Characterization Of Mouse Bone Marrow Mesenchymal Stem Cells And Their Exosomes Cultured Stably And Efficiently

Objective: In this study,we attempt to establish a stable and efficient culture method of mouse bone marrow Mesenchymal stem cell The exosomes after culture were extracted and characterized by ultra-high speed centrifugation,which is the simplest and most commonly used method This study provided the experimental basis and method for obtaining the effective exosomes of bone marrow Mesenchymal stem cell in mice.Methods: The culture of interblastated stem cells(BM-MSCs)in mice by applying an optimization method of "whole bone marrow,differential walling and assisting with low oxygen environment" is carried out,and stem cells are collected for transmission,and the use of flow cytometers to identify stem cell surface markers(CD117 antigens,CD31 antigens),Integrator family antigen marker molecule CD29,adhesion molecule family antigen label molecule CD44 and Ly-6 antigen family member Sca-1.Then the bone tissue,fat tissue and cartilage tissue induction,sissin red S identification into bone,oil red O staining into fat,Alyssin blue dyeing identified as cartilage condition,in addition,after 5 generations of culture to collect BM-MSCs of the upper liquid,the application of ultra-high-speed centrifugal separation method to extract exosomes,Electric mirror detects the physical structure and morphology of exosomes;Western blot identifies the characteristic positive protein markers of exosomes(HSP70 antigen,Syntenin1 antigen,CD9 antigen,CD63 antigen,and TSG101 antigen),negative protein marker Grp94 antigen);The nanoparticle tracking analyzer analyzes the diameter size and distribution law of BM-MSCs exosome cystic bubbles.Results: Extracted and cultured mice BM-MSCs growth state is good,can obtain a large number of stem cells,to the 3rd generation cells can reach more than 95% purity,and can be stable transmission to more than 15 generations,without changing the activity of stem cells,expression interstitial stem cell markers(CD117 antigen and CD31 antigen negative,CD29 antigen negative,CD29 antigen,CD44 antigen and Sca-1 antigen positive),and can be successfully induced to differentiate into bone,fat and cartilage tissue.The super-high-speed centrifugal isolated extracellular follicles have a typical cup-shaped cystic exosome structure,and Western blot identification shows that they have exosome characteristic protein markers(HSP70,Syntenin1,CD9,CD63 and TSG101 antigen-positive,while Grp94 antigen negative.Nanoparticle tracking analysis shows that BM-MSCs exosome cystic bubbles show a normal distribution,the size of 165 nm x 42 nm,these are in line with the characteristics of exosomes.Conclusion: The application of whole bone marrow culture method resulted in the least loss of stem cells in the initial stage of culture in mice,while the differential walling method in low oxygen(5% O2)environment made the mouse BM-MSCs multiply faster and purity more.In addition,the mice cultured by this method can be cultured by the generation to secrete exosome cystic bubbles,and can be extracted by a simple and efficient method of ultra-high-speed centrifugal separation.This provides a good method for the culture of inter-metastases stem cells in mice and the acquisition of exosomes,and lays the foundation for the further application of BM-MSCs exosomes in mice in basic research,which is worth promoting.