Cultivation of lacrimal gland acinar cells in a microgravity environment.

BACKGROUND: A rotary cell-culture system (RCCS) allows the creation of a microgravity environment of low shear force, high-mass transfer and three-dimensional cell culture of various cell types. The aim of the study was to evaluate the growth pattern and the secretory function of rabbit lacrimal gland acinar cells in a microgravity environment using an RCCS. METHODS: Lacrimal gland acinar cells from male New Zealand White rabbits were isolated and cultured in an RCCS up to 28 days. Cells were analysed by light and electron microscopy, and apoptosis was assessed by the TUNEL assay at days 7, 14, 21 and 28. Secretory function was tested by measuring the beta-hexosaminidase activity. RESULTS: After 7 days of culture, spheroidal aggregates were found inside the RCCS. The spheroids consisted of acinus-like cell conglomerates. Apoptotic centres inside the spheroids were observed at all time points by means of the TUNEL assay. Evaluation of the secretory function revealed beta-hexosaminidase release after carbachol stimulation which decreased over the culture period. CONCLUSION: A simulated microgravity environment promotes the development of three-dimensional cell spheroids containing viable acinar cells up to 28 days. Due to the evolving central apoptosis, it is unlikely that such simple three-dimensional cell communities can serve as tissue equivalents for clinical transplantation, but they promise opportunities for further applications in basic and applied cell research on lacrimal gland cells.

[Toxicity of a new moistening agent and preservative in vitro]. / Toxizität neuer Benetzungs- und Konservierungsmittel in vitro.

PURPOSE: The use of preservatives such as benzalkonium chloride (BAC) usually increases the toxicity of pharmaceutical tear substitutes. HP-guar has been recently introduced as a new artificial tear substitute and includes the preservative Polyquad (0.001%), which is considered to be non-toxic. We therefore examined the effect of preserved (cetrimide 0.01%) and unpreserved HPMC (hydroxypropylmethyl cellulose) and HP-guar in dose and time-response experiments in a human corneal and conjunctival epithelial cell culture model. METHODS: Immortalized human conjunctival and corneal epithelial cells were cultured in 96-well plates at 37 degrees C with 5% CO(2) and exposed to the test solutions. The ATP content was quantified by means of a luminescence-based ATP assay, intracellular esterase activity by double fluorescent viability staining (calcein AM/ethidium homodimer D-1) and cell migration by a colony dispersion assay. All experiments were performed in triplicate and repeated at least once. The significance of differences was determined with an unpaired two-sided t-test. RESULTS: HPMC with preservative severely reduced the ATP content at all concentrations tested. Unpreserved HPMC, however, showed an inhibition of ATP production only at 100% and good esterase activity. HP-guar with and without preservative were found to reduce ATP activity more than unpreserved HPMC, but the unpreserved solution was found to reduce cellular ATP levels significantly more than the preserved solution. CONCLUSIONS: The new preservative Polyquad induced significantly less cytotoxicity than cetrimide. However, even unpreserved HP-guar can induce cytotoxicity in vitro, while unpreserved HPMC remains a good alternative tear substitute with low cytotoxicity.

Amniotic membrane as a carrier for lacrimal gland acinar cells.

BACKGROUND: The secretion of the lacrimal gland provides 95% of the aqueous tears, which are essential for lubrication, nutrition and protection of the ocular surface. Long-term studies of acinar lacrimal gland cells in vitro are complicated by low proliferation rate and fast loss of cell function on plastic. Aim of this study was to evaluate the growth pattern and the secretory function of lacrimal gland acinar cells on amniotic membrane (AM) in a rabbit model. METHODS: Lacrimal gland acinar cells from Chinchilla Bastard and New Zealand White rabbits of both sexes were isolated and cultured on denuded amniotic membrane. Cells were analysed by light and electron microscopy. Secretory function was tested by measuring the beta-hexosaminidase activity. RESULTS: Three days after seeding to the amniotic membrane, the acinar cells had attached to each other and formed small cluster. Cell clusters consisted of 2-5 cell layers, and the cells showed fine granulation in their cytoplasm, typical for secreting cells. Between days 7 and 14 cell clusters increased in size, and acini-like structures with a central lumen were found. Cells showed polarity, with a basal nucleus and apical secretory granules. Between days 21 and 28 acini-like structures were still found inside the cell clusters. Accumulation of secretory material in the central lumen and desmosome formation connecting the apical cell structures was frequently evident. However, the number of cytoplasmatic granules decreased, and on parts of the AM, cell morphology changed to flat, spindle-shaped cells with a small nucleus. Stimulation with carbachol showed a strong beta-hexosaminidase release until day 7, with a decreasing secretory function detectable until day 21. CONCLUSION: Acinar lacrimal gland cells can be successfully cultured on amniotic membrane up to 28 days, with a secretory response to carbachol up to 21 days. This model may be used for further experimental work, to elucidate cellular mechanisms in normal and diseased lacrimal tissue.