Collagenase Blends for Gentle Tissue Dissociation
What is Collagenase?
Collagen is one of the structural components of extracellular matrix (ECM) that surrounds animal tissues and is critical for the physical maintenance of cells. To isolate functional and viable cells from animal tissue with minimal impact on the cells, enzymes that dissolve the ECM such as metalloproteinases are commonly used. Collagenase is such a metalloproteinase, and is the main reagent used by researchers in the tissue dissociation process to establish in vitro cultures and for therapeutic studies.
Although collagenase from Clostridium histolyticum is effective in the establishment of primary cell cultures in vitro, it can often be difficult to find the optimal conditions to completely isolate intact cells from their tested tissues. Most protocols require another digestion enzyme in addition to collagenase to improve the tissue dissociation process and make the process more specific for the desired tissue. There are several enzymes that are commonly used as secondary dissociation enzymes, including Deoxy-ribonuclease I (DNase I), Elastase, Hyaluronidase, and Dispase II.
Our dissociation blends combine collagenase with secondary dissociation enzymes to ensure gentle but effective extraction of cells from tissues. The efficacy of these blends to dissociate tissue is comparable or higher than individual enzymes themselves and these blends cut down the time spent by researchers in weighing out optimal amounts of individual enzymes for their experiments, thereby simplifying typical dissociation protocols.
Tissue Dissociation Blends Workflow
Tissue dissociation enzymes are necessary for removing the structural support of tissues and are essential for cell viability and the success of many downstream applications. A typical workflow for tissue dissociation includes organ perfusion, tissue dissociation with suitable enzyme blends, cell isolation and cell culture with optimal media, reagents, and antibiotics (Workflow 1).
Step 1. Organ Perfusion
Step 2. Tissue Dissociation
Step 3. Cell Isolation
Step 4. Culture Cells
Workflow 1. General Tissue Dissociation Blends workflow
Collagenase Blends Data
Collagenase-Deoxyribonuclease I Blend
Tissue dissociation by collagenase may result in cell lysis and subsequent release of DNA. This often causes cell clumping. Deoxyribonuclease I (DNase I) is endonuclease that degrades ds and ssDNA. Our Collagenase-Deoxyribonuclease I dissociation blend combines optimal concentrations of collagenase with DNase I (B20221) and prevents loss of cells from undesired clumping. The activity of the blend is higher when compared to that of the individual enzymes (Figure 1).
Collagenase-Hyaluronidase Blend
Hyaluronidase principally cleaves hyaluronic acid, the major component of the ECM. Our combination dissociation blend (B20222), consisting of collagenase and hyaluronidase, is effective for the isolation of functional and viable cells from breast, heart, and other tumors.
The activity of the Collagenase-Hyaluronidase blend is higher than that of the individual enzymes (Figure 2).
Collagenase-DNase I-Dispase II Blend
Dispase II is a neutral protease often used to prevent clumping in suspension cultures. It effectively cleaves the basement membrane but is gentle enough to preserve the viability of the epithelial cells.
The addition of dispase II to a combination of DNase I and collagenase (B20223) provides a mild and gently proteolytic action especially suitable for the preparation and subculture of most primary cells. While the activity of Collagenase in the blend was comparable, DNase I and Dispase II showed higher activity when combined in the blend than as individual enzymes. (Figure 3).
Figure 3.Collagenase-DNase I-Dispase II tissue dissociation blend (B20223) has similar collagenase activity but higher DNase I and dispase II activity compared to individual enzymes. A.) Enzyme activity in collagenase (C5138) alone or B.) in DNase I (D5025) alone or C.) in Dispase II (D4693) alone versus that of Collagenase-DNase I-Dispase II blend. * ttest ≤ 0.01
Collagenase-Elastase Blend
The main role of Elastase is the hydrolysis of elastin and the digestion of hemoglobin, casein, and fibrin. The combination of elastase with collagenase (B20224), can effectively isolate cells that are found in extensive intercellular fiber tissues.
The activity of the Collagenase-Elastase blend is comparable to that of the individual enzymes (Figure 4).
Tissue Dissociation Blends Benefits
Tissue dissociation blends have several benefits over using the individual dissociation enzymes, including saving time during the experiment. These blends are pre-mixed in the correct ratio for tissue dissociation, allowing researchers to spend less time measuring out enzymes for their experiments.
Our tissue dissociation blends also provide consistent quality and performance while ensuring lot-to-lot consistency and a reliable supply. The dissociation blends are lyophilized products and are stable at 25°C for up to 8 days. Tested and certified for shear protection, we provide comprehensive regulatory documentation for our tissue dissociation enzyme blends.
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