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 IV 型胶原酶，≥125 U/mg solid，具有低胰酶活性，特别适用于胰岛细胞制备。
使用方法
一、胶原酶储存液的配制
向每管 100 mg 的胶原酶中加入 1 ml 的相应缓冲液（根据特定的实验条件或者参考相应的文献资料确定），如 HBSS（Hank's 平衡盐溶液，含 Ca²+、Mg²+ 等），轻轻涡旋震荡使其充分溶解，制备成 100 mg/ml（即 100×）的储存液。用低蛋白结合性的 0.22 μm 的滤膜过滤除菌，分装成小份量，于 - 20℃避光冻存。
注：本品为粗提制品，溶解后体系中可能存在少量不溶性杂质，该类不溶物不影响产品生物活性，可正常使用。如果需要制备无菌溶液，建议先 4℃ 12000~15000 g 离心 5~10 min 取上清后再用 0.22 μm 滤膜过滤除菌或者先用 0.8 μm 滤膜过滤后再用 0.22 μm 滤膜过滤除菌，避免滤膜堵塞。
使用前于冰上解冻，避免反复冻融。其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。
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. 使用相应缓冲液（如 HBSS）洗涤组织块数次；
3. 加入足量的缓冲液，使其浸没组织，并加入胶原酶至需要的工作浓度；
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. 充分消化的组织可用不锈钢或尼龙网筛过滤，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℃继续孵解；
6. 使用不含胶原酶的缓冲液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。
三、器官灌注
1. 向 37℃预热的相应缓冲液（如 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℃进一步孵解；
4. 使用不含胶原酶的缓冲液洗涤收集的细胞数次；
5. 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. 于细胞培养皿上利用合适细胞培养基接种细胞。
Collagenase is a protease derived from Clostridium histolyticum. It specifically recognizes the Pro-X-Gly-Pro sequence and cleaves the peptide bond between glycine (Gly) and glycine (Gly) in this sequence, and this sequence occurs frequently in collagen. Collagenase is the only protease that can degrade the triple-helical natural collagen fibers widely present in connective tissue.This product is Type IV collagenase, with an activity of ≥125 U/mg solid and low trypsin activity, making it particularly suitable for islet cell preparation.
Usage Instructions
Ⅰ. Preparation of Collagenase Stock Solution
Add 1 ml of the appropriate buffer (determined according to specific experimental conditions or relevant literature) to each tube containing 100 mg of collagenase (e.g., HBSS [Hank's Balanced Salt Solution, containing Ca²+, Mg²+, etc.]). Gently vortex to dissolve thoroughly and prepare a 100 mg/ml (i.e., 100×) stock solution. Filter-sterilize using a 0.22 μm filter with low protein binding properties, aliquot into small portions, and store frozen at -20°C in the dark.Thaw on ice before use; avoid repeated freeze-thaw cycles. The typical working concentration for tissue and cell dissociation is 0.5~2.5 mg/ml, and 1~2 mg/ml for cartilage digestion. Determine the optimal working concentration based on specific experimental conditions or relevant literature.
Ⅱ. Tissue Isolation
- Use a sterile scalpel or scissors to cut the tissue into 3~4 mm tissue pieces;
- Wash the tissue pieces several times with the appropriate buffer (e.g., HBSS);
- Add sufficient buffer to submerge the tissue, then add collagenase to the required working concentration;
- Incubate at 37°C for 4~18 hours. Using a horizontal shaker and supplementing digestion with 3 mM CaCl₂ can improve digestion efficiency;
- Fully digested tissue can be filtered through a stainless steel or nylon mesh sieve and collected for later use. For incompletely dissociated tissue, add an appropriate amount of fresh collagenase working solution and continue incubation at 37°C;
- Wash the collected cells several times with collagenase-free buffer;
- Resuspend the cells in cell culture medium, and count the viable cell density using an automated cell counter or other methods;
- Seed the cells on cell culture dishes using a suitable cell culture medium.
Ⅲ. Organ Perfusion
- Add collagenase to the appropriate buffer (e.g., HBSS) preheated to 37°C; supplementing with 3 mM CaCl₂ helps improve isolation efficiency;
- Perfuse the target organ with the collagenase working solution at an optimized rate;
- Pass the recovered perfusion solution through a stainless steel or nylon mesh sieve to separate dissociated cells/small tissue fragments from larger clumps. Incompletely dissociated tissue should be further incubated with fresh collagenase working solution at 37°C;
- Wash the collected cells several times with collagenase-free buffer;
- Resuspend the cells in cell culture medium, and count the viable cell density using an automated cell counter or other methods;
- Seed the cells on cell culture dishes using a suitable cell culture medium.
ZJ26F1039889
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