Worthington Tissue Dissociation Guide

Methods and Materials: Cell Release Procedure

In order to transfer or pass cells in monolayer culture from one culture vessel to another it is necessary to release cells from the monolayer into suspension so that they can be easily handled by pipetting and diluting.

Releasing cells from the monolayer is almost always accomplished with purified trypsin by a procedure known as trypsinization. A usual trypsinization procedure is detailed in the inset below.

Trypsinization Procedure
Remove culture medium from cells. Add sterile trypsin solution (in BSS-balanced-salt solution, normally calcium-free Hanks. Allow trypsin solution to act on monolayer for several minutes at room temperature or 37°C (or longer at 4°C). Remove trypsin solution gently so as not to disturb cells. Add BSS or media (often with serum or trypsin inhibitor to inactivate residual trypsin) and agitate vessel to disrupt monolayer and suspend cells.

Some researchers have found that procedures using crystalline trypsin can provide increased viability in cells after they are released. Viability is usually determined by measuring cloning efficiency, i.e., the ability of a single cell to attach to the wall of a culture vessel and divide to produce a colony of cells which is visible to the naked eye after staining.

Optimization Techniques: General Guidelines

Tissue Tables

The Worthington Tissue Tables provide references useful to researchers interested in tissue dissociation and cell harvesting procedures. The references are organized by Tissue and Species type and linked to PubMed citations. The Cell type, Enzymes, and Medium for each reference is provided.

To search by specific criteria, use the Tissue References Search Tool.

Tissue References Search