Collagenase Type III, an endopeptide matrix metalloproteinase MMP-13 derived from the bacterium Clostridium histolyticum, preferentially degrades type III collagen and is commonly used in sensitive tissues such as breast, tumor, and uterus due to its mild activity.

Collagenase Type III Preparation and Usage
Collagenase Type III is dissolved in a buffer (commonly at concentrations of 0.1%-1mg/mL), and incubated with tissue samples at 37°C for 1-2 hours. Cells are then collected by centrifugation and washing. This method is commonly used to prepare primary cells from breast tissue [1].
Experimental Steps:
1. Storage Solution Preparation:
(1) Accurately weigh 100mg of collagenase and slowly add it to 1mL of Hank's Balanced Salt Solution (HBSS) containing Ca²⁺ and Mg²⁺. Shake to promote complete dissolution, preparing a 100mg/mL (100×) stock solution.
(2) Filter the solution through a 0.22μm filter to sterilize it. Use it immediately or aliquot it into small portions and store at -20°C to -5°C in a light-protected environment to maintain the solution's quality and activity.
(3) The required concentration should be determined according to specific experimental conditions or reference literature. Common concentrations for tissue and cell dissociation are 0.5-2.5mg/mL, and for cartilage digestion, concentrations of 1-2mg/mL are typically used.
2. Experimental Method:
(1) Clean the target tissue (such as breast tissue, colon cancer stem cells, etc.) thoroughly to remove any impurities.
(2) Dissolve Collagenase Type III in an appropriate buffer according to experimental needs, typically at a concentration of 1-2mg/mL.
(3) Place the tissue sample in the enzyme solution and incubate at 37°C for 1-2 hours. The incubation time can be adjusted depending on the tissue type and experimental requirements.
(4) Add an appropriate amount of stop solution (such as culture medium containing serum) to halt the enzyme activity.
(5) Separate the required cells using centrifugation or filtration Methods.