Isolation and characterization of equine microvascular endothelial cells in vitro.

The study outlines the method used to isolate equine microvascular endothelial cells for tissue culture in the study of horse diseases. It explores the optimal conditions for cell growth, highlighting that 10% whole fetal bovine serum and either fibronectin or laminin formed the most conducive environment for the growth of the isolated cells.

Cell Isolation and Culture Method

• The cells, equine microvascular endothelium (EMVE), were isolated from fresh omental tissue (fat tissue around abdominal organs) of horses and ponies.
• This tissue was diced and underwent collagenase digestion to break down the extracellular matrix and make the cells accessible.
• The product was then filtered and layered on 5% bovine serum albumin, a protein traditionally used in cell culture, to allow for gravity sedimentation.
• The bottom layer of this sedimentation process was taken, and the cells were placed onto fibronectin-coated flasks for culture growth.

Growth Medium Composition

• The cells were grown in Dulbecco modified Eagle medium, a common nutrient solution used for cell culture, containing 10% whole fetal bovine serum.
• The research highlights that the presence of 10% whole fetal bovine serum was the minimum requirement for adequate cell growth, and that this could be combined with either fibronectin or laminin as extracellular matrix substrates.
• The addition of other growth factors or serum supplements were evaluated but did not appear necessary for the growth of EMVE cells.

Cell Morphology and Metabolism

• These cultured cells adapted a ‘cobblestone’ appearance when they reached confluence (covered all the available surface area), a typical characteristic of endothelial cells.
• They were also found to stain positively for factor VIII-related antigen, indicating their role in the clotting pathway.
• The cells were able to metabolize acetylated low-density lipoprotein (LDL), a process necessary for the functionality of living cells.

Contamination

• The primary cell cultures had minimal contamination with fibroblast and smooth muscle cells, common contaminates in cell culture.
• These cultures were passable and could be maintained for 3 to 5 serial passages, providing a sufficient amount of material for experimental purposes.