胶原酶 (Collagenase)，化学名为胶原蛋白水解酶，是从溶组织梭菌芽孢杆菌提取制备而成，能够特异性识别胶原蛋白中的 Pro-X-Gly-Pro 序列，并切割该序列中的中性氨基酸 (X) 和甘氨酸 (Gly) 之间的肽键，常用于原代细胞制备[1][2]。不同类型胶原酶的应用为大家整理在下面啦~

表 1. 胶原酶的类型及应用。  

图 1. 从固体组织制备单细胞悬浮液的关键步骤^[3]。

? 胶原酶配制

向 100 mg 胶原酶中加入 4.5 mL HBSS，震荡使其充分溶解， 0.22 μm 滤膜过滤除菌后，立即使用或分装成小份量，于 -20°C 避光冻存，使用前在冰上解冻，避免反复冻融[4]。

? 组织分离

1、从生物体中解剖组织后，使用培养基/PBS/HBSS 清洗组织，随后用无菌手术刀或剪刀将组织切成小的组织块，HBSS 洗涤组织块[5][6]。

2、加入足量 HBSS，使其浸没组织块，并加入胶原酶至需要的工作浓度，于 37℃ 孵育。

表 2. 不同组织样本解离方案。

3、已分散开的细胞可使用细胞过滤器/尼龙网筛得，收集备用。

4、使用不含胶原酶的 HBSS 或培养基洗涤收集的细胞数次。

5、预冷的细胞培养液重悬细胞[13]。

? 实验技巧

MCE 除了胶原酶外，还提供如胰酶、分散酶、重组胰酶消化液等，均可应用于细胞消化及组织解离。 

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[6] Zhewen Hu,et al. Novel strategy for primary epithelial cell isolation: Combination of hyaluronidase and collagenase I. Cell Prolif. 2023 Jan;56(1):e13320.

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[8] Chaojun Tang ,et al. CLEC-2-dependent platelet subendothelial accumulation by flow disturbance contributes to atherogenesis in mice.Theranostics. 2021 Oct 3;11(20):9791-9804.

[9] Miguel F. Diaz ,et al. Bone marrow stromal cell therapy improves survival after radiation injury but does not restore endogenous hematopoiesis.Scientific Reports. (2020) 10:22211

[10] Nan Mei ,et al. The genotoxicity of acrylamide and glycidamide in big blue rats.Toxicol Sci. 2010 Jun;115(2):412-21.

[11] Chenwei Li ,et al. Identification of Pancreatic Cancer Stem Cells.Cancer Res (2007) 67 (3): 1030-1037.

[12] Suzanne Bertera ,et al. Human Hemangioblast-Derived Mesenchymal Stem Cells Promote Islet Engraftment in a Minimal Islet Mass Transplantation Model in Mice.Front. Med. 8:660877.

[13] Akihito Harusato,et al. Macrophage Isolation from the Mouse Small and Large Intestine.Methods Mol Biol. 2016:1422:171-80.