Add 100 µL of Ca2+ and Mg2+-containing HBSS (Hank’s Balanced Salt Solution, containing Ca2+ and Mg2+) to each tube of 100 mg of Collagenase II, vortex gently to dissolve it fully, and prepare 1 g/ml (1000×) for storage liquid. Then use a low-protein-binding 0.22 μm filter membrane to filter and sterilize, aliquot into small portions, and freeze at -20°C in the dark.
Thaw on ice before use, avoid repeated freezing and thawing. The commonly used concentration for tissue and cell dispersion is: 0.5-2.5mg/ml, and the commonly used concentration for cartilage digestion is 1-2mg/ml. It needs to be determined according to specific experimental conditions or by referring to the corresponding literature. Optimum working concentration.
1) Use a sterile scalpel or scissors to cut the tissue into 3-4mm tissue pieces;
2) Wash the tissue block several times with HBSS containing Ca2+ and Mg2+;
3) Add enough HBSS containing Ca2+ and Mg2+ to submerge the tissue block, and add collagenase to the required working concentration;
4) Incubate at 37°C for 4-18h. Using a horizontal shaker during digestion and supplementing digestion with 3mM CaCl2 can improve digestion efficiency.
5) The dispersed cells can be sieved with stainless steel or nylon mesh and collected for later use. For incompletely dissociated tissues, add an appropriate amount of fresh collagenase working solution and continue to incubate at 37°C;
6) Wash the collected cells several times with Collagenase II-free HBSS;
7) Resuspend the above cells in cell culture medium, and calculate the viable cell density using an automatic cell counter or other methods.
8) Inoculate cells on a cell culture dish with a suitable cell culture medium.
1) Add Collagenase II to the HBSS containing Ca2+ and Mg2+ preheated at 37°C, and add 3mM CaCl2 to help improve the separation efficiency;
2) Perfuse the corresponding organ with Collagenase II working solution at the optimized rate;
3) Pass the perfusate recovered in the above process through a stainless steel or nylon mesh sieve to separate dissociated cells or small fragments of tissue from larger clumps. Insufficiently dissociated tissues need to use fresh collagenase The working solution was further incubated at 37°C;
4) Wash the collected cells several times with Collagenase II-free HBSS;
5) Resuspend the above cells in cell culture medium, and calculate the viable cell density using an automatic cell counter or other methods.
6) Inoculate cells on a cell culture dish with a suitable cell culture medium.