title: A multi-organ chip co-culture of neurospheres and liver equivalents for long-term substance testing.
abstract: 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.
authors: Technische Universität Berlin, Institute of Biotechnology, Gustav-Meyer-Allee 25, Berlin 13355, Germany. Electronic address: eva-maria.materne@tu-berlin.de.
authors: Eva-Maria
authors: EM
authors: Materne
authors: Technische Universität Berlin, Institute of Biotechnology, Gustav-Meyer-Allee 25, Berlin 13355, Germany. Electronic address: anja.ramme@tu-berlin.de.
authors: Anja Patricia
authors: AP
authors: Ramme
authors: 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. Electronic address: aterrasso@itqb.unl.pt.
authors: Ana Paula
authors: AP
authors: Terrasso
authors: 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. Electronic address: mserra@ibet.pt.
authors: Margarida
authors: M
authors: Serra
authors: 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. Electronic address: marques@ibet.pt.
authors: Paula Marques
authors: PM
authors: Alves
authors: 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. Electronic address: anabrito@itqb.unl.pt.
authors: Catarina
authors: C
authors: Brito
authors: Scientific Research Centre Bioclinicum, Ugreshskaya ul. 2/85, 115088 Moscow, Russia. Electronic address: dimitri_sakharov@mail.ru.
authors: Dmitry A
authors: DA
authors: Sakharov
authors: Institute of General Pathology and Pathophysiology, Russian Academy of Medical Science, Baltiyskaya ul. 8, 125315 Moscow, Russia. Electronic address: tonevitsky@mail.ru.
authors: Alexander G
authors: AG
authors: Tonevitsky
authors: Technische Universität Berlin, Institute of Biotechnology, Gustav-Meyer-Allee 25, Berlin 13355, Germany. Electronic address: roland.lauster@tu-berlin.de.
authors: Roland
authors: R
authors: Lauster
authors: Technische Universität Berlin, Institute of Biotechnology, Gustav-Meyer-Allee 25, Berlin 13355, Germany; TissUse GmbH, Markgrafenstr. 18, 15528 Spreenhagen, Germany. Electronic address: uwe.marx@tu-berlin.de.
authors: Uwe
authors: U
authors: Marx
mesh_terms: D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods*; D006588:Hexanones / Q000633:toxicity; D006801:Humans; D008099:Liver / Q000166:cytology* / Q000187:drug effects; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D009474:Neurons / Q000378:metabolism*; D018874:Spheroids, Cellular / Q000166:cytology* / Q000187:drug effects; D046509:Tissue Culture Techniques / Q000379:methods
chemical_list: D006588:Hexanones; C011269:2,5-hexanedione
keywords: In vitro substance testing; Liver; Multi-organ-chip; Neurospheres; Organoids; Tissue engineering