Abstract
We introduce a novel technology to cryopreserve cells directly in monolayer utilizing a collagen vitrigel membrane as a scaffold. The membrane has excellent mechanical strength and can be easily handled by tweezers even after culturing cells on it. In this study, we used the membrane for direct preservation of primary hepatocytes, feeder cells, and ES cells. Freshly isolated hepatocytes (4x10^5 cells), mitomycin C treated mouse fibroblasts (feeder cells) or mouse embryonic stem (ES) cells were inoculated and cultured on a collagen vitrigel membrane (35mm in diameter) in a culture dish for 24hr to form colonies in monolayer. The cell colony-spread scaffold was transferred to a new dish with fresh preservation medium containing 10%DMSO. The dish was then gradually frozen to -80℃ and stored in liquid nitrogen for 1 week to 3 months. The cell adhesiveness and viability before and after cryopreservation were evaluated by cell morphology and calcein staining, respectively. The cells preserved by this method showed significant improvement compared to cells preserved without the collagen vitrigel membrane.
