Abstract
Sensor-based cellular microphysiometry is a technique that allows non-invasive, label-free, real-time monitoring of living cells that can greatly improve the predictability of toxicology testing by removing the influence of biochemical labels. In this work, the Intelligent Mobile Lab for In Vitro Diagnostics (IMOLA-IVD) was utilized to perform cellular microphysiometry on 3D multicellular spheroids. Using a commercial 3D printer, 3 × 3 microwell arrays were fabricated to maintain nine previously cultured HepG2 spheroids on a single BioChip. Integrated layers above and under the spheroids allowed fluidic contact between spheroids in microwells and BioChip sensors while preventing wash out from medium perfusion. Spheroid culturing protocols were optimized to grow spheroids to a diameter of around 620 μm prior to transfer onto BioChips. An ON/OFF pump cycling protocol was developed to optimize spheroid culture within the designed microwells, intermittently perfuse spheroids with fresh culture medium, and measure the extracellular acidification rate (EAR) and oxygen uptake rate (OUR) with the BioChips of the IMOLA-IVD platform. In a proof-of-concept experiment, spheroids were perfused for 36 h with cell culture medium before being exposed to medium with 1% sodium dodecyl sulphate (SDS) to lyse cells as a positive control. These microphysiometry studies revealed a repeatable pattern of extracellular acidification throughout the experiment, indicating the ability to monitor real-time metabolic activity of spheroids embedded in the newly designed tissue encapsulation. After perfusion for 36 h with medium, SDS exposure resulted in an instant decrease in EAR and OUR signals from 37 mV/h (± 5) to 8 mV/h (± 8) and from 308 mV/h (± 21) to −2 mV/h (± 13), respectively. The presented spheroid monitoring system holds great potential as a method to automate screening and analysis of pharmaceutical agents using 3D multicellular spheroid models.
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Abu-Absi SF, Friend JR, Hansen LK, Hu W-S (2002) Structural polarity and functional bile canaliculi in rat hepatocyte spheroids. Exp Cell Res 274:56–67. doi:10.1006/excr.2001.5467
Alépée N, Alepee N, Bahinski A et al (2014) State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology. Altex 31:441–477. doi:10.14573/altex1406111
Alexander F, Wiest J (2016) Automated transepithelial electrical resistance measurements of the EpiDerm reconstructed human epidermis model. Conf Proc IEEE Eng Med Biol Soc 2016:469–472. doi:10.1109/EMBC.2016.7590741
Astashkina A, Grainger DW (2014) Critical analysis of 3-D organoid in vitro cell culture models for high-throughput drug candidate toxicity assessments. Adv Drug Deliv Rev 69–70:1–18. doi:10.1016/j.addr.2014.02.008
Bale SS, Vernetti L, Senutovitch N et al (2014) In vitro platforms for evaluating liver toxicity. Exp Biol Med 239:1180–1191. doi:10.1177/1535370214531872
Baras ASAI, Baras ASAI, Schulman KA (2012) Drug development risk and the cost of capital. Nat Rev Drug Discov 11:347–348. doi:10.1038/nrd3722
Brischwein M, Grothe H, Wiest J, Zottmann M, Ressler J, Wolf B (2009) Planar ruthenium oxide sensors for cell-on-a-chip metabolic studies. Chem Anal-Wars 54:1193–1201
Edmondson R, Broglie JJ, Adcock AF, Yang L (2014) Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol 12:207–218. doi:10.1089/adt.2014.573
Eggert S, Alexander F, Wiest J (2015) An automated microphysiological assay for toxicity evaluation. Conf Proc IEEE Eng Med Biol Soc 2015:2175–2178. doi:10.1109/EMBC.2015.7318821
Eggert S, Alexander F, Wiest J (2017) Enabling 3D hepatocyte spheroids for microphysiometry. Conf Proc IEEE Eng Med Biol Soc 2017:1617–1620. doi:10.1109/EMBC.2017.8037148
Eklund SE, Taylor D, Kozlov E et al (2004) A microphysiometer for simultaneous measurement of changes in extracellular glucose, lactate, oxygen, and acidification rate. Anal Chem 76:519–527. doi:10.1021/ac034641z
Hartung T, Bruner L, Curren R et al (2010) First alternative method validated by a retrospective weight-of-evidence approach to replace the Draize eye test for the identification of non-irritant substances for a defined applicability domain. Altex 27:43–51. doi:10.14573/altex.2010.1.43
Huh D, Torisawa Y, Hamilton GA et al (2012) Microengineered physiological biomimicry: organs-on-chips. Lab Chip 12:2156–2164. doi:10.1039/c2lc40089h
