Abstract
Purpose::
The possibility to culture cells from ocular surface epithelium obtained by minimally invasive techniques, such as Impression Cytology (IC) or Brush Cytology (BC) has not been developed yet. This research intends to test different variables in order to recover the highest possible numbers of viable cells from IC and BC samples.
Methods::
Epithelial cells were obtained from superior bulbar or tarsal conjunctiva of healthy donors by IC (n=32) or BC (n=18). Polyether sulfone filters (pore size 0.20µm) were used for IC and CytobrushTM-plus GT for BC. By IC, a first sample was collected (superficial IC), followed by a second one at the exact same spot (deep IC). Cells were removed from filters by i) vortex agitation, ii) dispase digestion for 1, 5, 10 or 15 min, or iii) placing filters onto plastic culture dishes as ‘explants’. DMEM/F12 culture medium supplemented with 2 ng/ml EGF, 1 µg/ml insulin, 0.1 µg/ml cholera toxin, 0.5 µg/ml hydrocortisone, and 10% FBS was used to maintain cells. As controls, full-thickness biopsies (n=3) were taken after superficial and deep ICs at the same spot. Also, cell removal from filters was checked by staining with PAS-Papanicolau. BC was performed after superficial IC and cells were detached by shaking the brush in culture medium. This procedure was repeated three times. Detached cells from both IC filters and BC were counted and viability assessed using the Trypan Blue dye exclusion test.
Results::
Conjunctival biopsies showed that after two ICs, only the basal and sometimes one more layer remained, whereas 2-3 layers remained with only a superficial IC. For IC, dispase digestion for 10 min yielded the highest cell numbers (21 x 103) and the best viability (17.6%). However, by BC the average cell number recovered was 136 x 103 and the average viability was 21%.
Conclusions::
BC seems to be preferable over IC to recover viable cells. Additionally, other interdependent variables have shown to affect the retrieval of viable cells from conjunctival epithelium. BC seems to show promise as those viable cells could be further cultured and used for in vitro assays.
Keywords: conjunctiva • cell survival • clinical research methodology