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Mathias Roth, Kristina Spaniol, Gerd Geerling, Stefan Schrader; Hypoxia improves the maintenance of murine lacrimal gland MSC in vitro. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1495.
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© ARVO (1962-2015); The Authors (2016-present)
A possible causal treatment strategy for severe dry eye syndrome due to lacrimal gland insufficiency is the regeneration of lacrimal gland tissue by transplantation of in vitro expanded autologous or allogenic lacrimal gland mesenchymal stem cells (MSC). Aim of the present study was to evaluate the effects of low oxygen pressure on the expansion of murine lacrimal gland derived MSC in vitro.
MSC isolated from lacrimal glands of C57BL/6 mice were grown either under routine culture conditions (95% air, 21% O2 respectively, 5% CO2 , 37°C; “normoxia”) or under low oxygen pressure (95% N, 5% O2, 5% CO2, 37°C; “hypoxia”). Cells from both culture conditions were characterized and compared by flow-cytometry and immunhistochemistry. Proliferation was evaluated by CFU-F and additionally a defined number of cells was seeded using a cell sorter and counted by flow-cytometry after 7 and 14 days. Cells were differentiated into adipocytes and osteocytes and the differentiation potential was evaluated by Oil-Red-O- / Alizarin-Red staining and qPCR.
All cells showed positivity for the minimal criteria markers for MSC. Furthermore, expression of mesenchymal stemcell markers desmin, vimentin, musashi-1, nestin and SCA-1 was observed. Cells cultured in hypoxia showed a higher proliferation rate and a more homogenous morphology. Cells cultured in normoxia showed a more heterogenous morphology and expressed more stress fibers. CFU-F assays revealed a significantly higher number of colonies in cells cultured in hypoxia. Also the differentiation potential seemed to be higher in cells cultured in hypoxia, especially for differentiation into osteocytes.
In summary hypoxia improves the maintenance of murine lacrimal gland MSC during in vitro expansion. Thus a low oxygen culture system might be beneficial for the expansion of lacrimal gland MSC for regeneration of lacrimal gland tissue in vivo. However more studies are needed to further optimize the culture conditions and to evaluate the effects of hypoxia in in vivo experiments.
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