Preparing an appropriate culture medium for SCAP cells
Dental stem cells were extracted according to approved protocols in Laboratory under mycoplasma-free conditions. The cell passage range used was 6-12. After cultivation in a suitable medium, the cells were maintained for three to four days until the confluence reached approximately 80% in the dish bottom.
Passage and counting of SCAP cells
After observing the cultured cells under a microscope and ensuring their morphology, density, and lack of contamination, the cell culture medium was removed, and the cells were washed using an appropriate volume of PBS solution. Then, an appropriate volume of Trypsin-EDTA (sufficient to fully cover the cells) was added to the culture dish. The dish was placed in the incubator for 3 to 5 minutes, after which the cells were observed under a microscope. The reaction was stopped using double the volume of the culture medium compared to trypsin. Then, the cells were transferred to a centrifuge tube using a pipette. The tubes were centrifuged at 1800 rpm for 5 minutes. The supernatant was poured off, and the cellular pellet obtained was resuspended in 1 milliliter of the culture medium. For cell counting, 20 microliters of the cellular suspension were uniformly distributed in the counting chamber area between the cover glass and the counting chamber using an inverted microscope. Cell counting was performed in four designated areas as shown in Figure 3-1. The total cell count was obtained through the average number of cells counted in the four areas, using the following formula.
Freezing SCAP Cells
The required medium for freezing, consisting of 10% DMSO, 50% FBS, and 40% culture medium, was prepared. This medium should be freshly prepared a few minutes before freezing. When the cells reached approximately 80% confluence, they can be prepared for storage. The cells were treated with trypsin and after centrifugation at 1800 rpm, the freezing medium was gently added onto the cells. Then the vials were placed in the freezing chamber at -70°C. After 24 hours, these vials were transferred to a liquid nitrogen tank.
Thawing of SCAP (Stem Cells from Apical Papilla)
First, the culture medium was equilibrated to 37°C by placing it in a water bath. The desired vials were removed from liquid nitrogen, and the lid was immediately loosened halfway to allow gas to escape. Then, the vials were thawed in a 37°C water bath (some ice crystals may have remained in the sample at this stage). The thawed cells were gently transferred to a 4 mL culture medium-containing tube (the sample was completely thawed at this point) and centrifuged at 1800 rpm for 7 minutes. After removing the supernatant containing DMSO, the counted cells were transferred to a culture flask. Upon reaching 80% confluence and following the described steps, the cells were passaged in a T-150 flask. After preparing the proteinase K solution, the experiment was designed in six separate groups: Group 1: GMEM medium supplemented with GlutaMax and NEAA, used as the initial medium for collecting conditioned media from SCAP cells, was considered. Group 2: Only conditioned media from SCAP cells were used. Groups 3 to 6: Conditioned media from SCAP cells were treated with proteinase K at concentrations of 400, 300, 200, and 100 mg/ml, respectively. Subsequently, all media were incubated for one hour at 37°C.
Cultivation and passage of pigmented retinal cells
In order to prepare the material, an appropriate volume of KDMEM basal medium was added to the electrospun material to achieve a final concentration of 3.0 mg/ml. Then, the dishes were incubated overnight at the specified temperature. Prior to use, the culture medium was removed from the dishes. Human embryonic stem cells of the RH6 line were differentiated into photoreceptor precursors (PRE) following the protocol published by Karamali and colleagues. Subsequently, the cells were transferred onto the prepared dishes.Upon reaching confluence, for cell passaging, the culture medium was first removed, and after washing with PBS-, EGTA at a concentration of 5.0 mM was added to the cells for 15 minutes at 37°C. After removing EGTA, accutase enzyme was added to the cells and they were placed back in the incubator for another 15 minutes. The dissociated cells were collected, centrifuged, and then transferred onto dishes coated with the appropriate culture material and medium. Additionally, a rock inhibitor at a concentration of 10 µM was added during the culturing of these cells. The grouping of experimental categories was performed to examine the proliferation rate of retinal pigmented epithelial cells in environments containing different treatments. The experimental groups were divided into four categories as follows: In the positive control group, the cells were cultured in a medium without any adjacent cells. In the second group, the cells were cultured in a medium adjacent to dental stem cells (SCAP). In the third group, retinal pigmented epithelial cells were cultured in a medium adjacent to SCAP cells, which were pre-treated with proteinase K enzyme and then treated with PMSF (to deactivate the enzymatic activity of proteinase K). The fourth group was similar to the third group, except that heat treatment (at 90 degrees Celsius for 5 minutes) was used instead of PMSF to deactivate proteinase K. To assess the effects of the conditioned medium from SCAP cells on the proliferation of retinal pigmented epithelial cells, four experimental groups were designed: In the first group, retinal pigmented epithelial cells were cultured in the presence of a regular medium. In the second group, retinal pigmented epithelial cells were cultured in the presence of the conditioned medium from SCAP cells. In the third group, retinal pigmented epithelial cells were exposed to the conditioned medium from SCAP cells after digestion with proteinase K enzyme, followed by treatment with PMSF (to deactivate the enzymatic activity of proteinase K). The fourth group followed a similar procedure to the third group, except that heat treatment (at 90 degrees Celsius for 5 minutes) was applied to deactivate proteinase K instead of using PMSF. Then, Retinal pigmented epithelial cells were plated at a cell density of 5 × 10^4 cells per cm^2 in a 96-well plate. Subsequently, the desired media were added to the cells, and the metabolic activity of the cells was evaluated within the specified time intervals.
Statistic analyses
All descriptive and analytical analyses were conducted using the SPSS 22 software. To compare the study groups, the paired sample T-TEST statistical test was utilized. For evaluating the proliferation rate after reading on the Elisa device, the results were analyzed using a significance level of 0.05 (P < 0.05).