Dispase
Dispase is a neutral metalloprotease enzyme produced by Bacillus polymyxa. It is used extensively in cell biology and tissue engineering for its ability to cleave proteins in the extracellular matrix, thereby detaching cells from surfaces or tissues without damaging the cells themselves. Unlike trypsin, which degrades cell surface proteins, Dispase preferentially digests the adhesive proteins that hold cells together.
Mechanism of Action
Dispase functions by cleaving peptide bonds at the N-terminal side of hydrophobic amino acid residues, particularly in collagen IV, fibronectin, and other basement membrane proteins. This proteolytic activity disrupts cell-to-cell and cell-to-matrix adhesion, leading to the dissociation of tissues into individual cells or smaller cell aggregates. Its neutral pH optimum (around 7.0-8.0) contributes to its relatively gentle effect on cell viability.
Applications
Dispase is widely used in a variety of applications including:
- Cell Harvesting: Gentle removal of cells from culture flasks, microcarriers, and other surfaces without compromising cell viability.
- Tissue Dissociation: Separation of tissues into individual cells for cell culture, analysis, or transplantation. Examples include separating epidermal sheets from the dermis for skin grafting and isolating islets of Langerhans from the pancreas.
- Embryonic Dissection: Used in developmental biology to separate and manipulate embryonic tissues and cells.
- Stem Cell Research: Employed in the isolation and expansion of stem cells while preserving their pluripotency.
- Production of Cell Monolayers: Creating uniform cell layers for various in vitro assays.
Properties and Considerations
- Source: Typically obtained from Bacillus polymyxa.
- Activity: Measured in units, with different commercial preparations having varying levels of activity.
- Concentration: Optimal concentration varies depending on the specific application and tissue type.
- Incubation Time: The length of incubation is crucial and needs to be optimized to prevent cell damage.
- Inactivation: Dispase activity can be inhibited by EDTA (ethylenediaminetetraacetic acid), a chelating agent that binds to the metal ions required for its enzymatic activity.
- Sterility: Typically supplied as a sterile solution to prevent contamination of cell cultures.
- Cell Viability: While generally considered gentle, prolonged exposure or high concentrations can still affect cell viability. Cell viability should always be assessed after Dispase treatment.