膠原酶的類型和應(yīng)用及其組織解離方法介紹

 

膠原酶 (Collagenase)，化學(xué)名為膠原蛋白水解酶，是從溶組織梭菌芽孢桿菌提取制備而成，能夠特異性識別膠原蛋白中的 Pro-X-Gly-Pro 序列，并切割該序列中的中性氨基酸 (X) 和甘氨酸 (Gly) 之間的肽鍵，常用于原代細(xì)胞制備[1][2]。不同類型膠原酶的應(yīng)用為大家整理在下面啦~

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(shí)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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