To establish an 2D in vitro simulations, which model the interactions between retinal vascular endothelial cells (RMECs) and ganglion cells (RGC) under simulated diabetic retinopathy (DR) condition.

The rat RMECs and RGC were co-cultured in the Transwell (0.4 μm), cells were divided into the control group (5.5 mM ) and high glucose group (50.0 mM) according to the concentration of glucose in the culture medium. Cell morphology was observed by fluorescence inverted microscope imaging; cell proliferation assay, scratch and transwell migration assay as well as the lumen formation assay used to detect the capacity of the proliferation, migration, and lumen formation of RMECs and RGC. The apoptosis of cells was observed by PI/Hoechest experiment. The number of the cell migration, formed lumens and lumen intersections as well as the apoptosis rate in the two groups conformed to a normal distribution, were described as ±s, and compared by t testing. The optical density (OD) value of cell proliferation and cell migration rate in the two groups conformed to a non-normal distribution, were described using median and interquartile range, and comared by the Mann-Whitney U test.

In the co-culture model, the RGCs in the upper chamber showed adherent growth as a oval-like monolayer, while the RMECs in the lower chamber were more irregular, as a single layer of paving stones, and tightly connected between cells. The OD value of cell proliferation in RMECs of the high glucose group and control group were 1.163 (1.142, 1.188) and 0.812 (0.805, 0.817); the OD value of RGCs in two groups were 0.723 (0.709, 0.759) and 1.934 (1.803, 2.030). There were both statistically significant differences in the OD value of cell proliferation of two cells between two groups (Z=-6.069, -5.940; P<0.05). The migration ability of RMECs in the high glucose group and control group were [53.64 (37.69, 65.88)]% and [32.25(22.34, 42.85)%], which the former group better than latter group and there was a significant difference between two groups (Z=-2.841, P<0.05). The migration score of RMECs in two groups were (164±31) and (113±16); the apoptosis rate were (14.30±4.29)% and (9.65±3.43)%; the number of lumen formation were (23±5) and (19±4); lumen intersections were (11±3) and (13±2), respectively. There were all statistically significant differences in the migration score, the number of lumen formation, and the apoptosis rate between two groups (t=-4.653, -2.137, -2.929; P<0.05); non-significant difference in the lumen intersections between groups (t=1.424, P>0.05). The apoptosis rate of RGCs in two groups were (6.91±1.15) % and (4.87±1.70)% with statistically significant differences between two groups (t=-2.446, P<0.05).

The 2D co-culture model of RMECs and RGCs has shown that there were the same performance as that of single cell culture in vitro. Under the high glucose environment, the cell activity, apoptosis, migration ability, and lumen formation ability of RMECs and the cell activity and apoptosis of RGCs with synchronous physiological and pathological changes, which laying a model foundation for further mechanism research of DR.