膠原酶的類型和應用及其組織解離方法介紹
表 1. 膠原酶的類型及應用。
圖 1. 從固體組織制備單細胞懸浮液的關鍵步驟[3]。
▐ 膠原酶配制
向 100 mg 膠原酶中加入 4.5 mL HBSS，震蕩使其充分溶解， 0.22 μm 濾膜過濾除菌后，立即使用或分裝成小份量，于 -20°C 避光凍存，使用前在冰上解凍，避免反復凍融[4]。
▐ 組織分離
1、從生物體中解剖組織后，使用培養(yǎng)基/PBS/HBSS 清洗組織，隨后用無菌手術(shù)刀或剪刀將組織切成小的組織塊，HBSS 洗滌組織塊[5][6]。
2、加入足量 HBSS，使其浸沒組織塊，并加入膠原酶至需要的工作濃度，于 37℃ 孵育。
表 2. 不同組織樣本解離方案。
3、已分散開的細胞可使用細胞過濾器/尼龍網(wǎng)篩得，收集備用。
4、使用不含膠原酶的 HBSS 或培養(yǎng)基洗滌收集的細胞數(shù)次。
5、預冷的細胞培養(yǎng)液重懸細胞[13]。
▐ 實驗技巧
MCE 除了膠原酶外，還提供如胰酶、分散酶、重組胰酶消化液等，均可應用于細胞消化及組織解離。
[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.
