Harvesting of cells mechanically
This method is simple and easy. It involves gently scraping cells from the growth
surface into the culture medium using a rubber spatula that has a rigid polystyrene
handle with a soft polyethylene scraping blade (Fig. 2.5). This method is not suitable
for all cell types as the scraping may result in membrane damage and significant
cell death. Before adopting this approach it is important to carry out some test runs
where cell viability and growth are monitored in a small sample of cells following
harvesting.Harvesting of cells using proteolytic enzymes
Several different proteolytic enzymes can be exploited including trypsin, a proteolytic
enzyme that destroys proteinaceous connections between cells and between cells and
the surface of the flask in which they grow. As a result, harvesting of cells using this
enzyme results in the release of single cells, which is ideal for subculturing as each
cell will then divide and grow, thus enhancing the propagation of the cultures.
Trypsin is commonly used in combination with EDTA, which enhances the action of
the enzyme. EDTA alone can also be effective in detaching adherent cells as it chelates
the Ca^2 þrequired by some adhesion molecules that facilitate cell–cell or cell–matrix
interactions. Although EDTA alone is much gentler on the cells than trypsin, some cell
types may adhere strongly to the plastic, requiring trypsin to detach.
The standard procedure for detaching adherent cells using trypsin and EDTA
involves making a working solution of 0.1% trypsin plus 0.02% EDTA in
Ca^2 þ/Mg^2 þ-free phosphate-buffered saline. The growth medium is aspirated from
confluent cultures and washed at least twice with a serum-free medium such as
Ca^2 þor Mg^2 þ-free PBS to remove traces of serum that may inactivate the trypsin.
The trypsin–EDTA solution (approximately 1 cm^3 per 25 cm^2 of surface area) is then
added to the cell monolayer and swirled around for a few seconds. Excess trypsin–
EDTA is aspirated, leaving just enough to form a thin film over the monolayer.
The flask is thenincubated at 37C in a cell culture incubator for 2–5 min but
monitored under an inverted light microscope at intervals to detect when the cellsFig. 2.5Cell scrapers.53 2.5 Types of animal cell, characteristics and maintenance in culture