Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States; Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States.
Interdisciplinary Graduate School, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore.
Acta Biomater. 2018 Jun;73:217-227. doi: 10.1016/j.actbio.2018.02.009. Epub 2018 Feb 15.
A major challenge of maintaining primary hepatocytes in vitro is progressive loss of hepatocyte-specific functions, such as protein synthesis and cytochrome P450 (CYP450) catalytic activity. We developed a three-dimensional (3D) nanofibrous scaffold made from poly(l-lactide-co-glycolide) (PLGA) polymer using a newly optimized wet electrospinning technique that resulted in a highly porous structure that accommodated inclusion of primary human hepatocytes. Extracellular matrix (ECM) proteins (type I collagen or fibronectin) at varying concentrations were chemically linked to electrospun PLGA using amine coupling to develop an in vitro culture system containing the minimal essential ECM components of the liver micro-environment that preserve hepatocyte function in vitro. Cell-laden nanofiber scaffolds were tested in vitro to maintain hepatocyte function over a two-week period. Incorporation of type I collagen onto PLGA scaffolds (PLGA-C: 100 µg/mL) led to 10-fold greater albumin secretion, 4-fold higher urea synthesis, and elevated transcription of hepatocyte-specific CYP450 genes (CYP3A4, 3.5-fold increase and CYP2C9, 3-fold increase) in primary human hepatocytes compared to the same cells grown within unmodified PLGA scaffolds over two weeks. These indices, measured using collagen-bonded scaffolds, were also higher than scaffolds coupled to fibronectin or an ECM control sandwich culture composed of type I collagen and Matrigel. Induction of CYP2C9 activity was also higher in these same type I collagen PLGA scaffolds compared to other ECM-modified or unmodified PLGA constructs and was equivalent to the ECM control at 7 days. Together, we demonstrate a minimalist ECM-based 3D synthetic scaffold that accommodates primary human hepatocyte inclusion into the matrix, maintains long-term in vitro survival and stimulates function, which can be attributed to coupling of type I collagen.
Culturing primary hepatocytes within a three-dimensional (3D) structure that mimics the natural liver environment is a promising strategy for extending the function and viability of hepatocytes in vitro. In the present study we generate porous PLGA nanofibers, that are chemically modified with extracellular matrix proteins, to serve as 3D scaffolds for the in vitro culture of primary human hepatocytes. Our findings demonstrate that the use of ECM proteins, especially type I collagen, in a porous 3D environment helps to improve the synthetic function of primary hepatocytes over time. We believe the work presented within will provide insights to readers for drug toxicity and tissue engineering applications.
维持原代肝细胞体外培养的主要挑战是肝细胞特异性功能（如蛋白质合成和细胞色素 P450（CYP450）催化活性）的逐渐丧失。我们使用新优化的湿法静电纺丝技术开发了一种由聚（L-丙交酯-共-乙交酯）（PLGA）聚合物制成的三维（3D）纳米纤维支架，该技术产生了高度多孔的结构，可容纳原代人肝细胞。通过胺偶联将不同浓度的细胞外基质（ECM）蛋白（I 型胶原或纤维连接蛋白）化学连接到电纺 PLGA 上，以开发包含肝脏微环境最小必需 ECM 成分的体外培养系统，可在体外维持肝细胞功能。细胞负载的纳米纤维支架在体外进行测试，以维持两周内的肝细胞功能。与在未经修饰的 PLGA 支架中培养两周的相同细胞相比，将 I 型胶原掺入 PLGA 支架（PLGA-C：100μg/mL）可导致白蛋白分泌增加 10 倍，尿素合成增加 4 倍，以及肝细胞特异性 CYP450 基因（CYP3A4，增加 3.5 倍和 CYP2C9，增加 3 倍）的转录升高。使用胶原结合支架测量的这些指数也高于与纤维连接蛋白或由 I 型胶原和 Matrigel 组成的 ECM 对照三明治培养物偶联的支架。在相同的 I 型胶原 PLGA 支架中，CYP2C9 活性的诱导也高于其他 ECM 修饰或未修饰的 PLGA 结构，并且在第 7 天与 ECM 对照相当。总的来说，我们展示了一种基于最小 ECM 的 3D 合成支架，该支架可容纳原代人肝细胞纳入基质，在体外长期维持生存并刺激功能，这可归因于 I 型胶原的偶联。
在模拟天然肝脏环境的 3D 结构中培养原代肝细胞是延长肝细胞体外功能和活力的一种很有前途的策略。在本研究中，我们生成多孔的 PLGA 纳米纤维，并用细胞外基质蛋白进行化学修饰，用作原代人肝细胞体外培养的 3D 支架。我们的研究结果表明，在多孔 3D 环境中使用 ECM 蛋白，特别是 I 型胶原，有助于随着时间的推移改善原代肝细胞的合成功能。我们相信，本文将为读者提供有关药物毒性和组织工程应用的见解。
