A multi-organ chip co-culture of neurospheres and liver equivalents for long-term substance testing.

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.

[1]  L G Griffith,et al.  In Vitro Organogenesis of Liver Tissue a , 1997, Annals of the New York Academy of Sciences.

[2]  J. Trojanowski,et al.  Increased levels of fucosyltransferase IX and carbohydrate Lewisx adhesion determinant in human NT2N neurons , 2007, Journal of neuroscience research.

[3]  Jong Hwan Sung,et al.  A micro cell culture analog (microCCA) with 3-D hydrogel culture of multiple cell lines to assess metabolism-dependent cytotoxicity of anti-cancer drugs. , 2009, Lab on a chip.

[4]  P. Alves,et al.  Generation and genetic modification of 3D cultures of human dopaminergic neurons derived from neural progenitor cells , 2012 .

[5]  M. Dragunow,et al.  Adult human brain cell culture for neuroscience research. , 2010, The international journal of biochemistry & cell biology.

[6]  A. DeCaprio,et al.  Comparative neurotoxicity and pyrrole-forming potential of 2,5-hexanedione and perdeuterio-2,5-hexanedione in the rat. , 1988, Toxicology and applied pharmacology.

[7]  Uwe Marx,et al.  Integrating biological vasculature into a multi-organ-chip microsystem. , 2013, Lab on a chip.

[8]  Jarrod Bailey,et al.  An Analysis of the Use of Dogs in Predicting Human Toxicology and Drug Safety , 2013, Alternatives to laboratory animals : ATLA.

[9]  Wei-Shou Hu,et al.  Three-dimensional co-culture of hepatocytes and stellate cells , 2004, Cytotechnology.

[10]  Donald E Ingber,et al.  Microengineered physiological biomimicry: organs-on-chips. , 2012, Lab on a chip.

[11]  Lucas H. Hofmeister,et al.  Scaling and systems biology for integrating multiple organs-on-a-chip. , 2013, Lab on a chip.

[12]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[13]  Laurent Griscom,et al.  Improvement of HepG2/C3a cell functions in a microfluidic biochip , 2011, Biotechnology and bioengineering.

[14]  S. Koenig,et al.  Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells , 2009, In Vitro Cellular & Developmental Biology - Animal.

[15]  M. Coleman,et al.  The toxicity of hexanedione isomers in neural and astrocytic cell lines. , 2006, Environmental toxicology and pharmacology.

[16]  Uwe Marx,et al.  ‘Human-on-a-chip’ Developments: A Translational Cutting-edge Alternative to Systemic Safety Assessment and Efficiency Evaluation of Substances in Laboratory Animals and Man? , 2012, Alternatives to laboratory animals : ATLA.

[17]  O. Ladefoged,et al.  Acetone potentiation and influence on the reversibility of 2,5-hexanedione-induced neurotoxicity studied with behavioural and morphometric methods in rats. , 1994, Pharmacology & toxicology.

[18]  M. Dragunow.  The adult human brain in preclinical drug development , 2008, Nature Reviews Drug Discovery.

[19]  Po Ki Yuen,et al.  Perfusion-based microfluidic device for three-dimensional dynamic primary human hepatocyte cell culture in the absence of biological or synthetic matrices or coagulants. , 2010, Lab on a chip.

[20]  Uwe Marx,et al.  Chip-based liver equivalents for toxicity testing--organotypicalness versus cost-efficient high throughput. , 2013, Lab on a chip.

[21]  Uwe Marx,et al.  ‘ Human-ona-chip ’ Developments : A Translational Cutting-edge Alternative to Systemic Safety Assessment and Efficiency Evaluation of Substances in Laboratory Animals and Man ? , 2012 .

[22]  Hanry Yu,et al.  Towards a human-on-chip: culturing multiple cell types on a chip with compartmentalized microenvironments. , 2009, Lab on a chip.

[23]  T. Zeng,et al.  Correlation between Levels of 2, 5-Hexanedione and Pyrrole Adducts in Tissues of Rats Exposure to n-Hexane for 5-Days , 2013, PloS one.

[24]  Gwo-Bin Lee,et al.  Microfluidic cell culture systems for drug research. , 2010, Lab on a chip.

[25]  Charles C. Persinger,et al.  How to improve R&D productivity: the pharmaceutical industry's grand challenge , 2010, Nature Reviews Drug Discovery.

[26]  F. A. de Wolff,et al.  Effects of 2,5-hexanedione on calpain-mediated degradation of human neurofilaments in vitro. , 2000, Chemico-biological interactions.

[27]  Mandy B. Esch,et al.  The role of body-on-a-chip devices in drug and toxicity studies. , 2011, Annual review of biomedical engineering.

[28]  F. Sonntag,et al.  A dynamic multi-organ-chip for long-term cultivation and substance testing proven by 3D human liver and skin tissue co-culture. , 2013, Lab on a chip.

[29]  P. Alves,et al.  Integrating human stem cell expansion and neuronal differentiation in bioreactors , 2009, BMC biotechnology.

[30]  Michael L Shuler,et al.  Incorporation of 3T3‐L1 Cells To Mimic Bioaccumulation in a Microscale Cell Culture Analog Device for Toxicity Studies , 2008, Biotechnology progress.

[31]  Christian Trepo,et al.  Infection of a human hepatoma cell line by hepatitis B virus , 2002, Proceedings of the National Academy of Sciences of the United States of America.