Purchase this article with an account.
A.M. Wright, M. McKee, A. Renaud, P. Ranganathan; Cytometric Viability of Human Corneal Epithelial Cells Grown on Tissue Culture Plastic and a Biomaterial . Invest. Ophthalmol. Vis. Sci. 2005;46(13):871.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate cytometric methods to evaluate cellular viability using in vitro cell culture of SV–40 transformed human corneal cells (HCE–T) on tissue culture plastic (TCP) and a high oxygen permeable silicone hydrogel biomaterial (HiDk SiHy). Methods: Cell viability assay used were, Alamar BlueTM (AB), a resazurin based dye, and the Live/Dead Viability/Cytotoxicity Kit from Molecular Probes (LDVC) and the cytometric bead assay(CBA) for proinflammatory cytokines. HCE–T cells were seeded on TCP and HiDk SiHy(lotrafilcon A soft contact lenses). AB was added to the cells, incubated and fluorescence reading taken at exc =530nm, em =580 nm of the oxidized and reduced AB using a CytoFluor®, (PerSeptive Biosystems). Supernatants were harvested and frozen for cytokine analysis; IL–6, IL–8, TNF–α, IL1–ß, IL10 and IL12p70. HCE–T cells were removed, spun and characterized by double staining with calcein and ethidium homodimer –1 (CEH). Flow cytometric analysis was carried out at 488 nm on a BDFACSCailburTM 4 color unit cytometer. Subpopulations were identified and sorted by regions for live (green fluorescent) and dead (red fluorescent) cells. Benzalkonium chloride (BAC) was used as a positive control for the analysis of dead cells. Phorbol ester (PMA) was used as a stimuli to induce cytokines. Results:HCE–T cells grown on HiDk SiHy did not show any difference visually or with AB from controls grown on TCP. BAC exposed cells at or below 5.0 ppm showed AB cell viability above 50% at 24 hours. Cytokines were not upregulated for HCE–T cells grown on HiDk SiHy compared TCP. HCE–T cells grown on TCP were found to be 98% live cells. 10 ppm BAC treated cells were found 95% dead by region gating. Cells grown on the HiDk SiHy were found to be 80% live and 20% damaged cells. Conclusions: Alamar Blue and Live/Dead flow cytometry studies demonstrate the biocompatibility of SiHy BioM with HCE–T cells. No upregulation of proinflammatory cytokines was observed for HCE–T cells grown on HiDk SiHy further suggesting biocompatibility with the ocular environment. These tests with other toxicological tests provide valuable information for the evaluation of new biomaterials or solutions. HCE–T cells are biocompatible with HiDk SiHy and support the use of lotrafilcon A; a HiDk SiHy extended wear– high oxygen permeability contact lens; as a bandage contact lens for corneal protection and healing after insult or surgery.
This PDF is available to Subscribers Only