Cell culture is a foundational technique across biological research and biotechnology, allowing scientists to grow and study cells outside their natural environment. This controlled setting enables investigations into cellular processes, disease mechanisms, and drug responses. Working with cells in culture often requires manipulating them, which frequently involves detaching them from the surfaces they adhere to in their growth vessels. This process is a routine but delicate step in laboratory procedures.
The Need for Cell Detachment
Cells grown in culture often adhere to the surface of their containers, a characteristic known as anchorage dependence. To continue their growth, these adherent cells must be regularly detached and transferred to new, larger vessels in a process called passaging or subculturing. This prevents overcrowding, which can lead to nutrient depletion and accumulation of waste products, ultimately harming cell health.
Beyond routine maintenance, cell detachment is necessary for various experimental procedures. Researchers detach cells for accurate cell counting, cryopreservation (freezing for long-term storage), and preparing single-cell suspensions for assays like flow cytometry, where individual cells are analyzed. Detaching cells also facilitates transfection, a technique used to introduce foreign genetic material into cells.
What is Accutase?
Accutase is a proprietary blend of proteolytic and collagenolytic enzymes specifically designed for gentle cell detachment. Unlike traditional enzymes, Accutase is typically derived from non-mammalian and non-bacterial sources, such as crustaceans, reducing the risk of introducing animal-derived contaminants into cell cultures. It acts by breaking down the protein adhesions that anchor cells to the culture surface and to each other, including components of the extracellular matrix like collagen.
This enzyme solution is known for its gentle action, causing less damage to cell surface receptors and maintaining high cell viability after detachment. Accutase is particularly beneficial for sensitive cell types, such as human embryonic and induced pluripotent stem cells, neural stem cells, and primary cells, where preserving cell integrity and function is important. Its effectiveness at lower concentrations and the absence of a required inactivation step make it convenient for many applications, including flow cytometry and cell sorting. Accutase is often supplied as a ready-to-use solution and can be stored for approximately two months at 2-8°C once thawed.
What is Trypsin?
Trypsin is a serine protease enzyme widely used for detaching adherent cells in cell culture. Traditionally, it is derived from the pancreas of animals like pigs (porcine trypsin) or cows (bovine trypsin). Trypsin works by cleaving peptide bonds, specifically at the carboxyl side of lysine and arginine residues within proteins.
In cell culture, trypsin disrupts the protein connections that mediate cell-to-surface adhesion and cell-to-cell interactions, such as fibronectin, laminin, and collagen. While effective, trypsin can be potent and may damage cell surface proteins or compromise cell viability if exposed for too long or at high concentrations. Its activity requires inactivation, commonly achieved by adding serum (which contains protease inhibitors) or a specific trypsin inhibitor. Trypsin functions optimally at 37°C and a pH range of 7-9, and is often supplied with EDTA to enhance its dissociating effect by chelating divalent cations like calcium and magnesium, which are involved in cell adhesion.
Choosing Between Accutase and Trypsin
When selecting a cell detachment agent, several factors influence the choice between Accutase and Trypsin. Accutase is considered a gentler alternative, preserving cell viability and surface antigens more effectively, which is beneficial for sensitive cells like stem cells, primary cells, and neuronal cells. Trypsin, a more aggressive protease, can cause greater damage to cell surface proteins, potentially impacting downstream analyses like flow cytometry where surface marker integrity is important.
Regarding enzymatic activity and mechanism, Accutase is a mixture of proteolytic and collagenolytic enzymes, offering broader and more efficient dissociation at lower concentrations. Trypsin requires specific temperature conditions (around 37°C) for optimal activity, while Accutase is active over a wider temperature range, including room temperature incubation for 5-10 minutes.
A notable difference lies in inactivation: Accutase does not require a separate inactivation step, as its activity is sufficiently reduced by dilution with fresh culture medium or PBS. In contrast, Trypsin’s activity must be halted by adding serum or a specific trypsin inhibitor to prevent prolonged cell damage. Trypsin solutions may have varying stability and storage requirements depending on their formulation. While Accutase tends to be more expensive than traditional trypsin, its benefits in terms of cell health and simplified protocols can outweigh the cost for specific applications, especially when working with valuable or delicate cell lines.