, and 0.5 M IBMX; the insulin induction medium is DMEM supplemented with 10% fetal bovine serum and 1 μg / μL insulin. F12 medium or MEMα medium supplemented with 10% fetal bovine serum and 1 μg / μL insulin; the complete mesenchymal stem cell medium is MEMα medium supplemented with 20% fetal bovine serum, 1% Glutamax, and 1% penicillin-streptomycin antibiotics; the osteogenic induction differentiation medium is MEMα medium supplemented with 20% fetal bovine serum, 10 μL / mL β-glycerophosphate sodium, 2 μL / mL ascorbic acid, and 0.1 μL / mL dexamethasone.

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