Collagenase (Type D, animal free)

Storage Solution Preparation
1. Add 1 mL of HBSS (Hank's Balanced Salt Solution, containing Ca2+ and Mg2+) to 100 mg of collagenase, and shake to dissolve completely, preparing a 100 mg/mL (i.e., 100×) stock solution;
2. Filter-sterilize using a low-protein binding 0.22 μm filter membrane. Use immediately or aliquot into small portions and store at -20°C to -5°C in the dark;
3. Thaw on ice before use, avoiding 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 should be determined based on specific experimental conditions or by referring to relevant literature.
Tissue Dissociation
1. Cut the tissue into 3-4 mm pieces using a sterile scalpel or scissors;
2. Wash the tissue pieces several times with HBSS containing Ca2+ and Mg2+;
3. Add sufficient HBSS containing Ca2+ and Mg2+ to immerse the tissue pieces, and add collagenase to the required working concentration;
4. Incubate at 37℃ for 4-18 hours. Using a horizontal shaker and supplementing the digestion with 3 mM CaCl2 can improve digestion efficiency;
5. The dispersed cells can be collected using a stainless steel or nylon mesh sieve. For incompletely dissociated tissue, add an appropriate amount of fresh collagenase working solution and continue incubation at 37°C for further digestion;
6. Wash the collected cells several times with HBSS without collagenase;
7. Resuspend the cells in cell culture medium and calculate the viable cell density using an automated cell counter or other methods;
8. Seed the cells in cell culture dishes using appropriate cell culture medium. Organ Perfusion
1. Add collagenase to HBSS containing Ca2+ and Mg2+, preheated to 37°C. Adding 3 mM CaCl2 helps improve separation efficiency;
2. Perfuse the corresponding organ with the collagenase working solution at an optimized rate;
3. Pass the perfusate collected during the above process through a stainless steel or nylon mesh sieve to separate the dissociated cells or small tissue fragments from larger clumps. Tissue that is not sufficiently dissociated needs to be further incubated with fresh collagenase working solution at 37°C.
4. Follow steps 6-8 of the tissue separation protocol.