1. Experimental Design
A two-phase culture system was established to be used in CECs culture. Two-phase refers to the alternation of two media of different compositions: Proliferative (P) and resting (R) media. Our modified formula [21] of previously reported P media [14] contained Opti-MEM I (Gibco®; Thermo Fisher Scientific, Waltham, MA) supplemented with 8% fetal bovine serum (FBS; Cellgro, Manassas, VA) and 1% penicillin/streptomycin (Pen/Strep; Thermo Fisher Scientific), 20 ng/ml of nerve growth factor (NGF; Sigma-Aldrich Co.), 5 ng/ml of epidermal growth factor (EGF; Sigma-Aldrich Co.), 200 mg/l of calcium chloride (Sigma-Aldrich Co.), 20 µg/ml of ascorbic acid (Sigma-Aldrich Co.), 0.08% chondroitin sulfate (Sigma-Aldrich Co.). R media formulation was Opti-MEM I supplemented with 8% FBS and 1% Pen/Strep. Experimental design consisted of alternating media through several passages to evaluate combinations of the phases of our culture system as shown in Fig. 1. Eight sequences of alternating media were evaluated. The objective was to produce a maximum cell yield while conserving CECs characteristics.
Passage 0 consisted in recently isolated CECs cultured in a 12-well plate with P media until hexagonal morphology was observed (confluence beyond 80%). All passages herein mentioned followed a hexagonal morphology and > 80% confluence rule. At passage 1, CECs were splitted 1:2 and the medium was changed using R media. The culture continued up to passage 3. An Axiovert 40 CFL contrast microscope (CFL; Carl Zeiss AG, Oberkochen, Germany) featuring a PowerShot A640 digital camera (Canon Inc., Tokyo, Japan) was used to register the cell morphology.
2. Isolation And Culture Of CEC
This study was approved by the Institutional Ethics Committee (School of Medicine of Tecnologico de Monterrey), number 2017-005. All animals were treated according to the Guide for the Care and Use of Laboratory Animals.
The corneas were obtained from four 3-month-old New Zealand rabbits weighing ~ 3 kg. The rabbits were euthanized under general anesthesia with a combination of xylazine 5 mg/kg and 30 mg/kg of ketamine [WU2] (Pisa Farmaceutica, Guadalajara, México); followed by a lethal intraperitoneal injection of sodic pentobarbital (Pets Pharma, Estado de Mexico, Mexico). The corneas were excised, rinsed with 37 °C Phosphate Buffered Saline (PBS) pH 7.4 (Gibco, Thermo Fisher Scientific, Waltham, MA) with 1% streptomycin/penicillin antibiotics (Thermo Fisher Scientific), and placed in a sterile tissue culture dish.
All rabbit CECs were isolated using the “peel-and-digest” approach. Excised whole corneas were placed in a petri dish with PBS, all inside the Biosafety cabinet. Then, Trypan blue (0.04%, Milipore Sigma, Merck KGaA, Darmstadt, Germany) was poured on the endothelial side of the cornea for 1 min to stain CE borders. Trypan blue was then rinsed using PBS. The border of CE was gently pushed around the whole circumference. Descemet’s membrane with the intact endothelium (DM/CE) was carefully peeled from the corneal stroma and rinsed several times with 37° C PBS with 1% antibiotics. DM/CE complexes were incubated in Opti-MEM I 8% FBS and the 1% antibiotic combination overnight to stabilize the cells before culture. CE were incubated, while shaking at 170, rpm with 1 mg/ml of collagenase type I (Sigma-Aldrich Co., St. Louis, MO) at 37° C for 1 h. Cells were collected following centrifugation at 375 × g for 10 min and seeded into a 12-well culture dish.
3. Cell Yield Analysis
Cell yield was calculated to produce evidence of morphology changes when cells were continuously cultured in P media and when cultured occurred in alternation with R media. For this analysis, the quotient of cellular concentration (cells/ml) at the end of passage 2 divided by the cellular concentration at the end of passage 1 was calculated for each media sequence. The average and the standard error were calculated. A t test was used to analyze statistically significant differences between the calculated yields.
