Worthington Enzymatic Cell Harvest & Cell Adhesion and Harvest

Amigen Worthington Methods and Materials: Enzymatic Cell Harvesting

Most non-malignant cells grown in vitro move around and divide until they form a cell-thick monolayer that completely covers the surface of the culture vessel.When confluence is reached, motility and proliferation usually cease.Harvesting cells for research, processing or subculture requires dissociation and separation of monolayers.Limited treatment of the cell layer with trypsin is the most commonly used method.

Previously, trypsin preparations were thought to simply hydrolyze a protein-binding substance responsible for keeping cells firmly attached to their substrates, allowing cells to detach from the culture vessel.The mechanism of action of trypsin in cell harvesting is now thought to be more complex.

Ai MeijiWorthingtonMethods and Materials: Cell Adhesion and Harvest

During interphase, fibroblast-like cells in culture are distributed on the matrix in a characteristic spindle-shaped structure.Opinions are divided as to whether the actual area of ​​cell adhesion is distributed over most of the lower surface of the cell or in relatively narrow patches near the edge of the cell, mainly in the vicinity of wrinkling activity.In either case, these adherent regions appear to consist of clusters of attachment points, each about 1 µm in diameter.The individual attachment points are apparently distal parts of the matrix-bound cytoskeletal structure.

Within minutes after placing the cultured cells in a low temperature, chelating agent or trypsin solution, they drastically change shape by rounding and foaming.Electron micrographs show many long retracted fibers 0.25 - 0.5 µm in diameter extending from the surface of the round cell body to the enlarged terminal bulb attachment points previously located on the lower surface of the flat cells.

Cells remain attached to the matrix until the fibers are broken, either mechanically by tapping or shaking the culture vessel, or chemically by the continuous action of chelating agents and/or trypsin.(The low temperature alone was sufficient to enclose a circle, but not sufficient to detach. These conditions also greatly reduced the entry of trypsin into the cells.) Shortly after the cells were detached from the surface of the culture vessel, subcultured into new vessels containing trypsin-free medium,Cytoplasm flows into broken retracted fibers and repopulates them.Within an hour, the round cells began to take on their characteristic shape.

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.