Cellcoat

Cells from representative treatments were transferred into 500 µL of staining culture media, centrifuged for 5 minutes at 500 × g at 4°C, and resuspended in 50 µL of staining culture media. The resuspended cells were transferred to 384 glass bottom imaging plate (cell culture microplate 384 well, CLEAR^Ⓡ, Poly-L-lysine, cellcoat^Ⓡ, Greiner bio-one). Images were acquired using a 20 × objective on a ZEISS LSM900 confocal microscope and analyzed using ZEISS ZEN-black software.

Immortalized human
corneal epithelial cells were maintained in immortalized corneal epithelial
cell media (IM-CEpiCM) supplemented with fetal bovine serum (5%),
corneal epithelial cell growth supplement (1%), and penicillin–streptomycin
(1%). The cells were kept at 37 °C with 5% CO₂ and
were cultured as described by the supplier with minor modifications.
For subcultures, the cells were washed with PBS, and cells were detached
by incubating with a TrypLE Express enzyme (Gibco) for 3–5
min at 37 °C. The addition of the medium (IM-CEpiCM) and centrifugation
at 1000 rpm (Mega Star 600R, VWR, Sweden) for 5 min were performed
for collection of cell pellet followed by removal of the supernatant
and resuspension of the cell pellet in a fresh medium. Cells were
counted using an EVE Automated Cell Counter (NanoEntek) and seeded
at ∼10,000 cells/cm² in collagen type I coated T-75
flasks (CELLCOAT, Greiner bio-one). The medium was changed every 2–3
days, and the cells were subcultured when reaching ∼90% confluency.

Wnt-3α-expressing cells were purchased from ATCC (CRL-2647). Cells were cultured in Greiner Bio-One CELLCOAT™ tissue culture-treated T-75 flasks at an initial concentration of 1.5 × 10⁶ cells. The cells were cultured in 20 mL of Wnt-3α growth medium (WGM) (Supplementary Table S1) and passaged once 75% confluency was reached. We used 0.4 mg/mL G-418 for selection and cells were kept growing on selection medium for a week before producing conditioned medium. The cells were lifted using 3 mL of TrypLE (Life Technologies, Carlsbad, CA, USA) and passaged. The process was repeated until approximately 40–50 T75 flasks were generated. The media were replaced with 100 mL of harvest medium (HM) (Supplementary Table S1). The cells were then incubated in HM for one week without media changes. After one week, the media were collected, and tubes were centrifuged at 500× g for 5 min at 8 °C. Following this step, the media were filtered using filter cups (Stericup Quick Release, Millipore, Burlington, MA, USA). Lastly, 25 mL of the collected harvested medium was separated into 50 mL canonical tubes and utilized fresh or stored for no more than 2 weeks at 4 °C. Recombinant human Wnt-3α (R&D, 5036-WN010) was used at a final conc. of 100 ng/mL as a positive control, and Wnt activity of conditioned medium could be examined by the TOP/FOP assay as described [49 (link)].