Davidson Matthew D, Pickrell Joshua, Khetani Salman R
School of Biomedical Engineering, Colorado State University, Fort Collins, CO, United States; Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, United States.
School of Biomedical Engineering, Colorado State University, Fort Collins, CO, United States; Department of Mechanical Engineering, Colorado State University, Fort Collins, CO, United States.
Toxicology. 2021 Feb 15;449:152662. doi: 10.1016/j.tox.2020.152662. Epub 2020 Dec 24.
Given significant species-specific differences in liver functions, cultures of primary human hepatocytes (PHHs) are useful for assessing drug metabolism and to mitigate the risk of drug-induced hepatotoxicity in humans. While significant advances have been made to keep PHHs highly functional for 2-4 weeks in vitro, especially upon co-culture with both liver- and non-liver-derived non-parenchymal cells (NPCs), the functional lifespan of PHHs is 200-400 days in vivo. Therefore, it is desirable to determine culture conditions that can further prolong PHHs functions in vitro for modeling chronic drug exposure, disease pathogenesis, and to provide flexibility to the end-user for staggering drug incubations across multiple culture batches. Most PHH culture platforms utilize supraphysiologic levels of glucose and insulin and bovine-derived serum when including NPCs, which can alter PHH functions. Therefore, here we developed a culture medium containing physiologic levels of glucose (5 mM), insulin (500 pM), and human serum (10 % v/v) and tested its effects on micropatterned co-cultures (MPCCs) in which PHHs are organized onto collagen domains of empirically optimized dimensions and surrounded by 3T3-J2 murine fibroblasts that express liver-like molecules and induce higher PHH functions than liver-derived NPCs. Our physiologically-inspired culture medium allowed better retention of PHH morphology, polarity, and functions (albumin and urea, cytochrome-P450 activities, and sensitivity to insulin-mediated inhibition of gluconeogenesis) for up to 10 weeks relative to the traditional medium. Finally, PHHs in the physiologic medium displayed clinically-relevant responses to prototypical drugs for hepatoxicity and cytochrome-P450 induction. Ultimately, our physiologic culture medium could find broader utility for the continued development of PHH-NPC co-cultures for drug development, investigating the effects of patient-derived sera on PHH functions and disease phenotypes, and for use in cell-based therapies.
鉴于肝脏功能存在显著的物种特异性差异，原代人肝细胞（PHH）培养物可用于评估药物代谢，并降低药物诱导的人类肝毒性风险。虽然在使PHH在体外2至4周内保持高度功能方面已取得重大进展，特别是在与肝脏和非肝脏来源的非实质细胞（NPC）共培养时，但PHH在体内的功能寿命为200至400天。因此，需要确定能够在体外进一步延长PHH功能的培养条件，以模拟慢性药物暴露、疾病发病机制，并为终端用户提供灵活性，以便在多个培养批次中错开药物孵育时间。大多数PHH培养平台在包含NPC时会使用超生理水平的葡萄糖、胰岛素和牛源血清，这可能会改变PHH的功能。因此，我们在此开发了一种含有生理水平葡萄糖（5 mM）、胰岛素（500 pM）和人血清（10% v/v）的培养基，并测试了其对微图案共培养（MPCC）的影响。在MPCC中，PHH被组织在经验优化尺寸的胶原结构域上，并被表达肝脏样分子且比肝脏来源的NPC诱导更高PHH功能的3T3-J2小鼠成纤维细胞包围。相对于传统培养基，我们受生理启发的培养基能够在长达10周的时间内更好地保持PHH的形态、极性和功能（白蛋白和尿素、细胞色素P450活性以及对胰岛素介导的糖异生抑制的敏感性）。最后，生理培养基中的PHH对肝毒性和细胞色素P450诱导的原型药物表现出临床相关反应。最终，我们的生理培养基可能会在PHH-NPC共培养物的持续开发中找到更广泛的用途，用于药物开发、研究患者来源血清对PHH功能和疾病表型的影响以及用于基于细胞的治疗。
