Section IV Replicate Culture Methods - Single "Subculture" in Situ Using Glass Beads

In this laboratory we have studied the macromolecular changes accompanying release of contact inhibition by using a method by means of which a large fraction of cells in a confluent monolayer are released from contact inhibition of growth and division. This is accomplished without chemical treatment of the cells, without change of medium and in such a way that most cells are provided with free growth area. The procedure involves growing the cells to confluence on surfaces uniformly covered with glass beads 200 {microns} in diameter. When confluence has been attained, contact inhibition ma be released by discarding the beads leaving behind numerous spaces, throughout the culture. Removal of the beads dislodges few if any of the cells. After release of contact inhibition by removing the beads, the cultures double in cell number following the first round of DNA replications, and continue to grow until they are again contact inhibited. Cell types used have included several cell lines and strains including both primary cell cultures of neonatal rat heart cells and established 3T3 and 3T6 mouse fibroblast cell lines