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

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.