Characterization and short-term culture of cells recovered from human conjunctival epithelium by minimally invasive means

Abstract

Purpose

To characterize conjunctival cells obtained by brush cytology (BC) and establish short-term cultures.

Methods

Human tarsal and bulbar conjunctival cells were obtained by BC and transported in 3 different media: serum-free medium (DK-SFM) with low [Ca ²⁺], 10% fetal bovine serum (FBS) supplemented medium (FBSm10), and 20% FBS-supplemented medium (FBSm20). Recovered cells were counted and initial viability assessed. Flow cytometry established epithelial or immune lineage, viability, apoptosis, and cell cycle stage. To establish short-term cultures, tarsal conjunctival cells were seeded onto Permanox™ or denuded amniotic membrane (dAM) and cultured in the 3 media. Living adherent cells were assessed on Days 1, 2, and 5 by fluorescence microscopy.

Results

Initial cell recovery was significantly lower with DK-SFM than in the other two culture media. Flow cytometry showed that 3.8±0.4% of recovered tarsal cells were CD45+ leukocytes and 67.9±1.6% were CK7+ secretory epithelial cells. S-phase cells composed 3.5±0.3% of the recovered tarsal cells and 2.1±0.2% of the bulbar cells (p=0.0006). The percentage of viable, apoptotic, and dead cells was similar for tarsal and bulbar cells. Two different cell populations were observed in both locations. About 24% consisted of smaller, less complex cells with high viability, and the remainder was composed of larger, more complex cells with poor viability. Significantly more living cells were supported by FBSm10 on the dAM substratum (p =0.011) than by the other media on either dAM or Permanox.

Conclusions

Conjunctival BC recovers proliferating cells that can be maintained on dAM in FBSm10 for up to 5 days.