Gelatin Microcarrier Combined With Adipose-derived Mesenchymal Stem Cells To Construct 3D Cell Spheres In Tissue Engineered Nerves

Objective:1)To explore the advantages of 3D cell culture based gelatin microcarrier.2)To investigate the effect of gelatin microcarrier 3D cell spheres on nerve regeneration in tissue engineered nerves.Methods:The inguinal fat of SD suckling mice(48 hours)was shredded and digested and cultured into P1 generation.Then the gelatin microcarriers were combined with P1adipose-derived stem cells(ADSCs)to construct 3D cell spheres in bioreactors.In order to evaluate the advantages of 3D cell culture in vitro,we compared proliferation,apoptosis,maintenance of stem cell genes and stem cell function.50 x 10~4 ADSCs of P1generation were cultured in traditional 2-dimension(2D)and 3-dimension(3D),and the incubation time was 1,3,5 and 7 days respectively.Cells cultured in two-dimension(2D)and three-dimension(3-D)cultures were counted at these four time points.The expression of stem cell genes was detected by RT-PCR,and the apoptosis of cells cultured in two different ways was detected by FITC/PI.When comparing the functions of stem cells,the constructed 3-D cell spheres and 2-D cultured cells were placed in the upper layer of Transwell and co-cultured with dorsal root ganglion,respectively.The effects of 2-D and 3-D cells on the growth of dorsal root ganglion axons were compared.In vivo,a rat model of sciatic nerve defect(n=48)was constructed.The rats were randomly divided into four groups with a length of 15 mm.They were injected into 3-D cell spheres(3-D group),2-D cell(2-D group),chitosan conduit only(conduit group)and autologous nerve grafts(autigraft group)to bridge the nerve defect.After 12 weeks of operation,the effects of 3-D adipose stem cell spheres as seed cells injected into chitosan conduit to repair sciatic nerve defects in rats were evaluated by neuroelectrophysiology,wet weight recovery rate of gastrocnemius muscle and histological analysis(toluidine blue staining,immunofluorescence staining,transmission electron microscopy scanning).Results:Compared with the traditional 2D cell culture,the dynamic culture of 3D cells based on gelatin microcarriers has faster cell proliferation,slower apoptotic rate and stronger ability to maintain stem cell gene expression.The growth of DRG axons was accelerated by 3-D cell spheres more than by 2-D cells.At 12 weeks after operation,the results of electrophysiology,wet weight recovery rate of gastrocnemius muscle and pathological observation in the three-dimensional group and the autograft group were significantly better than those in the two-dimensional group and the conduit group.There was a significant difference between the three-dimensional group and the ANG group,and the latter was significantly better than the former,but there was no significant difference between the two groups.Conclusion:In this experiment,the dynamic cell culture based on gelatin microcarrier constructed 3D cell sphere has the advantages of fast cell proliferation,low apoptotic rate,better stem cell maintenance and improved stem cell function.It can be used as stem cells in tissue engineering nerve research,and its curative effect is obviously better than that of 2D cell group.