The Petri Dish-N2B27 Culture Condition Maintains RPE Phenotype by Inhibiting Cell Proliferation and mTOR Activation

Abstract

Objective: To develop a method for the rapid isolation of rat RPE cells with high yield and maintain its epithelial state in modified culture system.

Methods: The eyeballs were incubated with dispase. The retina was isolated with RPE attached and cut into several pieces. Following a brief incubation in growth medium, large RPE sheets can be harvested rapidly. RPE cells were divided into four groups and cultured for several weeks, that is, (1) in cell culture dishes with 10% FBS containing medium (CC dish-FBS), (2) in petri dishes with 10% FBS containing medium (Petri dish-FBS), (3) in cell culture dishes with N2 and B27 containing medium (CC dish-N2B27), and (4) in petri dishes with N2 and B27 containing medium (Petri dish-N2B27). Morphological and biological characteristics were investigated using light microscopy, Q-PCR, and western blot.

Results: The retina would curl inwardly during the growth medium incubation period, releasing RPE sheets in the medium. Compared with low density group (5,000 cells/cm²), RPE cells plated at high density (15,000 cells/cm²) can maintain RPE morphology for a more extended period. Meanwhile, plating RPE cells at low density significantly reduced the expression of RPE cell type-specific genes (RPE65, CRALBP, and bestrophin) and increased the expression of EMT-related genes (N-cadherin, fibronectin, and α-SMA), in comparison with the samples from the high density group. The petri dish culture condition reduced cell adhesion and thus inhibited RPE cell proliferation. As compared with other culture conditions, RPE cells in the petri dish-N2B27 condition could maintain RPE phenotype with increased expression of RPE-specific genes and decreased expression of EMT-related genes. The AKT/mTOR pathway was also decreased in petri dish-N2B27 condition.

Conclusion: The current study provided an alternative method for easy isolation of RPE cells with high yield and maintenance of its epithelial morphology in the petri dish-N2B27 condition.

Figure 1

Sequential dissection of DA rat retina to obtain primary RPE cells using dispase. (a) Eyes were dissected out from DA rats, and extraocular muscle attachments (black arrow) were removed from the eye under the dissecting microscope. (b) After incubation in the 2% dispase solution, eyes were washed in PBS. Then the retina/RPE complex (blue arrow) was isolated by removing the choroid (green arrow), the anterior cornea (red arrow), and lens (black arrow). The RPE monolayer was still attached to the retina. (c, d) The resulting retina/RPE complex was cut to small pieces and was further incubated in 10% FBS containing medium for 20 min at 37°C to allow the RPE (blue arrow) to detach from the neural retina (yellow arrow). (e) The detached RPE sheets were collected and digested in 0.1% trypsin solution and then gently pipetted up and down several times to achieve smaller patches of RPE cells. (f) Primary RPE cells of DA rats after 24 h on cell culture dish show high confluence and pigmentation. Scale bar: 100 μm.

Figure 2

Sequential steps of SD rat eye dissection to obtain primary RPE cells using dispase. (a) Eyes were dissected out from SD rats, and excess muscle attachments (black arrow) were removed in PBS. (b) After incubation in the 2% dispase solution, eyes were washed in PBS. Under dissecting microscope, the retina/RPE complex (blue arrow) was carefully isolated by removing the choroid (green arrow), cornea (red arrow), and lens (black arrow). (c, d) The resulting retina/RPE complex was cut to small pieces and was further incubated in 10% FBS containing medium for 20 min at 37°C to allow the RPE (blue arrow) to detach from the neural retina (yellow arrow). (e) The detached RPE sheets were collected and digested in 0.1% trypsin solution and then gently pipetted up and down several times to achieve smaller patches of RPE cells. (f) Primary RPE cells of SD rats after 24 h on cell culture dish show high confluence and pigmentation. Scale bar: 100 μm.

Figure 3

Morphology of the primary RPE cells cultured on cell culture dishes. (a–f) Images of SD rat RPE cells (cell density: 5,000 cells/cm² or 1,5000 cells/cm²) cultured on cell culture dishes with 10% FBS containing medium at different time points. Scale bar: 100 μm.

Figure 4

Expression levels of RPE cell type-specific genes and EMT-related genes in SD rat RPE cells at different time points. (a, b) QPCR analysis of RPE65, CRALBP, bestrophin, N-cadherin, fibronectin, and α-SMA in freshly isolated RPE tissues and in RPE cells cultured in 10% FBS containing medium (cell density: 1,5000 cells/cm²) for 3 days. (c–h) The expressions of RPE65, CRALBP, bestrophin, N-cadherin, fibronectin, and α-SMA genes in the two cell density groups at different time points. Low cell density group: 5,000 cells/cm². High cell density group: 1,5000 cells/cm². Data were expressed as mean ± SD. n = 4 in each group. ^∗p < 0.05.

Figure 5

The petri dish culture condition inhibits RPE cell migration and proliferation. (a–d) Images of RPE cells in the cell culture dish group and the petri dish group at different time points. Scale bar: 100 μm. (e) Calculation of RPE cell numbers in the cell culture dish group and the petri dish group at days 1, 3, 7, 10, and 14. Data were expressed as mean ± SD. n = 4 in each group.

Figure 6

Characteristics of SD rat RPE cells under different in vitro culture conditions. (a–d) Images of RPE cells cultured in different culture systems at 1 month. Scale bar: 100 μm. (e) QPCR analysis of RPE65, CRALBP, and bestrophin expressions in RPE cells from SD rats at 1 month in different culture conditions. (f) Expression levels of N-cadherin, fibronectin, and α-SMA in SD rat RPE cells at 1 month in different culture conditions. Cell density: 1,5000 cells/cm². Data were expressed as mean ± SD. n = 4 in each group. ^∗p < 0.05, the petri dish-N2B27 group versus the other three groups at 1 month in four culture conditions. (a) CC dish-FBS. (b) CC dish-N2B27. (c) Petri dish-FBS. (d) Petri dish-N2B27.

Figure 7

The petri dish-N2B27 culture condition inhibits AKT/mTOR pathway activation. (a) Protein levels of phospho-mTOR, total-mTOR, phospho-p70S6K, total-p70S6K, phospho-AKT, and total-AKT in freshly isolated RPE tissues and in RPE cells cultured in 10% FBS containing medium (cell density: 1,5000 cells/cm²) for 3 days. (b) Quantification of the western blot data from (a). (c) The expressions of phospho-mTOR, total-mTOR, phospho-p70S6K, total-p70S6K, phospho-AKT, and total-AKT in the two cell density groups at different time points. Low cell density group: 5,000 cells/cm². High cell density group: 1,5000 cells/cm². (d) Quantification of the western blot data from (c). Data were expressed as mean ± SD. n = 4 in each group. ^∗p < 0.05. (e) Western blot analysis of phospho-mTOR, total-mTOR, phospho-p70S6K, total-p70S6K, phospho-AKT, and total-AKT in SD rat RPE cells cultured in different culture conditions. Cell density: 1,5000 cells/cm². (f) Quantification of the western blot data from (e). Data were expressed as mean ± SD. n = 4 in each group. ^∗p < 0.05, the petri dish-N2B27 group versus the other three groups at 1 month in four culture conditions.