Collagenase, Type VIII

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.

Collagenase, Type VIII 是溶组织梭状芽胞杆菌来源的、含有胶原酶的混合酶，是非特异性蛋白酶和梭菌蛋白酶。Collagenase, Type VIII 可以水解 Type VIII collagen，可能用于研究动脉粥样硬化形成。Type VIII collagen 是内皮细胞分化和血管生成的调节剂，是平滑肌细胞等细胞粘附和迁移的底物，并可能在动脉粥样硬化中积累。内毒素激活 Collagenase, Type VIII 表达后，可减少 Type VIII collagen 产生，有潜力抑制动脉粥样硬化^[1]。

在体外，可用于释放大鼠附睾脂肪细胞和肝细胞。

储存液配制
1. 向100 mg 胶原酶中加入 1 mL HBSS (Hank’s 平衡盐溶液，含 Ca²⁺、Mg²⁺) ，震荡使其充分溶解，制备成 100 mg/mL (即100×) 的储存液；
2. 使用低蛋白结合性的 0.22 μm 滤膜过滤除菌，立即使用或分装成小份量，于 -20 ℃ 到-5 ℃ 避光冻存；
3. 使用前在冰上解冻，避免反复冻融。用于组织和细胞分散的常用浓度为：0.5-2.5 mg/mL，用于软骨消化的常用浓度为1-2 mg/mL，需要根据特定的实验条件或者参考相应的文献资料确定所需的最佳工作浓度。

组织分离
1. 使用无菌手术刀或剪刀将组织切成 3-4 mm 大小的组织块；
2. 利用含 Ca2+、Mg2+ 的 HBSS 洗涤组织块数次；
3. 加入足量的含 Ca2+、Mg2+ 的 HBSS，使其浸没组织块，并加入胶原酶至需要工作浓度；
4. 于 37℃ 孵育 4-18 h。消化时使用水平摇床以及用 3 mM 的 CaCl2 补充消化可以提高消化效率；
5. 已分散开的细胞可使用不锈钢或尼龙网筛筛得，收集备用。未完全解离的组织另外添加适量的新鲜胶原酶工作液于 37 ℃ 继续孵育，进一步消化；
6. 利用不含胶原酶的 HBSS 洗涤收集的细胞数次；
7. 细胞培养液重悬上述细胞，利用自动细胞计数器或其他方法计算活细胞密度；
8. 于细胞培养皿上利用合适细胞培养基接种细胞。

器官灌注
1. 向 37 ℃ 预热的含 Ca2+、Mg2+ 的HBSS中加入胶原酶，另添加 3 mM的CaCl2有助于提高分离效率；
2. 按照已优化的速率对相应的器官灌注胶原酶工作液；
3. 将上述过程中回收的灌注液流经不锈钢或尼龙网筛，从而将已解离的细胞或小片段组织块与较大团块分离开来，未充分解离的组织需利用新鲜胶原酶工作液于 37 ℃ 进一步孵育。