What Is Collagenase?

The chemical name of collagenase is collagenase, which can specifically hydrolyze the three-dimensional helical structure of natural collagen under physiological pH and temperature conditions without damaging other proteins and tissues. The chemical nature of collagenase is a protein. Therefore, it is very sensitive to temperature, pH, and various factors that cause protein denaturation. It is very susceptible to external conditions to change its conformation and properties.

Collagenase

Collagenase can be divided into two types: endogenous collagenase and medicinal collagenase according to the different ways in which it exists. Human-derived collagenase refers to the collagenase that the body itself has, such as

Collagenase solution is prepared, sterilized and stored like trypsin.

note:

Because type I collagenase molecular particles are larger than pancreatin, it is not easy to filter, so it can be sterilized by Tsai filter.

Calcium, magnesium ions and serum components will not affect the digestion of collagenase, so it can be prepared with BSS (such as D-hanks or PBS) or serum-containing culture solution.

1. Cut the rinsed and trimmed tissue into small pieces of about 1 ~ 2mm3

2. Place the tissue block into a centrifuge tube (conical flask), add 30-50 times the volume of collagenase solution, and tighten the lid (sealed flask).

3. Place the flask in a 37 ° C water bath or 37 ° C incubator and shake it every 3 minutes. It is better if it can be placed in a 37 ° C thermostatic water bath.

The digestion time is closely related to the type of tissue. For some tumor tissues or other dense connective tissues, the digestion time is 4 to 48 hours. For easily digestible tissues, digestion can be performed at 37 ° C for 15 to 45 minutes, or according to specific conditions. . If the tissue mass has dispersed and lost its shape, once shaken, it becomes a cell mass or a single cell, which can be considered to be fully digested. After the epithelial tissue is digested by collagenase, because the epithelial cells are tolerant to this enzyme, some cell clusters may not be completely dispersed, but the clustered epithelial cells are easier to grow than the dispersed single epithelial cells. Therefore, if there is no special need No further processing is required.

4. Collect the digestive fluid (sometimes containing individual tissue pieces and incompletely digested tissue debris need to be filtered with a 100 mesh stainless steel mesh), centrifuge at 1000 r / min for 5 min, remove the supernatant, and centrifuge and rinse with Hanks solution or serum-free culture solution. 1-2 times, remove the supernatant, add the culture solution to make a cell suspension, and inoculate the culture flask.