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Geeta K Vemuganti; . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):302.
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© ARVO (1962-2015); The Authors (2016-present)
Lacrimal gland dysfunction leading to tear film deficiency and instability is an important cause of ocular morbidity. Restoration of function by transplantation of autologous ex-vivo expanded stem cells could be explored as a long term management option. We have previously reported the presence of stem-like and functionally competent differentiated cells in in-vitro cultures of human lacrimal gland (Hu LG). The present study was aimed at identifying the proportion of stem cells in the 2D and 3D cultures of Hu LG.<br />
Fresh human lacrimal gland tissues (n=5) from patients undergoing exenteration were harvested for cultures after IRB approval. The gland was processed by enzymatic digestion using a cocktail of collagenase and hyaluronidase. The isolated cells were plated as groups of two-three cells on Matrigel coated plates for adherent cultures and ultralow attachment plates for non-adherent lacrisphere cultures. The growth media used was HepatoStim supplemented with l-glutamine, epidermal growth factor, fibroblast growth factor, N2 and antibiotics. The day 14-16 cultures were evaluated for stem cells through CD117 expression, BrdU label retention, clonality, proportion of cells in the G0/G1 phase by cell cycle analysis and secretory product quantification.
Both adherent monolayer and non-adherent lacrispheres showed the presence of stem cells. CD117 expression was seen in 0.2 ±0.05% of the cells under adherent conditions and this percentage increased four-fold to 0.8% when the cells were grown as lacrispheres. The cell cycle analysis also shows the presence of higher proportion of cells (76.9%) in the G0/G1 phase in lacrispheres as compared to adherent cultures (66.9%). Results of label retention studies show 27.9±4.3% of the cells in adherent culture as quiescent as compared to 9.3±0.41% in the lacrispheres. Clone formation was also seen in lacrispheres with a CFU of 3.1%. Secretory function evaluation showed a lesser amount of secreted components by the lacrispheres as compared to the adherent culture.
The present study provides evidence that 3 D lacrispheres are possibly a better source of stem cells which could be evaluated for the regeneration of the functionally compromised gland.
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