膠原酶的類型和應(yīng)用及其組織解離方法介紹
表 1. 膠原酶的類型及應(yīng)用。
圖 1. 從固體組織制備單細(xì)胞懸浮液的關(guān)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(fù)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(xì)胞可使用細(xì)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(xì)胞數(shù)次。
5、預(yù)冷的細(xì)胞培養(yǎng)液重懸細(xì)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(yīng)用于細(xì)胞消化及組織解離。
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