Collagenase (Type D, animal free)

Preparation
1. Add 1 mL of HBSS (Hank balanced salt solution containing Ca²⁺ and Mg²⁺) to 100 mg of collagenase, shake to fully dissolve it, and prepare a stock solution of 100 mg/mL (i.e., 100×).
2. Sterilize through a 0.22 μm filter membrane with low protein binding capacity. Use immediately or aliquot into small portions and store in the dark at -20°C to -5°C.
3. Thaw on ice before use to avoid repeated freezing and thawing. The commonly used concentration for tissue and cell dispersion is 0.5-2.5 mg/mL, and the commonly used concentration for cartilage digestion is 1-2 mg/mL. The optimal working concentration required should be determined based on specific experimental conditions or by referring to relevant literature.
Tissue separation
1. Use a sterile scalpel or scissors to cut the tissue into tissue blocks of 3-4 mm in size.
2. Wash the tissue blocks several times with HBSS containing Ca²⁺ and Mg²⁺;
3. Add an adequate amount of HBSS containing Ca²⁺ and Mg²⁺ to submerge the tissue block, and then add collagenase to the required working concentration.
Incubate at 37°C for 4 to 18 h. Using a horizontal shaker during digestion and supplementing digestion with 3 mM CaCl₂ can enhance digestion efficiency.
5. The dispersed cells can be sieved using stainless steel or nylon mesh screens and collected for future use. For the incompletely dissociated tissues, an appropriate amount of fresh collagenase working solution should be added and incubated at 37°C for further digestion.
6. Wash the collected cells several times with HBSS free of collagenase;
7. Resuspend the above-mentioned cells in cell culture medium and calculate the density of viable cells using an automatic cell counter or other methods;
8. Inoculate the cells on a cell culture dish using an appropriate cell culture medium.
Organ perfusion
1.Adding collagenase to HBSS preheated at 37°C and containing Ca²⁺ and Mg²⁺, along with 3 mM of CaCl₂, helps to enhance the separation efficiency.
2. Infuse the corresponding organs with collagenase working solution at the optimized rate;
3. The perfusion solution recovered during the above process should flow through a stainless steel or nylon mesh screen to separate the dissociated cells or small tissue fragments from the larger clumps. The infully dissociated tissues need to be further incubated at 37°C with fresh collagenase working solution.
4. The steps are the same as those for tissue separation 6-8.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.