Collagenase, Type VIII is a mixed enzyme derived from Clostridium histolyticum that contains collagenase, a nonspecific protease, and a clostripain. Collagenase, Type VIII can hydrolyze Type VIII collagen and may be used to study the formation of atherosclerosis. Type VIII collagen is a regulator of endothelial cell differentiation and angiogenesis, a substrate for cell adhesion and migration such as smooth muscle cells, and may accumulate in atherosclerosis. After endotoxin activates the expression of Collagenase, Type VIII, it can reduce the production of Type VIII collagen and has the potential to inhibit atherosclerosis[1].
In Vitro, Can be used to release rat epididymal adipocytes and hepatocytes.
Preparation of storage solution
1. Add 1 mL Hank’s Balanced Salt Solution (HBSS) with calcium and magnesium directly to 1 g vial of Collagenase.Vortex gently to ensure complete dissolution, and prepare a stock solution of 100 mg/mL (100X stock solution).
2. Filter sterilize 100X stock solution using a 0.22 μm filter with a low protein binding filtration unit. Use immediately or dispense into aliquots and store at -20°C to -5°C protected from light.
3. Thaw on ice prior to use. Commonly used concentrations for tissue and cell dispersion are 0.5-2.5 mg/mL and for cartilage digestion are 1-2 mg/mL, but the optimal working concentration required needs to be determined based on specific experimental conditions or by referring to the appropriate literature.
Dissociate Tissue
1. Mince tissue into 3-4 mm pieces with a sterile scalpel or scissors.
2. Wash the tissue pieces several times with HBSS containing calcium and magnesium.
3. Add sufficient HBSS with calcium and magnesium to submerge tissue. Add collagenase to required working concentration.
4. Incubate at 37°C for 4-18 hours. Increased efficiency is obtained using a rocker platform and supplementing the digest with 3 mM CaCl2.
5. Disperse cells by passing through a sterile stainless steel or nylon mesh. Remaining tissue fragments may be disaggregated by addition to fresh collagenase solution and further incubation at 37 °C.
6. Wash dispersed cells several times by centrifugation in HBSS w/o collagenase.
7. Resuspend cell pellet, after the final wash step, in culture medium. Determine viable cell density using a Automated Cell Counter (alternate automated or manual methods may be used). 8. Seed cells into culture vessels containing appropriate media.
Organ Perfusion
1. Add collagenase to prewarmed (37°C) HBSS with calcium and magnesium. Addition of 3 mM CaCl2 increases the efficiency of dissociation.
2. Perfuse organ at preoptimized rate for the particular organ.
3. Dispersed cells and tissue fragments are separated from larger pieces by passing the perfusate through a sterile stainless steel or nylon mesh. Remaining tissue fragments may be disaggregated by addition to fresh collagenase solution and further incubation at 37°C.
4. The steps are the same as for tissue isolation 6-8.
References:
[1]. Plenz GA, et al. Vascular collagens: spotlight on the role of type VIII collagen in atherogenesis. Atherosclerosis. 2003 Jan;166(1):1-11.