Reconstruction of conjunctival epithelium-like tissue using a temperature-responsive culture dish

Isolation and culture of rabbit conjunctival epithelial cells

All experimental procedures adhered to the guidelines of the Chinese Animal Administration and the Association for Research in Vision and Ophthalmology Statement for the use of animals in ophthalmic and vision research. The rCjECs were isolated and cultured as previously described [14]. Briefly, the conjunctiva originating from the palpebral and fornix regions of New Zealand white rabbits was carefully dissected. The entire sheet of conjunctival epithelial tissue was separated from the attaching Tenon’s tissue. The sheet was rinsed three times with PBS (1X; 130 mM NaCl, 3 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) containing 100 U/ml penicillin and then was incubated with Dispase II (2.4 units/ml; Sigma-Aldrich, St. Louis, MO) at 4 °C for 16 h. The detached epithelial layer was then scattered into single cells with 0.05% trypsin/EDTA for 10 min at 37 °C. The cells were then seeded on a cell culture dish (with I collagen coating, Millipore Corporation, Billerica, MA) in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F12 (1:1 DMEM/F12, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT), 5 μg/ml insulin, 5 μg/mL transferrin, 5 ng/ml selenium, 1% penicillin/streptomycin, 10 ng/ml human epidermal growth factor (hEGF; R&D Systems, Minneapolis, MN) and 100 ng/ml nerve growth factor (NGF; R&D Systems). After 2 days of culture, the non-adherent cells were removed by washing with PBS. When the adherent cells reached approximately 80% to 90% confluence, the cells were rendered single cells by trypsin/EDTA and subcultured for additional experiments.

Cell morphology and phenotype

The morphology of the rCjECs was viewed with phalloidin staining [15]. The subcultured cells were cultured on glass coverslips. When the cells reached 70% to 80% confluence, they were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich), permeabilized with 0.5% Triton X-100 (Sigma-Aldrich), and stained with Alexa Fluor 546 phalloidin (Life Technologies, Carlsbad, CA) for 30 min. The nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Life Technologies) for 30 min.

Periodic acid–Schiff (PAS) staining was performed for the detection and distribution of the mucosubstances [16]. When the subcultured cells cultured on glass coverslips reached 70% to 80% confluence, they were fixed with 4% PFA, and then the cells were stained using a PAS staining kit (Sigma-Aldrich) according to the manufacturer’s manual.

For the identification of the cell phenotype, immunocytochemistry staining was performed as described previously [17]. The subcultured cells were seeded onto glass coverslips, fixed with 4% PFA for 15 min, and permeabilized with 0.3% Triton X-100 in PBS. After blocking with 10% normal goat serum (Invitrogen), the cells were incubated with primary antibodies overnight at 4 °C: rabbit polyclonal anti-CK19 (1:200, Abcam, Cambridge, MA), mouse monoclonal anti-CK4 (1:200, Sigma-Aldrich), and mouse monoclonal anti-MUC5AC (1:200, Abcam). Subsequently, the samples were subjected to a fluorescent-labeled secondary antibodies (Alexa Fluor 546 goat anti-mouse/rabbit, BD Biosciences, San Jose, CA) for 1 h. The nuclei were counterstained with DAPI (Invitrogen, Molecular Probes, Eugene, OR). Negative control samples were processed in parallel but without a primary antibody. An Olympus BX51 (Tokyo, Japan) fluorescent microscope was used to capture the images.

Preparation of the temperature-responsive culture dish

A temperature-responsive culture dish was purchased from CellSeed (Tokyo, Japan). The 35-mm cell culture dish was first exposed to ultraviolet (UV) light for 30 min to disinfect the surface. A temperature-responsive polymer, PIPAAm, was grafted to the surfaces of the cell culture dish. For cell culture use, these surfaces were washed twice with PBS.

Preparation of the cell sheets

The subcultured cells were plated at a density of 1.0×10⁷ cells/dish and cultured on the surface of a 35-mm temperature-responsive culture dish. After culture for 4 days, the cells reached confluence. The same number of cells was then seeded on the confluent cells and cultured for 6 days. The culture medium was removed, and a hydrophilically modified polyvinylidene fluoride (PVDF) membrane was placed over the cell sheet. Fresh medium was added to prevent dryness. The cell sheet was harvested from the culture dish by reducing the culture temperature to 20 °C for 30 min.

