Primary culture and characterization of microvascular endothelial cells from Macaca monkey retina

Abstract

Purpose: To develop methods for the culture of microvascular endothelial cells (EC) from Macaca monkey retina and to investigate their propagation and survival in vitro.

Methods: Endothelial cells from capillary fragments were cultured on fibronectin-coated dishes in QB-58 serum-free medium containing 20 microliters/ml bovine retinal extract, 90 micrograms/ml heparin, 10% fetal bovine serum, and 10% monkey serum. Non-EC were removed manually. Endothelial cell-specific properties were assessed by endocytosis of acetylated low-density lipoprotein (ac-LDL) and by immunocytochemical staining. The response to growth factors was assayed by 3H-thymidine incorporation. The synthesis of matrix macromolecules was studied by metabolic labeling with 3H-proline and identification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis-immunoblotting.

Results: Under these culture conditions, migrating cells emerged from capillary fragments after 1 to 2 days and formed large colonies by 1 week. Cells exhibited a mean doubling time of 44.5 hours during the first 3 to 5 days of culture and 23 hours at 6 to 8 days in culture, and they formed a confluent monolayer by 12 to 14 days. These cells demonstrated uptake of ac-LDL, expressed von Willebrand factor and the cell adhesion protein CD31, and did not contain smooth muscle alpha-actin. Before purification, 92% of the cells in primary cultures were identified as EC. The EC could be maintained in vitro for more than 1 month without the addition of growth factors; however, basic fibroblast growth factor and vascular endothelial growth factor each stimulated cell replication. Secreted extracellular proteins included fibronectin, collagen types I and IV, laminin, and SPARC (secreted protein, acidic, and rich in cysteine).

Conclusions: This study is the first description of the culture and propagation of purified retinal EC from Macaca monkey, a widely accepted model for the human retina. These cultures will be highly relevant to studies of abnormal vascular disease in the human eye.