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。
[1] Zhewen Hu,et al. Novel strategy for primary epithelial cell isolation: Combination of hyaluronidase and collagenase I.Cell Prolif. 2023 Jan;56(1):e13320.
[2] Meital Charni-Natan,et al. Protocol for Primary Mouse Hepatocyte Isolation.STAR Protoc. 2020 Aug 13;1(2):100086.
[3] Andrew Reichard,et al.Best Practices for Preparing a Single Cell Suspension from Solid Tissues for Flow Cytometry. Cytometry A. 2019 Feb;95(2):219-226.
[4] Manouela V. Valtcheva,et al. Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures. Nat Protoc. 2016 October ; 11(10): 1877-1888.
[5] Hussein Shoaito,et al. The Role of Peroxisome Proliferator–Activated Receptor Gamma (PPARγ) in Mono(2-ethylhexyl) Phthalate (MEHP)-Mediated Cytotrophoblast Differentiation.Environ Health Perspect. 2019 Feb;127(2):27003.
[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.
[7] Anna A. Nushtaeva ,et al. Establishment of primary human breast cancer cell lines using “pulsed hypoxia” method and development of metastatic tumor model in immunodeficient mice. Cancer Cell International volume 19, Article number: 46 (2019).
[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.