Abstract
Purpose :
The scarcity of in vitro models is an important drawback to study conjunctiva pathophysiology. The Immortalized Human Conjunctival Epithelial Cell line IM-HConEpiC is commercially available, but has not been widely used in research, yet. The aim of this study is to characterize this cell line and to use it as a source of conjunctival extracellular vesicles (EVs).
Methods :
IM-HConEpiC cells were cultured with DMEM/F12 supplemented medium. Total protein was isolated in RIPA buffer and used for Western blot (WB). RNA was isolated in RLT lysis buffer and used for reverse transcription-PCR. Cells grown in slides were fixed and used for immunofluorescence (IF) microscopy. The expression of cytokeratin (CK) 7, CK8+18, CK19, E-cadherin, ZO-1, mucin (MUC) 4, and MUC5AC was analyzed by WB, IF, and/or PCR. IM-HConEpiC cells (passages 7-20) were also used to obtain EVs. Cells were grown to 70% confluence and treated with EVs free-FBS supplemented medium for 48 h. Medium was collected and EVs obtained by differential ultracentrifugation (UC) using two different methods (M): M1, at 4°C, with SW28 rotor, and M2, at 20°C and combination of SW28/SW60Ti rotors. All EVs were resuspended in PBS/trehalose and stored at -80°C for further analysis. The ExoStep Kit (ImmunoStep) was used for the detection and determination of the concentration of EVs by flow cytometry using a Beckman Coulter Navios cytometer. A Beckman Coulter CytoFLEX LX flow cytometer was used to individually characterize the EV content. EVs were morphologically analyzed by atomic force microscopy.
Results :
IM-HConEpiC cells expressed CK7, CK8+18, CK19, E-cadherin, ZO-1, MUC4, and MUC5AC, as demonstrated by WB, IF and/or PCR. EVs were isolated from IM-HConEpiC with the two methods. The CytoFLEX results showed that IM-HConEpiC-EVs expressed the EV markers: CD9, CD63, CD81 and CD147 and that the majority of the EV subpopulation was made up of small EVs, with 66.06±2.23% (M1) and 62.63±0.4% (M2) having a similar violet SSC to 80nm Apogee beads.
Conclusions :
Cultured IM-HConEpiC cells express typical conjunctival epithelial markers, suggesting that this cell line is a valuable tool for in vitro research. Although the EVs were properly isolated from the conditioned supernatants by UC, an exhaustive analysis is necessary to elucidate the abundance and composition of the different populations.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.