Culture of Human Conjunctival Epithelial Cells Recovered by Brush Cytology

Abstract

Purpose:: To use minimally invasive techniques to recover and subsequently culture viable cells from the ocular surface. To our knowledge, this goal has not been accomplished at the ocular level, although some attempts are being made at other mucosal sites.

Methods:: Brush cytology to recover conjunctival epithelial cells was performed in superior tarsal conjunctiva of 54 healthy donors (31 females, 23 males; mean age 63 ± 14.8 years). Recovered cells were counted and initial viability was assessed by Trypan Blue dye exclusion test. Cells were seeded onto Permanox^TM or denuded amniotic membrane (dAM). Three different transport and culture media were used: medium 1 [Ca²⁺ 0.09 mM]= serum-free DK-SFM supplemented with EGF, bFGF and insulin; medium 2 [Ca²⁺ 1.16 mM]= DMEM/F12 supplemented with 2 ng/ml EGF, 1 µg/ml insulin, 0.1 µg/ml cholera toxin, 0.5 µg/ml hydrocortisone, 10% FBS; medium 3 [Ca²⁺ 1.2 mM]= DMEM/F12 supplemented with 10 ng/ml EGF, 5 µg/ml insulin, 0.1 µg/ml cholera toxin, 0.5 µg/ml hydrocortisone, 20% FBS. Cell viability of plated cells was assessed after 1, 2, and 5 days in culture using the Calcein/EthDIII kit by fluorescence microscopy. Three independent experiments were performed for each condition. As control, the IOBA-NHC cell line from healthy human conjunctival epithelium was used. Recovered cells were characterized by flow cytometry using cytokeratin 7-FITC as epithelial marker and CD45-ECD as panleukocyte marker. ANOVA analysis was performed for statistical comparisons.

Results:: The average number of recovered cells was 13 x 10⁴ (initial mean viability: 20 ± 3.3 %). Of those, 95% were epithelial and 5% were leukocytes, as determinated by flow cytometry. Initial cell recovery was significantly lower with culture medium 1 than with the other media. Medium 2 was significantly better in terms of supporting cell viability in culture, regardless of the substrate used. Multifactorial analysis showed a significantly higher number of viable cells with culture medium 2 and dAM at day 1. Under these conditions, cell survival ratio in attached cells was 68.2% at day 2 and 48.0% at day 5; this survival decrease from day 1, however, was not statistically significant.

Conclusions:: Brush cytology and the appropiate culture medium recovers enough quantity of viable to stablish initial cell cultures. Whether these cells will further survive and proliferate is yet to be determined. These results open the possibility to remove and culture diseased ocular surface epithelial cells from any patient and at different time-points.

Keywords: conjunctiva • cell survival • calcium