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Isobel Massie, Kristina Spaniol, Gerd Geerling, Marco Metzger, Stefan Schrader; Formation of functionally competent 3D lacrimal gland cell spheroids - towards regeneration of lacrimal gland tissue. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Lacrimal gland (LG) dysfunction can lead to severe dry eye syndrome (DES), which can cause severe pain and visual impairment. Treatments for DES are palliative, so reconstruction of LG tissue is desirable. Since native LG comprises at least epithelial, mesenchymal and endothelial cells, we aimed to form functional 3D LG spheroids comprising these 3 cell types.
Explants were used to initiate epithelial and mesenchymal stem cells (MSC) cultures from porcine LG in 21 and 5% O2 respectively. Endothelial cells were isolated from human foreskin. Isolated cells were characterised using immunocytochemistry, flow cytometry, and multi-lineage differentiation assays. A total of 1x106 epithelial, endothelial, and MSC cells were mixed (ratio 10:8:2) and plated onto tissue culture plastic or 50% v/v Matrigel. Macroscopic images were captured over time for 1 week. Spheroids were stained with haematoxylin and eosin and thicknesses measured. Functional capacity was assessed by β-hexosaminidase assay with carbachol (CCH) as stimulant, and lactoferrin ELISA out to day 3. Data are N=3 +/- SD.
In 2D, LG epithelial cells were Rab3D and HexA+, endothelial cells were von Willebrand factor and CD31+. LG MSC cells were CD31-, CD44 and CD105+ and culture in adipo- or osteogenic differentiation media triggered formation of lipid droplets or calcium deposits respectively. When the cell mix was plated onto plastic, there was no cell contraction/spheroid formation. After 18 hours on Matrigel, cells contracted to form spheroids with en face areas of 0.11+/-0.002cm2 (p<0.05 cf. initial seeding area, 1.1cm2); there was no further contraction after this (p>0.05). Spheroid thickness was 149+/-54µm. Both 3D spheroids and 2D monolayer cultures responded to CCH (~2.5-fold increase over baseline), but activity for both fell over time. Baseline and CCH activities were ~2-fold greater from spheroids cf. monolayer (p<0.01 on days 1 and 2). Lactoferrin production from spheroids was increased cf. monolayer on day 2 only (p<0.05).
Secretory active 3D spheroids comprising the 3 main LG cell types were formed that had increased secretory capacity cf. equivalent monolayer cultures, underlining that 3D culture promotes function. These spheroids may be useful in treatment of DES, either by triggering regeneration of existing LG tissue or by forming the cellular component of a reconstructed LG.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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