He used to cells harvested trypsin & release procedure

Amygen Worthington Trypsin for Cell Harvest:

In 1916, Rous and Jones used "trypsin powder of Merck, Bruler, and Kahlbaum" to digest a plasma clot in which living cells grew to obtain a cell suspension for subculture.In 1934, Vogelaar and Erlichman were the next researchers to use the digestive enzymes in crude trypsin preparations to liquefy coagulated plasma from human fibroblasts grown before subculture.Scherer, Syverton, and Gey introduced trypsin techniques similar to those used today in 1953 to harvest then-newly cultured HeLa cell lines for subculture and biochemical analysis.These workers tested recrystallized trypsin and NF 1:250 trypsin for cell harvesting and found that purified trypsin was more potent and less toxic to cells.Nevertheless, NF 1:

Relatively crude pancreas preparations such as NF 1:250 trypsin are still used for cell harvesting today, although they exhibit considerable batch-to-batch variability and contain foreign substances and other enzymatic activities.Impurities in crude trypsin can cause unnecessary damage to cells and reduce cloning efficiency.Many of these difficulties can be eliminated by using higher purity crystalline trypsin.

Any contaminants present in the NF 1:250 material do not appear to be required for cell harvest activity, as purified trypsin is very efficient for monolayer dissociation and crude NF 1:250 trypsin plus soybean trypsinInhibitors are ineffective.

McKeehan and Ham report that the viability and proliferative potential of single cells in low serum medium are significantly improved when harvested with crystalline trypsin at low temperature (i.e., 4°C).

 

WorthingtonCell Release Program:

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

Release of cells from a monolayer is almost always accomplished using purified trypsin through a process called trypsinization.The usual trypsin digestion procedure is detailed in the inset below.

Amigen WorthingtonTrypsin Digestion Procedure:

1. Remove medium from cells.

2. Add sterile trypsin solution (in BSS balanced salt solution, usually calcium-free Hanks.

3. Let the trypsin solution work on the monolayer for a few minutes at room temperature or 37°C (or 4°C longer).

4. Gently remove the trypsin solution so as not to disturb the cells.

5. Add BSS or medium (usually containing serum or trypsin inhibitor to inactivate residual trypsin) and agitate the vessel to disrupt the monolayer and suspend the cells.

Some researchers have found that a procedure using crystallized trypsin can improve cell viability after release.Viability is usually determined by measuring cloning efficiency, the ability of a single cell to attach to the walls of a culture vessel and divide to produce colonies of cells that are visible to the naked eye after staining.

Note: Worthington's tissue dissociation enzymes are used interchangeably for most of the preparations cited.

Worthington core enzymes: including: collagenase, deoxyribonuclease I, elastase, papain, ribonuclease, trypsin, etc.