Preparation Of Tissue Engineered Conjunctiva With Favorable Cell Activity By A 3D Dynamic Perfusion System In Vitro

Objective: To use the highly active conjunctival epithelial cells(CECs)cultured by CHIR-99021 as seed cells,combined the self-assembling peptide hydrogels and the 3D dynamic perfusion system to accomplish the reconstruction of tissue engineered conjunctiva(TEC).Methods:(1)Differentiation and detection of CECs induced by CHIR-99021: Rabbit conjunctival epithelial cells(r CECs)were cultured with CHIR-99021(5μM、10μM、20μM) and traditional culture medium,respectively,the growth morphology,cell activity and goblet cell number were compared.(2)Preparation and identification of decellularized conjunctival matrix(DCM): The rabbit conjunctival tissue was decellularized with phospholipase A2,and then the DCM was stained by immunohistochemistry and immunofluorescence to detect the decellularization efficiency;rehydration rate and in vitro degradation experiments were carried out to release the physicochemical properties of the DCM.(3)Construction of TEC in vitro by3 D dynamic culture system: The self-assembling polypeptide hydrogel RAD16-I was mixed with r CECs and planted on the surface of DCM.The cell lamination,cell differentiation and intercellular connection of TEC were detected by HE staining and immunofluorescence staining.Results:(1)Compared with traditional culture medium,the CECs density increased and the goblet cell-specific MUC5 AC expression was up-regulated in the group supplemented with CHIR-99021,among which,5μM CHIR-99021 had better effect.(2)After the decellularization,the upper cortex of the conjunctiva was completely removed while the lamina propria was retained,there was no statistical difference in water content and in vitro degradation rate between DCM and natural conjunctiva after rehydration(P > 0.05).(3)r CECs were mixed with RAD16-I and planted on the surface of DCM to form stable 3-5 layers of cells,compared with the traditional static culture group,the cells in the 3D perfusion dynamic culture group were evenly distributed in the inside of the scaffold,and more cells(P < 0.05).Conclusions:(1)Compared with traditional culture medium,CHIR-99021 can induce r CECs to differentiate into goblet cells and promote the expression of MUC5AC;(2)Phospholipase decellularization method can completely remove components of cells,retain the collagen structure of extracellular matrix,and prepare DCM suitable for TEC;(3)RAD16-I mixed with r CECs could rapidly constructe three-dimensional multilayer cell structure,compared with the traditional static culture,the TEC with the help of perfusion dynamic culture system had more cells and more uniform cell distribution.