Localisation of epithelial cells capable of holoclone formation in vitro and direct interaction with stromal cells in the native human limbal crypt.

1.

Ethics Statement

All human tissue was handled according to the tenets of the Declaration of Helsinki and written consent was acquired from next of kin of all deceased donors regarding eye donation for research. Research consent was obtained via the Moorfields Eye Hospital Lions Eye Bank (U.K) http://www.moorfields.nhs.uk/Aboutus/Clinicalsupportservices/Eyebank and Lions Eye Institute (Florida, U.S) http://www.fleb.org/ . All experiments were approved by the National Research Ethics Service, South west 3 REC, reference 10/H0106/57. 2.

Isolation and Culture of Limbal Epithelial Cells

Limbal palisades, which surround the LCs were macroscopically identified under a dissecting microscope. After dissection, crypt-rich (from superior and inferior limbus) and non-crypt rich (from temporal and nasal limbus) limbal biopsies isolated from the same donor were transferred separately into a solution containing 1.2 U/mL dispase II (Roche diagnostics GmbH, Mannheim, Germany) in corneal epithelial cell culture medium (CECM) containing a 1∶1 ratio of DMEM:F12, 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL Fungizone, epidermal growth factor (EGF) 10 ng/mL (Life technologies, Paisley, UK), hydrocortisone (0.4 μg/mL), insulin (5 μg/mL), adenine (0.18 mM), transferrin (5 μg/mL), T3 (2 nM), cholera toxin (0.1 nM) (Sigma-Aldrich, Dorset, UK) and incubated for 2 hours at 37°C.

Limbal epithelial cells

(LECs) were isolated from the crypt-rich and non-crypt rich biopsies by gently scraping the epithelium using the point of thin forceps. LECs were then pre-expanded for 7 days in T25 flasks on a 3T3 feeder layer that had been previously growth arrested with 4 μg/mL mitomycin C (Sigma-Aldrich, Dorset, UK) for 2 hours. CECM culture medium was changed three times a week and the co-cultures maintained at 37°C in a humidified atmosphere containing 5% CO 2 in air. 3.

Clonal Analysis

Cadaveric human corneas were obtained from three donors (age range 51–71 years; mean 58.3 years). In total, 124 clones isolated from the crypt-rich and non-crypt limbal biopsies from three donors were analyzed for their in vitro growth potential. When they had reached 50% confluence in primary 3T3-LECs co-cultures, the LECs were isolated using serial trypsinization. The 3T3 feeder cells were detached and removed using 0.05% trypsin-0,02% EDTA, before 0.5% trypsin-0.2% EDTA (Life technologies, Paisley, UK) was used to detach and prepare a single cell suspension of LECs. For each donor, 250 single LECs initially isolated from crypt-rich or from non-crypt limbal biopsies were seeded onto 55 mm 2 plates containing 1.4×10 6 growth-arrested 3T3s. Plates were then checked under an inverted microscope to confirm seeding of a single LECs suspension on top of growth arrested 3T3s. After 7 days in culture, small colonies (clones) of approximately 1 mm diameter were randomly selected and isolated using 8 mm sterile cloning cylinders (Sigma-Aldrich, Dorset, UK) and 0.5% trypsin-0.2% EDTA. Limbal epithelial cells from these single cell colonies (clone) were again seeded onto a 55 mm 2 plate containing 1.4×10 6 growth-arrested 3T3s. Epithelial cells were expanded for up to 12 days and plates fixed with 4% paraformaldehyde (PFA) (VWR International Ltd. Leicestershire, UK) and stained with 2% rhodamine. Each plate was then scored as holoclone, meroclone or paraclone depending on the percentage of aborted colonies [20] . When 0–5% of the total colonies were terminally differentiated, the clone was scored as a holoclone. When more than 95% of colonies were terminally differentiated, the clone was scored as a paraclone. Finally, when >5% but