Kang LF, Chung BG, Langer R, Khademhosseini A (2008) Microfluidics for drug discovery and development: from target selection to product lifecycle management. Drug Discov Today 13:1–13. doi:10.1016/j.drudis.2007.10.003
Kelm JM, Fussenegger M (2004) Microscale tissue engineering using gravity-enforced cell assembly. Trends Biotechnol 22:195–202. doi:10.1016/j.tibtech.2004.02.002
Kola I, Landis J (2004) Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov 3:711–716. doi:10.1038/nrd1470
Lee M-Y, Park CB, Dordick JS, Clark DS (2005) Metabolizing enzyme toxicology assay chip (MetaChip) for high-throughput microscale toxicity analyses. Proc Natl Acad Sci USA 102:983–987. doi:10.1073/pnas.0406755102
Marx U, Andersson TB, Bahinski A et al (2016) Biology-inspired microphysiological system approaches to solve the prediction dilemma of substance testing. Altex 33:272–321. doi:10.14573/altex.1603161
Matsusaki M, Case CP, Akashi M (2014) Three-dimensional cell culture technique and pathophysiology. Adv Drug Deliv Rev 74:95–103. doi:10.1016/j.addr.2014.01.003
McConnell HM, Owicki JC, Parce JW et al (1992) The cytosensor microphysiometer: biological applications of silicon technology. Science 257:1906–1912. doi:10.1126/science.1329199
Mehta G, Hsiao AY, Ingram M et al (2012) Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J Control Release 164:192–204. doi:10.1016/j.jconrel.2012.04.045
Mueller D, Krämer L, Hoffmann E et al (2014) 3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies. Toxicol Vitr 28:104–112. doi:10.1016/j.tiv.2013.06.024
Ramaiahgari SC, den Braver MW, Herpers B et al (2014) A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies. Arch Toxicol 88:1083–1095. doi:10.1007/s00204-014-1215-9
Rubin EH, Gilliland DG (2012) Drug development and clinical trials—the path to an approved cancer drug. Nat Rev Clin Oncol 9:215–222. doi:10.1038/nrclinonc.2012.22
Schmid YRF, Bürgel SC, Misun PM et al (2016) Electrical impedance spectroscopy for microtissue spheroid analysis in hanging-drop networks. ACS Sens 1:1028–1035. doi:10.1021/acssensors.6b00272
Soldatow VVY, Lecluyse ELEEL, Griffith LLG, Rusyn I (2013) In vitro models for liver toxicity testing. Toxicol Res (Camb) 2:23–39. doi:10.1039/C2TX20051A
Wang P, Xu G, Qin L et al (2005) Cell-based biosensors and its application in biomedicine. Sens Actuators B Chem 108:576–584. doi:10.1016/j.snb.2004.11.056
Waring MJ, Arrowsmith J, Leach AR et al (2015) An analysis of the attrition of drug candidates from four major pharmaceutical companies. Nat Rev Drug Discov 14:475–486. doi:10.1038/nrd4609
Weiss D, Brischwein M, Grothe H et al (2013) Label-free monitoring of whole cell vitality. Conf Proc IEEE Eng Med Biol Soc 2013:1607–1610. doi:10.1109/EMBC.2013.6609823
Weltin A, Slotwinski K, Kieninger J et al (2014) Cell culture monitoring for drug screening and cancer research: a transparent, microfluidic, multi-sensor microsystem. Lab Chip 14:138–146. doi:10.1039/c3lc50759a
Weltin A, Hammer S, Noor F et al (2017) Accessing 3D microtissue metabolism: lactate and oxygen monitoring in hepatocyte spheroids. Biosens Bioelectron 87:941–948. doi:10.1016/j.bios.2016.07.094
Wen Y, Yang S-T (2008) The future of microfluidic assays in drug development. Expert Opin Drug Discov 3:1237–1253. doi:10.1517/17460441.3.10.1237
Wiest J, Namias A, Pfister C et al (2016) Data processing in cellular microphysiometry. IEEE Trans Biomed Eng 63:2368–2375. doi:10.1109/TBME.2016.2533868
Wilkening S, Stahl F, Bader A (2003) Comparison of primary human hepatocytes and hepatoma cell line HepG2 with regard to their biotransformation properties. Drug Metab Dispos 31:1035–1042. doi:10.1124/dmd.31.8.1035
Acknowledgements
F. Alexander would like to thank the Whitaker International Program for their financial support of this work. The authors would also like to thank the Deutscher Tierschutzbund—Akademie für Tierschutz (German Animal Welfare Federation—Animal Welfare Academy).
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Alexander, F., Eggert, S. & Wiest, J. A novel lab-on-a-chip platform for spheroid metabolism monitoring. Cytotechnology 70, 375–386 (2018). https://doi.org/10.1007/s10616-017-0152-x
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DOI: https://doi.org/10.1007/s10616-017-0152-x