Cell proliferation and viability

To investigate cell proliferation, Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay and 5-bromo-2’-deoxyuridine (BrdU) staining were performed. Briefly, the conjunctival epithelial cell sheets were dissociated with trypsin-EDTA, and then the cells were seeded on 24-well plates at a density of 2×10⁴ cell/well. After 1, 3, and 5 days of cell culture, the cells were washed with PBS and incubated with 10% of CCK-8 containing DMEM/F12. After incubation for 3 h, 200 μl DMEM/F12 containing CCK-8 was transferred to 96-well plates. The absorbance of each well was measured at 450 nm with a microplate reader (ELX800, BioTek, Winooski, VT) At least six samples were measured at each time point [18]. A BrdU label retention assay was also performed. Briefly, the conjunctival epithelial cell sheets were dissociated with trypsin-EDTA, and then the cells were seeded on glass coverslips at a density of 2×10⁶ cell/well. After the cells were attached on the glass coverslips, they were then incubated with fresh DMEM/F12 medium containing with 10 μM BrdU (Sigma-Aldrich) for 4 h at 37 °C in a humidified 5% CO₂ atmosphere. After fixation with 4% PFA, the samples were incubated with 2 N HCl at 37 °C for 1 h to denature the DNA, neutralized in 0.1 M borate buffer, permeabilized with 0.3% Triton X-100 in PBS, and blocked with normal goat serum. After overnight incubation at 4 °C with a rabbit anti-BrdU polyclonal antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), the cells were incubated with a fluorescent-labeled secondary antibody (Alexa Fluor 488 goat anti-rabbit, BD) for 1 h at room temperature in the dark. The nuclei were counterstained with DAPI (Invitrogen). The cells were imaged using an Olympus BX51 fluorescent microscope [19].

A Live/Dead^® Viability/Cytotoxicity Kit (Life Technologies, Carlsbad, CA) was used to investigate cell viability in the conjunctival epithelial cell sheets. The staining was based on the differential permeability of live and dead cells. When the cells cultured on temperature-responsive culture dish reached confluence, the live cells were stained with green fluorescent calcein-acetoxymethyl ester (CAM), a cytoplasm stain, and dead cells were stained with red fluorescent ethidium homodimer-2 (EthD-2). A fluorescent microscope (Olympus BX51) was used to analyze the images of the cell staining patterns [20]. The number of live and dead cells (green and red fluorescence, respectively) was counted in three different areas.

Identification of goblet cells, histology, and immunostaining

After being cultured for 10 days, conjunctival epithelial cell sheets were harvested from the surface of the temperature-responsive culture dish by decreasing the culture temperature to 20 °C. Conjunctival epithelial cell sheets were embedded in Tissue-Tek optimum cutting temperature (OCT) compound (Sakura Seiki, Tokyo, Japan) and processed into 6 μm frozen sections on cryofilm. The samples were then stained with a PAS staining kit (Sigma-Aldrich) according to the manufacturer’s manual. Hematoxylin and eosin (H&E) staining was used to assess the degree of stratification of the conjunctival epithelial cells [21]. Briefly, the sections were fixed with 4% PFA at room temperature for 5 min, washed three times with distilled-deionized water (DDW), and then stained with H&E. Specific cell marker expression was visualized in the immunostaining experiments. Primary antibodies of CK4, CK19, and MUC5AC were used to stain the frozen sections of the conjunctival epithelial cell sheets. The biotin-conjugated secondary antibodies (4 mg/ml; Vector Laboratories, Burlingame, CA) were used to visualize the specific marker. Briefly, after fixation with 4% PFA for 30 min, the sections were blocked for 1 h in a blocking solution (PBS containing 10% (v/v) normal goat serum (Invitrogen), 0.3% Triton X-100 (Sigma-Aldrich), and 0.1% NaN₃ (Sigma-Aldrich)) at room temperature for 1 h. After washing, the primary antibodies (CK4, CK19, and MUC5AC) at a dilution of 1:200 were incubated at 4 °C overnight. The next day, after extensive washing, the secondary antibody was incubated for 2 h at room temperature. Primary antibodies were not added in the negative control group. Positive control samples (native rabbit conjunctival tissue) were processed in parallel. The images were observed with a microscope (Olympus BX51).

