Bovine Serum Albumin Contributes To Spheroid Formation Of Adipose-derived Stem Cells And Its Applications In The Healing Of Oral Mucosal Defects

Objective:Cell culture is the most important and fundamental techniques in the fields of biology and medicine.At present,two-dimensional adherent culture and three-dimensional spherical culture are the existing in vitro cell culture methods.Compared with 2D adherent culture,3D spherical culture can significantly improve the proliferation,anti-apoptotic,anti-inflammatory and paracrine functions of mesenchymal stem cells（MSCs）.Among mesenchymal stem cells,adipose-derived stem cells（ADSCs）are abundant and can be easily obtained from subcutaneous adipose tissue by liposuction.In this study,therefore,ADSCs was used and first found that they can form 3D spheroids under the stimulation of bovine serum albumin（BSA）.Then the biological properties in vitro and applications of 3D spheroids in vivo are further explored.They provide references for the treatment of oral mucosal defect diseases.Methods:1.ADSCs extraction,culture and spheroid formation（1）ADSCs extraction and culture:The ADSCs were obtained from the subcutaneous adipose tissue after liposuction.They were identified by flow cytometry（FC）.Mesenchymal Stem Cell Medium（MSCM）is used for the amplification and passage of ADSCs.（2）Spheroids formation:The ADSCs at passages 3-7 were dissociated into single cells,washed twice with Phosphate balanced solution（PBS）,and resuspended in DMEM/F12medium supplemented with 1%BSA,1%P/S.The initial cell density was approximately50,000 cells/cm².Incubating for 3-4 days in a 5%CO₂incubator.Morphological characteristics of ADSCs spheroids are observed by inverted research microscope.Spheroid formation was recorded by the Essen Bio Science Incu Cyte（?）Zoom Live-Cell Analysis System.2.Basic biological properties and formation mechanism of ADSCs spheroidsThe experiment was divided into Control group（DMEM/F12）and BSA（DMEM/F12+1%BSA）group.（1）Proliferation ability:Detection was performed using Cell counting kit-8（CCK-8）.Proliferation-related genes were quantified by Real-time Quantitative polymerase chain reaction（RT-PCR）,and the differences in cell proliferation ability between the two groups were analyzed.（2）Cell viability:Cell viability was detected using live and dead cell viability detection kits,and quantitative analysis was performed after recording with a laser confocal microscope.（3）Inflammation assay:Inflammation-related genes such as:interleukin-10（IL-10）,transforming growth factor（TGF-β）,interleukin-1β（IL-1β）and tumor necrosis factor-a（TNF-a）were quantified using RT-PCR by macrophage inflammatory assay.The IL-1RN was quantified by RT-PCR and Western Blot（WB）.（4）Formation mechanism:The formation mechanism of ADSCs spheroids was studied by RT-PCR,WB and immunofluorescence（IF）.3.Establishment of SD rat model of oral mucosal defect and cell transplantation:The SD rats aged 7-8 weeks were selected,and a 3mm diameter skin biopsy punch was used to establish a full-thickness annular defect of the mucosa behind the third mucosal fold of the hard palate.The cells are then injected to the edge of the defect.The therapeutic potential of ADSCs spheroids was evaluated by stereomicroscopy,hematoxylin-eosin staining（H&E）,Masson staining and immunohistochemistry（IHC）.Results:1.Live cell imaging showed that ADSCs aggregated and formed ADSCs spheroids at3-4 days with the stimulation of 1%BSA.2.The general biological characteristics showed that compared with Control group,the BSA group cells proliferated significantly,maintain cell viability,increased expression of anti-inflammatory genes IL-10 and TGF-βand decreased expression of pro-inflammatory factors IL-1βand TNF-a.In addition,the PCR and WB results showed that compared with Control group,the expression of IL-1RN in BSA group was significantly increased（P<0.05）.3.To explore the formation mechanism of ADSCs spheroids,RT-PCR,WB and IF experiments were used,respectively.The PCR results showed that compared with Control group,the expression of YAP genes in BSA group was significantly increased.The IF results further showed that proportion of nuclear YAP of BSA group was significantly higher（P<0.05）.Compared with Control group,WB results demonstrated that the ration of p-YAP/t-YAP was significantly reduced（P<0.05）in BSA group.4.The results of animal experiments showed that the healing speed of the oral mucosa of the rats in the ADSCs spheroids group was significantly faster than that in the PBS group.Conclusion:The above experiments show that 1%BSA can promote the formation of ADSCs spheroids.The ADSCs spheroids exhibit the advantages of pro-proliferation,anti-inflammatory and maintenance cell viability.Here,we explored that BSA regulated the formation of ADSCs spheroids by activating the Hippo-YAP pathway.The animal experiments demonstrate that ADSCs spheroids can accelerate the healing of oral mucosal defects.Therefore,ADSCs spheroids are expected to become a novel therapeutic strategy for oral mucosal defect diseases.