The mouse liver tissue rapid dissociation kit, a key tool in cell biology research, achieves efficient and gentle dissociation of liver tissue through the synergistic action of specific enzyme components, while preserving cell viability and surface antigen integrity. These kits typically contain multiple enzymes, each targeting different extracellular matrix components in the liver tissue to collectively complete the tissue dissociation process.
Collagenase is one of the key enzymes in the kit. Its mechanism of action involves specifically cleaving collagen fibers in liver tissue. The liver's extracellular matrix is rich in type I and type III collagen, which form the main structural support of the tissue. Collagenase hydrolyzes the triple helix structure of collagen, disrupting its mechanical stability and loosening intercellular connections. This process creates conditions for the subsequent action of other enzymes while avoiding cell damage that might result from mechanical dissociation.
Neutral proteases in the kit play a role in breaking down intercellular adhesion proteins. Unlike collagenase, neutral proteases primarily target non-collagenous intercellular connection molecules, such as fibronectin and laminin. These proteins are equally important in maintaining tissue integrity, but their degradation requires different enzyme cleavage sites. Neutral proteases further weaken cell-cell interactions and promote cell separation by breaking down these adhesion proteins. Their enzyme activity peaks at physiological temperatures, ensuring efficient dissociation under mild conditions.
DNase I is added to address potential problems caused by DNA fragments released during dissociation. Hepatocytes release large amounts of free DNA during dissociation; these DNA fragments form a sticky network through electrostatic interactions, causing cell aggregation and affecting the quality of single-cell suspensions. DNase I degrades these DNA fragments, reducing electrostatic adsorption between cells and significantly decreasing aggregation. This effect is crucial for obtaining high-purity, high-activity single-cell suspensions, especially in subsequent applications such as single-cell sequencing, where cell aggregation directly impacts data quality.
The enzyme components in the kit do not act in isolation but achieve synergistic dissociation through a cascade of reactions involving "network breaking, bridging, and obstacle clearing." Collagenase first disrupts the collagen fiber network, followed by neutral protease breaking down intercellular adhesion proteins, resulting in initial cell separation. DNase I then removes DNA barriers released during dissociation, ensuring complete cell dispersion. This triple-enzyme synergistic mechanism maintains stable, high cell viability, far exceeding the effectiveness of traditional single-enzyme digestion methods.
In addition to the synergistic effect of the enzyme components, the kit optimizes the dissociation environment through dynamic pH control technology. The pretreatment solution uses weakly alkaline conditions to gently open tight intercellular junctions; the stop solution rapidly inactivates residual enzyme activity through an acidic environment, preventing over-digestion. This "slow rise-sudden fall" pH curve design ensures both enzymatic efficiency and avoids metabolic disturbances caused by prolonged exposure of cells to extreme pH environments.
The introduction of a temperature-responsive protection system further enhances the kit's performance. Trehalose complex forms a thermally stable protective layer at the dissociation temperature, reducing cellular oxidative stress; recombinant albumin dynamically binds free metal ions, inhibiting non-specific protease cleavage; and heparin analogues prevent the release of platelet-activating factor, reducing cell activation. These protective components work together to ensure that dissociated hepatocytes maintain a high proliferative capacity under cryogenic preservation conditions.
The mouse liver tissue rapid dissociation kit achieves efficient and gentle dissociation of liver tissue through multiple mechanisms, including multi-enzyme synergy, dynamic pH regulation, and temperature-responsive protection. Its core enzyme components each perform their specific functions, working together to complete the transformation from tissue blocks to single-cell suspensions, providing high-quality cell samples for subsequent cell culture, single-cell sequencing, and other research.
