Materne Eva-Maria, Ramme Anja Patricia, Terrasso Ana Paula, Serra Margarida, Alves Paula Marques, Brito Catarina, Sakharov Dmitry A, Tonevitsky Alexander G, Lauster Roland, Marx Uwe
Technische Universität Berlin, Institute of Biotechnology, Gustav-Meyer-Allee 25, Berlin 13355, Germany.
iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras 2780-901, Portugal; ITQB-UNL, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal.
J Biotechnol. 2015 Jul 10;205:36-46. doi: 10.1016/j.jbiotec.2015.02.002. Epub 2015 Feb 9.
Current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, often do not accurately predict drug toxicity, leading to high attrition rates in clinical studies (Paul et al., 2010). The phylogenetic distance between humans and laboratory animals is enormous, this affects the transferability of animal data on the efficacy of neuroprotective drugs. Therefore, many neuroprotective treatments that have shown promise in animals have not been successful when transferred to humans (Dragunow, 2008; Gibbons and Dragunow, 2010). We present a multi-organ chip capable of maintaining 3D tissues derived from various cell sources in a combined media circuit which bridges the gap in systemic and human tests. A steady state co-culture of human artificial liver microtissues and human neurospheres exposed to fluid flow over two weeks in the multi-organ chip has successfully proven its long-term performance. Daily lactate dehydrogenase activity measurements of the medium and immunofluorescence end-point staining proved the viability of the tissues and the maintenance of differentiated cellular phenotypes. Moreover, the lactate production and glucose consumption values of the tissues cultured indicated that a stable steady-state was achieved after 6 days of co-cultivation. The neurospheres remained differentiated neurons over the two-week cultivation in the multi-organ chip, proven by qPCR and immunofluorescence of the neuronal markers βIII-tubulin and microtubule-associated protein-2. Additionally, a two-week toxicity assay with a repeated substance exposure to the neurotoxic 2,5-hexanedione in two different concentrations induced high apoptosis within the neurospheres and liver microtissues, as shown by a strong increase of lactate dehydrogenase activity in the medium. The principal finding of the exposure of the co-culture to 2,5-hexanedione was that not only toxicity profiles of two different doses could be discriminated, but also that the co-cultures were more sensitive to the substance compared to respective single-tissue cultures in the multi-organ-chip. Thus, we provide here a new in vitro tool which might be utilized to predict the safety and efficacy of substances in clinical studies more accurately in the future.
当前用于药物研发的体外和动物试验无法模拟人体的全身器官复杂性，因此常常无法准确预测药物毒性，导致临床研究中的高淘汰率（保罗等人，2010年）。人类与实验动物之间的系统发育距离巨大，这影响了动物数据在神经保护药物疗效方面的可转移性。因此，许多在动物身上显示出前景的神经保护疗法在应用于人类时并未取得成功（德拉古诺夫，2008年；吉本斯和德拉古诺夫，2010年）。我们展示了一种多器官芯片，它能够在组合培养基回路中维持源自各种细胞来源的3D组织，弥合了全身和人体试验之间的差距。在多器官芯片中，人人工肝微组织和人神经球在流体流动下进行了两周的稳态共培养，成功证明了其长期性能。对培养基进行的每日乳酸脱氢酶活性测量以及免疫荧光终点染色证明了组织的活力以及分化细胞表型的维持。此外，培养组织的乳酸产生和葡萄糖消耗值表明，共培养6天后达到了稳定的稳态。在多器官芯片中培养的两周时间里，神经球一直保持为分化的神经元，这通过神经元标志物βIII - 微管蛋白和微管相关蛋白 - 2的定量聚合酶链反应（qPCR）和免疫荧光得以证实。此外，一项为期两周的毒性试验，对神经毒性物质2,5 - 己二酮进行两种不同浓度的重复暴露，结果显示培养基中乳酸脱氢酶活性大幅增加，表明神经球和肝微组织内出现了高度凋亡。共培养物暴露于2,5 - 己二酮的主要发现是，不仅可以区分两种不同剂量的毒性特征，而且与多器官芯片中的各自单组织培养相比，共培养物对该物质更敏感。因此，我们在此提供了一种新的体外工具，未来可能用于更准确地预测临床研究中物质的安全性和有效性。
