Optimizing Protocols for the Derivation of Primary Cultures from Mammalian Tissue

Abstract

The isolation of primary cell cultures from fresh mammalian tissue is a major interest of our laboratory. Thus, the ability to generate cancer cell lines from cancer tissue, or to generate non-malignant cultures form the host stroma surrounding a tumor, allows us to develop new laboratory models to study the behavior of cancer cells and its microenvironment. We report our proof of principle study on optimizing the derivation of primary culture, using rat tissues as a model. Thus, rat kidney and rat skin samples were isolated, minced with surgical blades, and then incubated with an enzyme digestion cocktail (Collagenase III- 4mg/mL, Collagenase IV-2mg/mL, Hyaluronidase-2mg/mL, in phosphate buffered saline, pH 7.4). Thereafter, the digested tissues were plated in tissue culture dishes in serum (20%) containing media. Three weeks after plating, very few cells were visible in any of the preparations. However, four weeks after plating we noted the appearance of colonies in rat kidney preparations. These colonies have since rapidly grown into larger colonies of fibroblast-like cells. These results demonstrate that we have an effective protocol for the derivation of fresh primary cultures from fresh tissues. Our plans for further optimization of the isolation procedure, for the eventual derivation of primary cultures from human and dog tumors (and from their benign stromal components such as fibroblasts and endothelial cells), will be discussed