Long term 2D-culture of primary mouse liver sinusoidal endothelial cells (LSEC)

Abstract

Liver sinusoidal endothelial cells (LSEC) are unique and specialized cell type with high endocytic activity of bloodborne waste macromolecules. LSEC lack basal lamina and present a unique structural feature called fenestrations, which facilitate passive filtering and exchange of nutrients with other liver cells. Developing LSEC cell lines is challenging with regards to preserving their morphology and functions. Therefore, the aim of this study is to optimize the conditions of a long-term 2D-culture of LSEC and examine the changes (morphology and endocytosis) over a prolonged period of time, up to 21 days. New primary cells were isolated for each experiment from mouse liver. After conducting two pilot experiments and optimizing the initial conditions, several methods were applied to study LSEC in a long-term culture. Three different assays were performed to study the viability of LSEC. In addition, the purity of LSEC cultures was assessed by immunostaining. Two different microscopic techniques; scanning electron microscopy (SEM) and fluorescence microscopy, were utilized to investigate changes in fenestrations and cytoskeleton (actin and tubulin). Different concentrations of cytochalasin B, which prevents actin polymerization, were added to the cells to study the role of the actin cytoskeleton in LSEC fenestrations in long-term culture. Furthermore, a quantitative endocytosis assay using radiolabelled formaldehyde-treated serum albumin (FSA) was used to examine changes in endocytosis in the long-term culture. A qualitative endocytosis assay was also conducted by treating the cells with fluorescently labelled FSA, ribonuclease B (RNase B) and aggregated gamma globulin (AGG) to examine endocytosis mediated via stabilin-, mannose- and Fc-gamma IIb2 receptors, respectively. Initial experiments revealed LSEC to have better survival for up to 8 days on fibronectin-coated plastic well plates in endothelial cell growth media (EGM). Microscopy imaging showed that a heterogeneous population of LSEC maintained monolayer and partially fenestrated morphology for up to 8 days. Both the qualitative and semi-quantitative analysis showed a decrease in the fenestration number and an increase in the fenestration size from day 3. In addition, an increase in the number of fenestrations was observed in LSEC treated with cytochalasin B. FSA endocytosis showed a steep decrease in endocytosis in long-term culture for LSEC cultured in RPMI media. However, the endocytosis in LSEC cultured in EGM initially decreased until day 5, but recovered on day 8 and remained stable until day 11. Fluorescent imaging demonstrated that LSEC maintained the endocytosis via the three types of receptors examined, stabilin-, mannose- and Fc gamma IIb2 receptors, for up to 8 days. Viability assessments demonstrated a decrease in cell number with time, and an increase in the cell volume but no changes in the metabolic activity per cell with time. Furthermore, the purity assay showed high-purity LSEC cultures. The results of this study suggest that optimizing the conditions for prolonged preservation of mLSEC morphology and function in long-term culture is possible. The main outcome showed that EGM provides allows for LSEC to maintain high viability and partially fenestrated morphology for up to 8 days and preserves endocytic functions for up to 11 days.