Enzyme-immobilized hydrogels to create hypoxia for in vitro cancer cell culture
Enzyme-immobilized hydrogels to create hypoxia for in vitro cancer cell culture
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DOI：
10.1016/j.jbiotec.2017.03.007
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发表时间：
2017-04-20
影响因子：
4.1
通讯作者：
Lin, Chien-Chi
中科院分区：
文献类型：
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作者：
Dawes, Camron S.;Konig, Heiko;Lin, Chien-Chi
低氧是控制细胞命运过程许多方面的关键条件。在低氧细胞培养中，最常见的做法是将细胞保存在有可控气体入口的培养箱中(即，缺氧室)。在这里，我们描述了酶固定化水凝胶的设计和表征，以在环境条件下创造溶液低氧，用于体外癌细胞培养。具体地说，将葡萄糖氧化酶(GOx)与聚乙二醇二丙烯酸酯(PEGDA)进行丙烯化反应，并通过光聚合形成固载GOx的聚乙二醇二丙烯酸酯水凝胶。我们首先评估了可溶性Gox对溶液低氧(O2<；5%)的诱导效果，发现未改性和丙烯酸化的Gox即使在空气中氧气从气液界面扩散的环境条件下也能维持至少24小时的低氧。然而，由于酶活性的丧失，可溶性Gox在24 h后逐渐失去耐缺氧的能力。另一方面，GOX固定化水凝胶能够在水凝胶中产生至少120小时的低氧，这可能是由于酶的聚乙二醇化和固定化增强了蛋白质的稳定性。作为概念验证，这种Gox固定化水凝胶系统被用于体外培养Molm14(急性髓系白血病细胞系)和Huh7(肝细胞癌细胞系)的低氧培养。在GOX固定化水凝胶中培养的细胞至少存活了24小时，低氧相关基因，包括碳酸氢酶9(CA9)和赖氨酸氧化酶(LOX)的表达显著上调。这些结果证明了利用酶固定化水凝胶为体外癌细胞培养创造低氧环境的潜力。(C)2017爱思唯尔B.V.保留所有权利。
Hypoxia is a critical condition governing many aspects of cellular fate processes. The most common practice in hypoxic cell culture is to maintain cells in an incubator with controlled gas inlet (i.e., hypoxic chamber). Here, we describe the design and characterization of enzyme-immobilized hydrogels to create solution hypoxia under ambient conditions for in vitro cancer cell culture. Specifically, glucose oxidase (GOX) was acrylated and co-polymerized with poly(ethylene glycol)-diacrylate (PEGDA) through photopolymerization to form GOX-immobilized PEG-based hydrogels. We first evaluated the effect of soluble GOX on inducing solution hypoxia (O-2 < 5%) and found that both unmodified and acrylated GOX could sustain hypoxia for at least 24 h even under ambient air condition with constant oxygen diffusion from the air-liquid interface. However, soluble GOX gradually lost its ability to sustain hypoxia after 24 h due to the loss of enzyme activity over time. On the other hand, GOX-immobilized hydrogels were able to create hypoxia within the hydrogel for at least 120 h, potentially due to enhanced protein stabilization by enzyme 'PEGylation' and immobilization. As a proof-of-concept, this GOX-immobilized hydrogel system was used to create hypoxia for in vitro culture of Molm14 (acute myeloid leukemia (AML) cell line) and Huh7 (hepatocellular carcinoma (HCC) cell line). Cells cultured in the presence of GOX-immobilized hydrogels remained viable for at least 24 h. The expression of hypoxia associated genes, including carbonic anhydrase 9 (CA9) and lysyl oxidase (LOX), were significantly upregulated in cells cultured with GOX-immobilized hydrogels. These results have demonstrated the potential of using enzyme-immobilized hydrogels to create hypoxic environment for in vitro cancer cell culture. (C) 2017 Elsevier B.V. All rights reserved.