Reverse transcription and qPCR

Total RNA was extracted from the conjunctival epithelial cell sheets using the RNeasy Mini Kit (Qiagen, Valencia, CA). The concentration and purity of the total RNA were determined spectrophotometrically at OD260 nm and OD280 nm. Quantitative-PCR (qPCR) analyses were performed with A260/A280 ratios between 1.9 and 2.1. cDNA was synthesized using a PrimeScript^TM RT reagent kit (TaKaRa, Dalian, China). qPCR was performed in a 20-μl solution containing a 10 μl reaction mixture, 1 μl cDNA, 2 μl primers (Table 1), and 7 μl ddH₂O. The reaction was conducted using a 7500 Real-Time PCR Detection System (Applied Biosystems, Irvine, CA) and activated at 95 °C for 10 min and 40 cycles of amplification (15 s at 95 °C and 1 min at 60 °C). Each sample was tested in triplicate [22]. The Pfaffl method was used to analyze the relative gene expression [23]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, endogenous control) was selected to normalize the gene expression.

Statistical analyses

The experimental results were expressed as the mean ± the standard derivation (SD). All experiments were performed in triplicate unless otherwise specified. Statistical analyses were performed using a two-sample equal variance Student t test, and a p value of less than 0.05 was considered to be statistically significant.

Morphology and phenotype of cultured rabbit conjunctival epithelial cells

The confluent rCjECs appeared to be compact, uniform, tightly joined, and the typical cobblestone shape (Figure 1A). As observed in the phalloidin staining (Figure 1B), the majority of the cultured cells appeared to be non-goblet epithelial cells with a small squamous morphology; a few cells contained vesicles in the cytoplasm and had the typical shape of goblet cells. PAS staining showed that a few cells were strongly stained, indicating the presence of mucin-secreting goblet cells (Figure 1C).

Immunofluorescence staining of CK4, CK19, and MUC5AC was used to identify the rCjECs. As shown in Figure 1D–F, the specific markers of these conjunctival epithelial cells were expressed in cultured cells, and the positive rate for MUC5AC-positive cells was approximately 21.5% ±1.65%. The results indicated that these cultured cells were conjunctival epithelial cells containing goblet cells.

Assessment of conjunctival epithelial cell sheets

After the rCjECs were seeded and cultured on a temperature-responsive culture dish for 4 days, the attached cells reached confluence, and the morphology of the conjunctival epithelial cells remained (Figure 2A). As shown in Figure 2B, strong PAS staining was found in a few cells, indicating the presence of conjunctival goblet cells. The cell sheets were harvested as a contiguous cell sheet by reducing the temperature to 20 °C without using a digestive enzymatic treatment (Figure 2C). H&E staining showed that the conjunctival epithelial cell sheets displayed a coherent, stratified structure, which was approximately four to five cell layers similar to the native conjunctival epithelium (Figure 2D).

To further study the cell sheets, immunohistochemistry was performed. As revealed in the immunostaining results (Figure 2E–G), the staining of CK4 and CK19, conjunctival putative specific cytokeratin, showed that numerous CK4-positive and CK19-positive cells were located on the conjunctival epithelial cell sheets. Examination of the MUC5AC staining showed that the goblet cells that occurred singly or in clusters were surrounded by conjunctival epithelial cells.

Proliferation and viability of conjunctival epithelial cells

BrdU staining was performed to reflect the cell proliferation. As shown in Figure 3A, the immunofluorescence analysis of BrdU was positive in part of the cells, and the proliferation rate was approximately 38.4±1.75%, indicating the cell sheets cultured on the temperature-responsive culture dish maintained proliferative ability. CCK-8 analysis was performed to observe the proliferation of the cells. Figure 3B shows that the cells proliferated well, and the number of the cells increased with the duration of the culture, indicating that the cell sheets retained proliferative cells, similar to what was observed for the BrdU staining.

Live and dead staining was used to evaluate the toxicity of the temperature-responsive culture dish. The results of the live and dead staining showed a low percentage of dead cells in the conjunctival epithelial cell sheets (Figure 3C).

Gene expression

The putative specific gene expression levels of the rCjECs were examined with qPCR. With the extension of the culture time, the expression of CK4 mRNA was apparently increased in the conjunctival epithelial cell sheets, and the expression of MUC5AC mRNA, which was of the most interest to us, was not notably decreased (Figure 4). The results demonstrated that CK4 and MUC5AC mRNA were stably expressed in the conjunctival epithelial cell sheets, indicating that the temperature-responsive culture dish did not have a negative effect on the proliferation and differentiation of conjunctival cells, especially goblet cells.