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
  1. Remove culture medium from cells.
  2. Add sterile trypsin solution (in BSS-balanced-salt solution, normally calcium-free Hanks.
  3. Allow trypsin solution to act on monolayer for several minutes at room temperature or 37°C (or longer at 4°C).
  4. Remove trypsin solution gently so as not to disturb cells.
  5. 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.

More Information: Worthington Trypsin

Next: Optimization Techniques: General Guidelines

Tissue Tables (references, grouped by tissue type and species)

Adipose/Fat Adrenal Bone Brain
Cartilage Colon Endothelial Epithelial
Eye Heart Intestine Kidney
Liver Lung Lymph nodes Mammary
Miscellaneous Muscle Neural Pancreas
Parotid Pituitary Prostate Reproductive
Scales Skin Spleen Stem
Thymus Thyroid/Parathyroid Tonsil Tumor

Note: We have not limited the references listed to only those papers using Worthington enzymes. Generally speaking, the tissue dissociation enzymes offered by Worthington can be used interchangeably for most preparations cited.