Objective  (1) To explore the proper density and the cellular biological characteristics when corneal epithelial cells and oral mucosal epithelial cells were seeded on temperature-responsive Nunc UpCell Suface.The carrier-free cell sheets fabricated on the temperature-responsive culture surfaces. (2) To investigate the proliferation effects of different concentration maxadilan to keratocytes. The influence of recombinant maxadilan to keratin CK3 expression of oral mucosal epithelial cells when co-culturing keratocytes were also studied.
Methods  (1) Different densities of primary corneal epithelial cells and oral mucosal epithelial cells were seeded on Nunc UpCell surface and tissue culture plate (TCP) surface of 6 well plates and incubated. Phase contrast microscope examination was performed daily to observe morphological differences in different cellular densities. Acridine orange (AO) staining was used to evaluated the cell viability. HE staining was carried to observe the paraffin longitudinal section of the cell sheets. SEM examination was carried out to observe the cell junction and the structure. (2) RT-PCR assay was used to detect the expression of PAC1 receptor in keratocytes. Western-Blot assay was used to detect the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells. The proliferation effects of keratocytes in 1nM-500nM maxadilan using DMEM medium with 0.3% FBS or 10% FBS were assessed by CCK-8 method. Real-Time PCR assay was established for detection of CK3 gene expression of four groups as bellow: rabbit mucosal epithelial cells co-culture with keratocytes through transwell culture (group one), group one +100nM recombinant maxadilan (group two), rabbit mucosal epithelial cells (group three) and corneal epithelial cells (group four) as control group.
Results  (1) There were not obvious discrimination in different densities of primary corneal epithelial cells and oral mucosal epithelial cells seeded on Nunc UpCell surface and TCP surface during the early stage. With the time prolongs, the cells of 5×10⁴/ml density presented retardation, the cells of 2×10⁵/ml density displayed apoptosis state, while the cells of 1×10⁵/ml density were easy to form cobble-like morphology and colony formation. There were multilayer cells and cells reached confluence on day 8. Cell sheets were harvested by reducing the culture temperature to 20℃-25℃. HE staining showed that the cell sheets were carrier-free. SEM showed tight junction of the cells. (2) RT-PCR assay demonstrated the expression of PAC1 receptor in keratocytes. Western-Blot detected the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells at protein level.The CCK8 assay revealed that maxadilan promoted the proliferation of keratocytes. Real-Time PCR demonstrated that maxadilan promoted the expression of CK3 in oral mucosal epithelial cells when co-culture keratocytes.
Conclusions  (1) Cell sheets obtained from Nunc UpCell surface show carrier-free without exogenous materials, which corresponds the requirements of reconstructing physiological condition of cornea cellular layers. (2) There is the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells. Maxadilan, an agonist of PAC1 receptor, can promote the proliferation of keratocytes and the expression of CK3 of oral mucosa epithelial cells. So Maxadilan is to the benefit of seeding cells of corneal tissue engineering.