Abstract
Purpose :
In order to improve the very limited access to primary cells and thus the research on the aqueous form of dry eye, we have successfully established a lacrimal gland epithelial cell line. 3D cell cultures are particularly suitable for better and more precise characterization in order to test and better understand the function and properties of the cells in vitro. In the present study, we therefore tested different 3D cell cultures for our generated lacrimal gland epithelial cell line.
Methods :
Several types of 3D cell culture were tested. Spheroids were cultured in ultra-low attachment plates and in extracellular matrix. The spheroids were observed for 29 days. After 7 days, the spheroids were harvested and embedded in paraffin before sectioning and staining. Staining included DAPI (immunofluorescence), Azan and hematoxylin-eosin. In addition, spheroids were seeded in extracellular matrix, some in medium mixed with various growth factors such as EGF and FGF, and some in conventional culture medium. These spheroids were observed for 4 days. RT-PCR was performed to validate the continuous expression of lacrimal gland epithelial genes detected in 2D cell culture.
Results :
Cultivation on ultra-low attachment plates allows easy and rapid growth of large numbers of spheroids under normal cell culture conditions. The spheroids showed compaction to smaller diameters and reduced translucency over a period of 29 days. When the spheroids were reseeded onto 2D cell culture plates, the 2D cell growth remained unchanged. Immunofluorescence showed DAPI staining throughout the spheroids. Azan and hematoxylin-eosin staining showed a higher density in the outer layers of the spheroids, indicating the formation of an epithelial barrier to the outside. Azan staining showed no connective tissue. After 2 days, budding was induced in the spheroids grown in an extracellular matrix with growth factors. They also showed outgrowing cells, indicating the partially epithelial and mesenchymal character of the cell line. RT-PCR confirmed the continuous expression of lacrimal gland epithelial genes.
Conclusions :
This project proposes simple methods to generate a 3D in vitro model using the newly established human lacrimal gland epithelial cell line. These spheroids are a useful tool for a better understanding of the processes in the human lacrimal gland. They can also be used to test potential drugs and simulate pathological conditions.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.