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K. Kasper, S. Schrader, D. Deininger, G. Geerling; Cultivation of Rabbit Acinar Lacrimal Gland Cells on Denuded and Undenuded Amniotic Membrane With and Without Co-Cultivation of 3T3 Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4182.
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© ARVO (1962-2015); The Authors (2016-present)
The human lacrimal gland provides about 95% of the aqeous tears, which are essential for the integrity of the ocular surface. In a cell culture model of primary rabbit acinar lacrimal gland cells amniotic membrane (AM) was already used as a carrier with good results on long term cultivation, morphology and cell function. Aim of this study was to compare the effect of denuded and undenuded AM with and without co-cultivation of growth arrested 3T3 fibroblasts on the proliferation and secretory function of rabbit lacrimal gland acinar cells. Furthermore, lacrimal gland cells were seeded directly on a 3T3 feeder layer and characterised by immunostaining for pan-cytokeratin.
Lacrimal gland acinar cells from New Zealand White rabbits were isolated and cultured in modified HSM-medium. AM was denuded by incubation with dispase and scraping of the epithelial cells. 3T3 fibroblasts were growth arrested by the use of MMC. Cell morphology was analysed by light- and electron-microscopy. Secretory response to carbachol was tested by measuring the ß-hexosaminidase activity. The detection of cytokeratin was performed with an antibody that binds to cytokeratin 5, 6, 8, 17 and 19 (pan-cytokeratin MNF116).
In all groups a secretory response was found up to 28 days . There was an increased secretory response and more multilayering in cells cultured on denuded AM compared to undenuded AM. The co-cultivation with a 3T3 fibroblast feeder layer did not alter morphology and function of the cultured lacrimal epithelial cells. Electron microscopy showed good cell-to-cell contacts and apical ductuli . After 7 days of culture pan-cytokeratin positive cell colonies were found surrounded by actin positive feeder layer fibroblasts.
Denuding of the AM but not co-cultivation with 3T3 fibroblasts seems to be advantageous for expanding lacrimal gland epithelial cells. Further optimization of culture conditions is required to maintain and expand functional lacrimal gland cells in vitro with the eventual aim to engineer a functional lacrimal gland construct.
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