Optimization Of Conditions Of Porous Bovine Acellular Dermal Matrixn Microspheres And Their Application In Alleviating The Senescence Of Canine Bone Marrow Mesenchymal Stem Cells

Three-dimensional(3D)cell culture of cells can simulate the natural microenvironment of cells in tissues and organs,and simulate the complex cell-cell and cell-extracellular matrix(ECM)interactions in vivo,which is more conducive to cell growth and proliferation.Three-dimensional cultures based on porous microspheres can promote the entry and exit of nutrients,growth factors,and oxygen,and have excellent prospects for application in regenerative medicine.Bovine acellular dermal matrix(BADM)is a natural biological material prepared by decellularizing bovine dermis,which retains the inherent biological activity of the natural matrix.Porous microspheres prepared using BADM can better induce cell adhesion.Mesenchymal stem cells(MSCs)need to be expanded in vitro,but long-term twodimensional(2D)culture may lead to senescence of MSCs due to differences between the in vitro environment and in vivo microenvironment.We prepared porous microspheres using a solution of BADM hydrogel and applied them to three-dimensional culture of canine bone marrow-derived mesenchymal stem cells(cBMSCs).We also studied the storage and cell harvesting methods for porous BADM microspheres,aiming to explore new materials and methods to alleviate the senescence of cBMSCs.In this experiment,we used natural biological material BADM hydrogel to prepare porous microspheres using a water/oil/water(W/O/W)emulsion crosslinking method,and compared the effects of different preparation conditions on the performance of the resulting porous microspheres on cell growth and senescence.We screened freeze-drying protective agent formulations and tested the performance of freeze-dried porous BADM microspheres to determine the most suitable storage method.We further studied the method of degrading porous BADM microspheres and successfully harvested viable cBMSCs.The specific experimental results are as follows:1.Long-term 2D culture can lead to the senescence of cBMSCs.P3,P5 and P8 cBMSCs showed good growth in vitro 2D culture,but with increasing passage number,cell morphology gradually enlarged,senescence-associatedβ-galactosidase(SA-β-Gal)activity significantly increased,expression of biomarkers of senescence genes p16 and p21 significantly increased,levels of cytokines VEGF,FGF and EGF secretion significantly reduced,and obvious senescent phenotypes appeared.2.Porous BADM microspheres have been successfully developed,which can promote cell growth.Using frozen bovine skin as raw material,we prepared BADM hydrogel and prepared well-shaped,defect-free,moderately hard and high-performance porous BADM microspheres through pore-forming,W/O/W emulsion crosslinking and filtration and washing steps.cBMSCs can grow and proliferate on them.3.Three-dimensional culture based on porous BADM microspheres can be applied to alleviate cBMSCs senescence.We examined the senescence status of cells under 2D and 3D culture conditions and found that the senescence of cells was alleviated under three-dimensional culture based on porous BADM microspheres,and the effect was better than that of BADM microspheres.4.Long-term storage conditions for porous BADM microspheres have been established.Through the screening of the formula of freeze-drying protectants,we found that a10% trehalose + 10% skim milk powder freeze-drying protectant formula can maintain the integrity of the structure of porous microspheres after freeze-drying.The freeze-dried porous BADM microspheres can be restored to their roundness after 3 minutes of rehydration and can be stored at 4°C for more than three months without affecting the performance of the porous BADM microspheres.This is a favorable method for longterm preservation.5.A method for degrading porous BADM microspheres for downstream cell therapy have been developed.We screened 100 mg/mL papain + 0.25% EDTA as the degradation reagent and successfully harvested cells from the porous BADM microspheres.After filtration,washing,and resuspension in culture medium,there was no residual microcarrier in the cell suspension.The degraded cells can grow normally,and after four days of culture,their viability can be restored to normal.In summary,this study successfully developed porous BADM microspheres for three-dimensional culture,which can effectively alleviate the senescence of canine bone marrow mesenchymal stem cells caused by long-term in vitro two-dimensional culture.At the same time,we explored the long-term storage conditions and degradation methods of porous BADM microspheres,providing new materials and methods for the application of canine bone marrow mesenchymal stem cell therapy.