A Non-enzymatic Method for Subculturing Monolayer Cell Cultures 1

Abstract

Cell cultures grown as monolayers conventionally may be removed from the surface of the culture vessel by physical or by chemical methods. Physical methods, such as scraping the cells from their growth substrate, generally yield heterogenous suspensions ranging from single cells to cell aggregates of varying size to monolayer membranes. Furthermore, many cell types, such as human fibroblasts, do not yield readily to removal by physical means. Most chemical methods for the removal of cell monolayers employ proteolytic enzymes. Although chemical techniques tend to produce more uniform single cell suspensions, the possibility exists that repeated exposure of cell cultures to proteolytic enzymes may have deleterious effects upon cell membranes. However, it is also possible that cell membranes are effectively “rejuvenated” with each division; in which case one could consider that individual cells are exposed to proteolytic enzymes only once and that membrane damage therefore might not be cumulative.

Here we introduce a new physical method for subculturing monolayer cell cultures, one in which the cells are not removed from occupied space, but the occupied space is removed and replaced with available and accessible space.

Materials and Methods. Cell cultures of human fibroblasts were initiated by trypsinization of neonatal foreskins obtained at the time of circumcision. They were maintained at 37° and 5% CO₂. Cells were fed twice a week using Eagle's minimum essential medium fortified with calf serum, penicillin, streptomycin, and Mycostatin. Cultures were doubled when the cell monolayers became confluent.

The standard enzymatic method for subculturing monolayers is as follows: spent culture medium is aspirated from plastic tissue culture dishes (Falcon No. 3002) as completely as possible and the cultures are washed briefly with 2 ml of phosphate buffered saline. The saline is aspirated and 1 ml of 0.25% trypsin in calcium- and magnesium-free Hanks' balanced salt solution is added to the dish.