CN-121991889-A - Isolated culture and identification method for swine myogenic mesenchymal stem cells and fat progenitor cells

CN121991889ACN 121991889 ACN121991889 ACN 121991889ACN-121991889-A

Abstract

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

Inventors

LI FENGNA
CHEN SISI
GUO QIUPING
PENG CAN
DUAN YEHUI
Zhong Yinzhao
YIN YULONG

Assignees

中国科学院亚热带农业生态研究所

Dates

Publication Date: 20260508
Application Date: 20260106

Claims (8)

1. A method for isolated culture and identification of porcine mesenchymal stem cells and fat progenitor cells, comprising the steps of: (1) Dissociating the porcine muscle tissue with a porcine muscle tissue dissociation solution and via a tissue processor to obtain P0 generation muscle primary cells comprising a mixture of mesenchymal stem cells and adipose progenitor cells; (2) Sorting and purifying the P0 generation muscle primary cells obtained in the step (1) by utilizing a flow cytometry based on a specific antibody combination to obtain sorted and purified mesenchymal stem cells and fat progenitor cells, wherein the specific antibody combination is CD45, CD31, CD29, CD140a, CD90, CD105 and CD73; (3) And culturing the separated and purified mesenchymal stem cells and fat progenitor cells, and then identifying.
2. The method for separating, culturing and identifying the mesenchymal stem cells and the fat progenitor cells of the pig according to claim 1, wherein the pig muscle tissue dissociation liquid in the step (1) is prepared by adding type I collagenase and anhydrous calcium chloride into a DMEM F12 culture medium, the concentration of the type I collagenase in the DMEM F12 culture medium is 2.8+/-0.5 mg/mL, the concentration of the anhydrous calcium chloride in the DMEM F12 culture medium is 5+/-0.5 mM, and the dissociation program condition of the automatic tissue processor during treatment is that the temperature is 37 ℃, the time is 50-70min, and the rotating speed is 1800-2200rpm.
3. The method for isolated culture and identification of porcine mesenchymal stem cells and fat progenitor cells according to claim 2, wherein the concentration of the type I collagenase in the DMEM F12 medium is 2.8mg/mL, the concentration of the anhydrous calcium chloride in the DMEM F12 medium is 5mM, and the dissociation procedure conditions of the automated tissue processor are that the temperature is 37 ℃, the time is 60min and the rotation speed is 2000rpm.
4. The method of claim 1, wherein the specific antibody combination in step (2) comprises a mesenchymal stem cell-targeted antibody combination and an adipose progenitor cell-targeted antibody combination, wherein the mesenchymal stem cell-targeted antibody combination is CD45, CD31, CD90, CD105 and CD73, and the adipose progenitor cell-targeted antibody combination is CD45, CD31, CD29 and CD140a.
5. The method of claim 4, wherein the target mesenchymal stem cell phenotype of the antibody combination of mesenchymal stem cells is CD45-CD31-CD90+CD105+CD73+, and the target adipose progenitor cell phenotype of the antibody combination of adipose progenitor cells is CD45-CD31-CD29+CD140a+.
6. The method for isolated culture and identification of the mesenchymal stem cells and the fat progenitor cells of the pig according to claim 1, wherein the culture in the step (3) comprises fat-forming induced differentiation culture and bone-forming induced differentiation culture, wherein the culture medium for the fat-forming induced differentiation culture comprises fat progenitor cell complete culture medium, fat-forming induced differentiation culture medium and insulin-forming induced differentiation culture medium, the culture medium for the bone-forming induced differentiation culture comprises mesenchymal stem cell complete culture medium and bone-forming induced differentiation culture medium, the fat-forming induced differentiation culture is that the mesenchymal stem cells and the fat progenitor cells are inoculated to the fat progenitor cell complete culture medium and then put into the fat-forming induced differentiation culture medium for culture, then put into the insulin-forming induced differentiation culture medium for culture, finally put into the fat progenitor cell complete culture medium for maintenance culture and then carry out identification, and the bone-forming induced differentiation culture is that the mesenchymal stem cells are inoculated to the mesenchymal stem cell complete culture medium for culture, then put into the bone-forming induced differentiation culture medium for induction culture and then carry out identification.
7. The method for isolated culture and identification of mesenchymal stem cells and fat progenitor cells of claim 6, wherein the identification after the adipogenic differentiation culture is characterized in that the adipogenic differentiation result is identified by an oil red O staining method, positive staining indicates that fat drops exist in cells, and the identification after the osteogenic differentiation culture is characterized in that the osteogenic differentiation result is identified by an alizarin red staining method, and positive staining indicates that calcium nodules exist in extracellular matrix.
8. The method for isolated culture and identification of porcine mesenchymal stem cells and fat progenitor cells according to claim 6, wherein the fat progenitor cell complete medium is DMEM F12 medium supplemented with 20% fetal bovine serum, 1% glutamax, 1% neaa, 1% sodium pyruvate, 2ng/mL bFGF, 1% penicillin-streptomycin diabody; the adipogenic differentiation medium is DMEM F12 medium added with 10% fetal bovine serum, 1 mu g/mu L of insulin, 2.5mM dexamethasone, 0.5M IBMX or MEM alpha medium added with 10% fetal bovine serum, 1 mu g/mu L of insulin, 2.5mM dexamethasone, 0.5M IBMX, the insulin induction medium is DMEM F12 medium added with 10% fetal bovine serum, 1 mu g/mu L of insulin or MEM alpha medium added with 10% fetal bovine serum, 1 mu g/mu L of insulin, the mesenchymal stem cell complete medium is MEM alpha medium added with 20% fetal bovine serum, 1% Glutamax, 1% penicillin-streptomycin double-resistant, and the osteogenic differentiation medium is MEM alpha medium added with 20% fetal bovine serum, 10 mu L/mL of beta-sodium glycerophosphate, 2 mu L/mL of ascorbic acid, 0.1 mu L/mL of dexamethasone.