4. Morphological Analysis
CECs were observed (Axioivert 40CFL inverted microscope, Zeizz, Germany) and photo documented daily. Scale was set on each photograph and 40 cells per daily image were delimited with free shape ROI tool from Image J. Perimeter (P), area (A), circularity (C), aspect radio (AR) and roundness (R) indexes were obtained using Measure (Analyze menu). Basal parameters were also measured from cells observed in pictures obtained from recently isolated Descemet’s membrane. Statistical analysis was performed through a one-way ANOVA complemented with an all pairwise multiple comparison procedure by Holm-Sidak method (P = 0.001). Microsoft Excel (2007, Redmond, WA) was used for data processing. Statistical analysis and graph creation were carried out using Systat Sigma Plot (V. 11, San Jose, CA).
5. Protein Production Analysis
Cultured cells were detached using trypsin and then and centrifuged at 500 x g for 5 min at 4 °C to obtain a cell lysate for total protein analysis. The pellet was washed three times with 1X PBS buffer and incubated 20 min with extraction buffer (Abcam ab193970) on ice. The solution was centrifuged at 18,000 x g for 20 minutes at 4 °C. The supernatant was used for the total protein concentration analysis. Colorimetric bicinchoninic acid assay (BCA Pierce 23225) was used. A standard curve was prepared using albumin provided in the kit. The concentration of total protein was measured at 562 nm in a spectrophotometer. The assay was done in triplicates using a dermal fibroblast cell line as control. Statistical analysis was performed with a t-test using Microsoft Excel (2007, Redmond, WA).
6. Procollagen I Alpha 1 Analysis
Pro-collagen I-α1 was determined as a measure of the ability of the cells to produce collagen 1-α1, ELISA Kit (Abcam ab210966) was used with the cell lysates described before. The procedure was followed in accordance with the kit manufacturer and the absorbance was measured at 562 nm in a microplate reader. The assay was done in triplicates using a dermal fibroblast cell line as control. Statistical analysis was performed with a t-test using Microsoft Excel (2007, Redmond, WA).
7. Biomarker Immunodetection (ZO1 And ATPase)
Immunocytochemistry was performed on basal whole cornea and on CECs passage 2 cultured with PRR media sequence combination to analyze the presence ZO-1 (Thermo Fisher, 61–7300, Waltham, MA), and Na/K-ATPase (Abcam, ab176163, Cambridge, UK). The procedure consisted of overnight cell stabilization over coverslips with poly-D lysine (Sigma-Aldrich, P7280), fixation with 4% paraformaldehyde, nonspecific bonding blockage with 5% bovine serum albumin (BSA; Sigma-Aldrich, A-7030), overnight 4 °C incubation with primary antibodies (ZO-1 5 µg/ml and Na/K-ATPase 1:100), and incubation with Alexa Fluor 488 secondary antibody (Abcam, ab150077) for 1 h at room temperature. Fluroshield Mounting Medium with 4′,6-Diamidino-2′-phenylindole dihydrochloride (DAPI; Abcam, ab104139) counterstain was used. Epifluorescence was registered with a widefield fluorescence microscope (Zeiss Imager Z1) with an AxioCam HRm (Zeiss) camera (Göttingen, Germany). Whole width cornea sections were used as controls [15].
8. ATPase Activity Analysis
A colorimetric enzymatic assay was used to measure the ATPase activity (Mybiosource MBS8243226). Cells (CEC and dermal fibroblasts) were harvested when confluence was reached and sonicated for the detection in accordance with the manufacturer procedure. The final absorbance was registered at 660 nm. The assay was done in triplicates using a dermal fibroblast cell line as control. Statistical analysis was performed with a t-test using Microsoft Excel (2007, Redmond, WA).