Intermittent Starvation Extends the Functional Lifetime of Primary Human Hepatocyte Cultures
Intermittent Starvation Extends the Functional Lifetime of Primary Human Hepatocyte Cultures
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DOI：
10.1093/toxsci/kfaa003
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发表时间：
2020-04-01
影响因子：
3.8
通讯作者：
Khetani, Salman R.
中科院分区：
文献类型：
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作者：
Davidson, Matthew D.;Khetani, Salman R.
原代人肝细胞（PHH）培养物对于减轻人类患者的药物不良反应风险已变得不可或缺。与去分化的单一培养物相反，与非实质细胞共培养可维持PHH功能2-4周。然而，由于PHH在体内的功能寿命为200-400天，因此期望进一步延长PHH在体外的功能，以模拟慢性药物暴露和疾病进展。禁食对生物体的寿命和肝脏等组织的健康有好处。我们假设模拟动态禁食/饥饿的培养方案可以激活饥饿途径并延长PHH功能寿命。为了模拟饥饿，从微图案化共培养物（MPCC）的培养基中间歇地去除血清和激素，所述微图案化共培养物（MPCC）含有组织到胶原结构域上并被3 T3-J2鼠成纤维细胞包围的PHH。每周2天的饥饿最佳延长PHH功能寿命为6+周的MPC与下降后3周的非饥饿控制。饥饿2天也增强了PHH单一培养物2周的功能，表明对PHH的直接影响。在MPC中，饥饿激活5'腺苷一磷酸激活蛋白激酶（AMPK），限制成纤维细胞过度生长到PHH岛，从而维持肝脏极性。饥饿对MPCCs的影响通过使用二甲双胍激活AMPK或通过丝裂霉素C抑制成纤维细胞生长而部分重现。最后，饥饿的MPC表现出较低的假阳性药物毒性试验和较高的药物诱导的细胞色素P450活性与非饥饿的控制，即使在5周后。总之，间歇性血清/激素饥饿延长PHH功能寿命，使临床相关的药物筛选。
Primary human hepatocyte (PHH) cultures have become indispensable to mitigate the risk of adverse drug reactions in human patients. In contrast to dedifferentiating monocultures, coculture with nonparenchymal cells maintains PHH functions for 2-4 weeks. However, because the functional lifespan of PHHs in vivo is 200-400 days, it is desirable to further prolong PHH functions in vitro toward modeling chronic drug exposure and disease progression. Fasting has benefits on the longevity of organisms and the health of tissues such as the liver. We hypothesized that a culturing protocol that mimics dynamic fasting/starvation could activate starvation pathways and prolong PHH functional lifetime. To mimic starvation, serum and hormones were intermittently removed from the culture medium of micropatterned cocultures (MPCCs) containing PHHs organized onto collagen domains and surrounded by 3T3-J2 murine fibroblasts. A weekly 2-day starvation optimally prolonged PHH functional lifetime for 6+ weeks in MPCCs versus a decline after 3 weeks in nonstarved controls. The 2-day starvation also enhanced the functions of PHH monocultures for 2 weeks, suggesting direct effects on PHHs. In MPCCs, starvation activated 5' adenosine monophosphate-activated protein kinase (AMPK) and restricted fibroblast overgrowth onto PHH islands, thereby maintaining hepatic polarity. The effects of starvation on MPCCs were partially recapitulated by activating AMPK using metformin or growth arresting fibroblasts via mitomycin-C. Lastly, starved MPCCs demonstrated lower false positives for drug toxicity tests and higher drug-induced cytochrome-P450 activities versus nonstarved controls even after 5 weeks. In conclusion, intermittent serum/hormone starvation extends PHH functional lifetime toward enabling clinically relevant drug screening.