Dispase 1

Subcutaneous fat was removed from the skin with a scalpel, and the whole skin was incubated in Hank’s balanced salt solution (HBSS; 1.25 mM CaCl₂ and 0.81 mM MgSO₄; Sigma) containing 10% CS and dispase I (25 U/ml, Wako) for 20 min at 37 °C. Dermal cell suspensions were obtained by scraping the skin gently and incubating in HBSS containing 10% CS, collagenase I (1.25 mg/ml, Life Technology), collagenase II (0.5 mg/ml, Millipore), collagenase IV (0.5 mg, Sigma), hyaluronidase (0.1 mg/ml, Sigma) and DNase I (50 U/ml, TaKaRa) for 30 min at 37 °C under gentle shaking. The cells were then filtered through 70-μm strainers, collected by centrifugation (300 × g, 5 min) and washed with HBSS containing 10% CS.

Primary TTFs were isolated from 12–16-week-old male C57BL/6 mice. Tail tips were washed in PBS, minced, and incubated in DMEM containing 1000 U/ml dispase I (Wako) and 2 mg/ml collagenase type 2 (Worthington) at 37 °C for 30 min. After digestion, an equal amount of DMEM containing 10% FBS and 1% PS was added, followed by gentle pipetting. The tissue/medium mixture was filtered through a 100 μm nylon Cell Strainer. The filtered cells were collected by centrifugation and resuspended in fresh DMEM supplemented with 10% FBS and 1% PS, and seeded in plastic dishes. Attached TTFs were used 4-5 days after incubation.

Eyes of Japanese white rabbits were purchased from Funakoshi Corporation (Tokyo, Japan). Rabbit corneal epithelial cells were prepared and cultured according to a modified version of the previously reported method31 (link). In brief, limbal tissues were removed from the eyes, and the half-layers of endothelial side were then mechanically removed by surgical scissors. The remaining corneal tissues were transferred to minimum essential keratinocyte serum-free medium (KSFM; Life Technologies, California, USA) ([Ca²⁺] = 0.4 mM) containing dispase I (Wako Pure Chemical Industries, Osaka, Japan) at 250 PU/mL and then incubated at 4 °C for 24 hours and 37 °C for 1 hour. The corneal epithelial cells were then peeled and further incubated with TrypLE^TM select (Life Technologies, California, USA) at 4 °C for 30 minutes and 37 °C for 5 minutes to dissociate adhesion between the cells. The prepared rabbit CECs were suspended in serum-free CnT-20 medium (CELLnTEC, Santa Cruz, CA, USA) containing 1% Antibiotic-Antimyotic (Life Technologies Corporation, Carlsbad, CA USA) to isolate and grow CECs and then plated in collagen-coated tissue culture plates (AGC Techno Grass Co., Ltd., Shizuoka, Japan). The rabbit CECs were then cultured at 37 °C with 5% CO₂, and the medium was replaced with fresh medium every 48 hours until the cells reached subconfluence.

Japanese white rabbit eyes were purchased from Funakoshi Corporation (Tokyo, Japan). Rabbit corneal epithelial cells were prepared and cultured according to a previously reported method35 (link). Briefly, the corneas were removed from the eyes, and the half-layers of the endothelial side were then mechanically removed by use of surgical scissors. The remaining corneal tissues were transferred to a minimum essential keratinocyte serum-free medium (Life Technologies, Carlsbad, CA) containing dispase I (Wako Pure Chemical Industries, Osaka, Japan). The tissues were then incubated at 4 °C for 24 hours and at 37 °C for 1 hour. The resultant corneal epithelium was then peeled and further incubated with TrypLE^TM Select (Life Technologies) trypsin replacement enzyme at 4 °C for 30 minutes and at 37 °C for 5 minutes to dissociate the cells. The prepared rabbit CECs were suspended in CnT-20 medium (CELLnTEC, Santa Cruz, CA) and plated in collagen-coated tissue culture plates (AGC Techno Glass Co., Ltd., Shizuoka, Japan).

Conventional organoids and cyst-enriched organoids were dissociated into single cells in the same way as the monolayer culture. These cells were embedded with Matrigel and cultured in expansion medium supplemented with 10 μM Y-27632 for 4 days. Organoid growth was measured using CellTiter-Glo^® 3D Cell Viability Assay (Promega) after solubilizing Matrigel with dispase (Dispase I, FUJIFILM Wako Pure Chemical). For the Imaging analysis was performed with cellSens (OLYMPUS) according the manufacturer. The formation of an organoid was defined to be when the major axis of its structure was measured to be over 100 μm in the bright-field image.