Description

Isolated culture and identification method for swine myogenic mesenchymal stem cells and fat progenitor cells Technical Field The invention belongs to the technical field of cell culture, and particularly relates to a method for separating, culturing and identifying pig myogenic mesenchymal stem cells and fat progenitor cells. Background The existing tissue mesenchymal stem cell separation method mainly focuses on tissue sources such as bone marrow, fat, umbilical cord, placenta and the like. In contrast, myogenic mesenchymal stem cells and adipose progenitor cells occupy a lower proportion in skeletal muscle tissue, and related studies are limited. At present, the methods for separating and culturing tissue mesenchymal stem cells and fat progenitor cells are more and can be roughly divided into four methods, namely a density gradient centrifugation method, an adherence culture screening method, an immunomagnetic bead sorting method and a flow cell sorting method according to the technical characteristics of the tissue mesenchymal stem cells and the fat progenitor cells. Density gradient centrifugation typically involves slowly adding a cell suspension to the top of a Percoll or other separation medium, depending on the density differences, to effect cell separation. The culture and screening method of adherence carries out preliminary screening according to the cell adherence capacity and time difference. The immunomagnetic bead method and the flow cell sorting method realize sorting by recognizing cell surface specific antigens, and the obtained cells have higher purity, but have larger influence on cell activity, have limited yield and require special equipment, so that the method has not been widely applied to pig related researches. The method is more commonly used, and the characteristics that mesenchymal stem cells and fat progenitor cells are easier to adhere to impurities such as mature muscle fiber fragments, lymphocytes, erythrocytes and the like are utilized, so that the impurities can be effectively removed, target populations are enriched, but the purity of the obtained cells is limited, and subsequent experiments are easy to be interfered by mixed cells. The flow cell sorting rule realizes the sorting of target cells according to the difference of cell sizes or surface markers. In the prior art, the research of combining the adherent culture with the flow cell sorting is carried out for separating the muscle stem cells, however, a separation culture method for the swine myogenic mesenchymal stem cells and the fat progenitor cells is not established in a system at present. The pig and human have many similarities in anatomy and physiology, which brings great advantages for the pig as a human medical research model, and has important significance for revealing the disease occurrence mechanism, searching the drug target, carrying out drug effect evaluation and the like. However, pigs as non-model organisms lack specific antibodies of the corresponding species, species-specific reagents, and have insufficient control data in cell experiments. Human or mouse antibodies are often used for reference in the current research, but due to the difference of species, part of antibodies cannot be effectively combined with swine antigens, so that various control experiments are required to be designed and verified to confirm the effectiveness of the antibodies. Disclosure of Invention The invention aims to overcome the defects of the prior art and provides a method for separating, culturing and identifying pig myogenic mesenchymal stem cells and fat progenitor cells. In order to achieve the above purpose, the technical scheme provided by the invention is as follows: The method for separating, culturing and identifying the mesenchymal stem cells and the fat progenitor cells of the pigs comprises the following steps: (1) Dissociating the pig muscle tissue by adopting a pig muscle tissue dissociation liquid and a tissue processor to obtain P0 generation muscle primary cells containing a mixture of mesenchymal stem cells and fat progenitor cells; (2) Sorting and purifying the P0 generation muscle primary cells obtained in the step (1) by utilizing a flow cytometry based on a specific antibody combination to obtain sorted and purified mesenchymal stem cells and fat progenitor cells, wherein the specific antibody combination is CD45, CD31, CD29, CD140a, CD90, CD105 and CD73; (3) And culturing the separated and purified mesenchymal stem cells and fat progenitor cells, and then identifying. Preferably, the pig muscle tissue dissociation solution in the step (1) is prepared by adding type I collagenase and anhydrous calcium chloride into a DMEM F12 culture medium, wherein the concentration of the type I collagenase in the DMEM F12 culture medium is 2.8+/-0.5 mg/mL, the concentration of the anhydrous calcium chloride in the DMEM F12 culture medium is 5+/-0.5 mM, and the dissociation program conditions during the trea