Abstract
Purpose :
The exact cellular mechanism of lacrimal gland (LG) function is not fully understood, especially in the case of ductal cells (LGDC). These cells are only available in limited quantities for scientific research mainly due to the limitations in their accessibility from the LG. In addition, until recently there was no method to maintain LGDC culture for extended periods of time. The development of organoid technology over the last decade has opened up the possibility to not only maintain but also to propagate and culture different cell types. Our aim was to establish three-dimensional LGDC cultures in order to investigate their ability to reproduce the morphological and functional characteristics of LGDCs.
Methods :
Ductal sections from the LG of 12-16 weeks old FVB/N mice were isolated using the isolation method previously developed in our laboratory. Ion channels and transporters of organoids were detected by immunofluorescent staining, and video microscopy was used for functional analysis.
Results :
The effect of two growth factors (fibroblast-derived growth factor 10 and the epithelial-derived growth factor), previously described as key molecules in the development of the LG, were investigated. The most effective concentration was 100 ng/ml and 500 ng/ml, respectively. Immunostaining were performed on the organoid structures to detect membrane transporters and receptors characteristic to the duct cells. Cystic fibrosis transmembrane conductance regulator and aquaporin 4 water channel protein, vasoactive intestinal polypeptide 1 and 2 receptors could be detected on the organoid cells through passage 0 to passage 4. In functional assays, phenylephrine (10 μM), carbachol (100 μM) and 8-bromo-cAMP (100 μM) were used as secretagogues. All these compounds could induce secretory response (shrinkage) of the organoid structures.
Conclusions :
Method for maintaining and propagating LG duct epithelial cells has been successfully established. The organoids proved to be similar in structure to the original isolated ductal cells but differed from them in the polarization of the epithelial cells. The cell cultures maintained their morphological specificity and functionality throughout the passages thus they are considered suitable for further experiments.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.