胶原酶（Collagenase）来源于溶组织梭菌（Clostridium histolyticum），是一种蛋白酶，能够特异性识别 Pro-X-Gly-Pro 序列并切割该序列中中性氨基酸（X）和甘氨酸（Gly）之间的肽键，且该序列在胶原中出现频率很高。胶原酶是唯一可降解广泛存在于结缔组织中、具有三股螺旋结构的天然胶原纤维的蛋白酶。
本品为 I 型胶原酶，酶活≥125 U/mg solid，可用于上皮、肝、肺、脂肪和肾上腺等组织和细胞的解离。
使用方法
（一）胶原酶储存液的配制
向每管 100mg 的胶原酶中加入 1ml 含 Ca²⁺、Mg²⁺的 HBSS（Hank's 平衡盐溶液，含 Ca²⁺、Mg²⁺），轻轻旋涡震荡使其充分溶解，制备成 100 mg/ml（即 100×）的储存液。用低蛋白结合性的 0.22 μm 滤膜过滤除菌，分装成小份量，于 - 20℃避光冻存。
使用前于冰上解冻，避免反复冻融。常用浓度：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；需根据特定实验条件或参考相应文献资料确定最佳工作浓度。
（二）组织的分离
1. 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. 加入足量含 Ca²⁺、Mg²⁺的 HBSS，使其浸没组织块，并加入胶原酶至所需工作浓度；
4. 于 37℃孵育 4~18 h；消化时使用水平摇床，并用 3 mM 的 CaCl₂补充消化，可提高消化效率；
5. 已分散的细胞用不锈钢或尼龙网筛筛取，收集备用；未完全解离的组织需另外添加适量新鲜胶原酶工作液，于 37℃继续孵育；
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. 在细胞培养皿上，用合适的细胞培养基接种细胞。
（三）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，分离已解离的细胞或小片段组织块与较大团块；未充分解离的组织需用新鲜胶原酶工作液于 37℃进一步孵育；
4. 利用不含胶原酶的 HBSS 洗涤收集的细胞数次；
5. 用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；
6. 在细胞培养皿上，用合适的细胞培养基接种细胞。
Collagenase is derived from Clostridium histolyticum and is a type of protease. It can specifically recognize the Pro-X-Gly-Pro sequence and cleave the peptide bond between the neutral amino acid (X) and glycine (Gly) in this sequence, and this sequence appears very frequently in collagen. Collagenase is the only protease that can degrade natural collagen fibers with a triple-helix structure, which are widely present in connective tissues.
This product is Collagenase Type I with an enzyme activity of ≥125 U/mg solid, and it can be used for the dissociation of tissues and cells such as epithelium, liver, lung, fat, and adrenal gland.
Usage Instructions
(I) Preparation of Collagenase Stock Solution
Add 1 ml of HBSS (Hank's Balanced Salt Solution) containing Ca²⁺ and Mg²⁺ to each tube of 100 mg collagenase, gently vortex to dissolve it fully, and prepare a stock solution with a concentration of 100 mg/ml (i.e., 100×). Filter and sterilize using a 0.22 μm filter membrane with low protein binding property, aliquot into small portions, and store at -20℃ in the dark.
Note: This product is a crude preparation. A small amount of insoluble impurities may remain in the system after dissolution. Such insoluble matter does not affect the biological activity of the product, and the product can be used normally. If a sterile solution is required, it is recommended to centrifuge at 12 000‑15 000 g for 5‑10 min at 4 °C, collect the supernatant, and then sterile‑filter through a 0.22 μm membrane. Alternatively, pre‑filter with a 0.8 μm membrane followed by sterile‑filtration using a 0.22 μm membrane to prevent membrane clogging.
Thaw on ice before use, and avoid repeated freezing and thawing. Common concentrations: the common concentration for tissue and cell dispersion is 0.5~2.5 mg/ml, and the common concentration for cartilage digestion is 1~2 mg/ml; the optimal working concentration should be determined according to specific experimental conditions or by referring to relevant literature.
(II) Tissue Separation
1. Cut the tissue into tissue blocks of 3~4 mm in size using a sterile scalpel or scissors;
2. Wash the tissue blocks several times with HBSS containing Ca²⁺ and Mg²⁺;
3. Add a sufficient amount of HBSS containing Ca²⁺ and Mg²⁺ to submerge the tissue blocks, and add collagenase to the required working concentration;
4. Incubate at 37℃ for 4~18 hours; using a horizontal shaker during digestion and supplementing digestion with 3 mM CaCl₂ can improve digestion efficiency;
5. Sieve the dispersed cells using a stainless steel or nylon mesh sieve, collect and set aside; for tissues that are not completely dissociated, add an appropriate amount of fresh collagenase working solution and continue incubation at 37℃;
6. Wash the collected cells several times with HBSS without collagenase;
7. Resuspend the above cells with cell culture medium, and calculate the viable cell density using an automatic cell counter or other methods;
8. Inoculate the cells on a cell culture dish using a suitable cell culture medium.
(III) Organ Perfusion
1. Add collagenase to HBSS containing Ca²⁺ and Mg²⁺ preheated to 37℃; additionally 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 perfusion solution recovered in the above process through a stainless steel or nylon mesh sieve to separate the dissociated cells or small tissue fragments from larger clumps; for tissues that are not fully dissociated, further incubate at 37℃ with fresh collagenase working solution;
4. Wash the collected cells several times with HBSS without collagenase;
5. Resuspend the above cells with cell culture medium, and calculate the viable cell density using an automatic cell counter or other methods;
6. Inoculate the cells on a cell culture dish using a suitable cell culture medium.
ZJ26F0939195
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