Abstract
Purpose :
To assess toxicity and biocompatibility of ophthalmic materials, solutions and therapeutics, stratified human corneal epithelial cell (HCEC) cultures provide a better mimic of the ocular surface. Flow cytometry has been used extensively to assess cell response in in vitro monolayer models, but few studies have explored its use with stratified models. This study was undertaken to characterize the multilayers of a stratified HCEC model. Following a two-stage digestion protocol, apoptosis and expression of intracellular adhesion molecule (ICAM-1), integrin α3, and β1 on cells from the “superficial” and “deep” layers were characterized.
Methods :
HPV-immortalized HCEC were cultured on curved inserts with keratinocyte serum-free medium supplemented with growth factors (KM) on both apical (top) and basal (bottom) of the inserts for 7 days (Postnikoff et al, 2014). On day 7, cell differentiation was induced by exposing the monolayer to an air-liquid interface; cells were fed only on the basal side with 2% FBS in DMEM/F12 (DF) for 7 days, with DF changed daily. On day 14, stratified cultures were exposed to one of the following conditions for 2, 6 and 24 hrs: DF or KM on the basal side with or without KM on the apical side. Some samples were exposed to Benzalkonium chloride (BAK) (0.002% final concentration) or wore a PBS-soaked contact lens. The superficial and deep layers were detached via incubation with TryplExpress for 30 and 90 min respectively. After each digestion stage, cells were collected, washed, and incubated with caspase and antibodies and samples were analysed by flow cytometry.
Results :
Regardless of conditions, significant differences in ICAM-1, α3 and β1 expression were observed between cells from the superficial and deep layers. KM on the apical side did not affect receptor expression. Incubation with or without a PBS-soaked lens had similar receptor expression while exposure to BAK led to significant differential changes in receptor expression, apoptosis and necrosis in cells from the superficial and deep layers.
Conclusions :
A two-stage digestion protocol was successfully implemented with a stratified HCEC model. Flow cytometry represents an effective tool to characterize apoptosis and expression of membrane receptors on cells from different layers. The proposed methodology enables to quantitatively assess cell response in biocompatibility studies with stratified HCEC models.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.