Simple Paper-based Liver Cell Model for Drug Screening

Abstract

Investigation of the potential adverse effects of chemicals and drugs is essential during the drug development process. In vitro cell model systems have been developed over the past years towards such toxicity investigation. 96-well plate is the common platform for screening drug toxicity due to its simplicity. However, this platform only offers 2D cell culture environment and lacks the flow of solutions, which fails to provide the suitable environment for the cells to adequately metabolize the drugs, for the media to replenish, and for the metabolites and wastes to be removed. Microfluidic chips populated with human or animal cells, known as organ-on-a-chip (OOC), can reconcile many issues of in vitro cell models, such as the lack of extracellular matrix and flow as well as the species difference. However, OOC can be complicated to fabricate and operate. To bridge this gap, we utilized paper as a primary substrate for OOC, considering its fibrous structure that can mimic natural extracellular matrix, as well as a syringe pump and filter that are commonly available in most laboratories. Paper microfluidic model was designed and fabricated by wax printing on nitrocellulose paper, seeded and proliferated with liver cells (primary rat hepatocytes and HepG2 cells), and two paper substrates were stacked together to complete the paper model. To this paper-based liver cell model, the following drugs were added: Phenacetin (pain reliever and fever reducer), Bupropion (antidepressant), Dextromethorphan (antidepressant), and phosphate-buffered saline (PBS) as a control, all under a physiologically relevant flow rate. The combination of these drugs with Fluconazole (antifungal drug) was also investigated. Cell count, cell morphology, protein production, and urea secretion after drug treatment confirmed that the model successfully predicted toxicity within 40 minutes. This simple, paper-based liver cell model provided enhanced and faster cell response to drug toxicity and showed comparable or better behavior than the cells cultured in conventional 2D in vitro models.

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References

  1. 1.

    Zhang, J., Zhao, X., Liang, L., Li, J., Demirci, U. & Wang, SQ. A decade of progress in liver regenerative medicine. Biomaterials157, 161–176 (2018).

    CAS  Article  Google Scholar 

  2. 2.

    LeCluyse, E.L., Bullock, P.L. & Parkinson, A. Strategies for restoration and maintenance of normal hepatic structure and function in long-term cultures of rat hepatocytes. Adv. Drug Delivery Rev.22, 133–186 (1996).

    CAS  Article  Google Scholar 

  3. 3.

    DelRaso, N.J. In vitro methodologies for enhanced toxicity testing. Toxicol. Lett.68, 91–99 (1993).

    CAS  Article  Google Scholar 

  4. 4.

    Soldatow, V.Y., LeCluyse, E.L., Griffith, L.G. & Rusyn, I. In vitro models for liver toxicity testing. Toxicol. Res. (Cambridge, U. K.)2, 23–39 (2013).

    CAS  Google Scholar 

  5. 5.

    Kang, I.-K., Kim, G.J., Kwon, O.H. & Ito, Y. Coculture of hepatocytes and fibroblasts by micropatterned immobilization of β-galactose derivatives. Biomaterials25, 4225–4232 (2004).

    CAS  Article  Google Scholar 

  6. 6.

    Renault, C., Li, X., Fosdick, S.E. & Crooks, R.M. Hollow-channel paper analytical devices. Anal. Chem.85, 7976–7979 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    Phenacetin. Available at: https://www.ncbi.nlm.nih.gov/books/NBK304337/?report=classic. (Accessed: 21st March 2020).

  8. 8.

    Bupropion. Available at: https://livertox.nlm.nih.gov/Bupropion.htm. (Accessed: 21st March 2020)

  9. 9.

    Dextromethorphan: MedlinePlus Drug Information. Available at: https://medlineplus.gov/druginfo/meds/a682492.html. (Accessed: 21st March 2020)

  10. 10.

    Dextromethorphan: Side Effects, Dosages, Treatment, Interactions, Warnings. Available at: https://www.rxlist.com/consumer_dextromethorphan/drugs-condition.htm. (Accessed: 21st March 2020)

  11. 11.

    Anderson, I.B. Chapter 60. Dextromethorphan. in Poisoning & Drug Overdose (ed. Olson, K.R.) (McGraw-Hill: New York, 2012).

    Google Scholar 

  12. 12.

    Fluconazole. Available at: https://livertox.nlm.nih.gov/Fluconazole.htm. (Accessed: 21st March 2020)

  13. 13.

    Jacobson, M.A., Hanks, D.K. & Ferrell, L.D. Fatal acute hepatic necrosis due to fluconazole. Am. J. Med.96, 188–190 (1994).

    CAS  Article  Google Scholar 

  14. 14.

    Puhl, G., Schaser, K.D., Vollmar, B., Menger, M.D. & Settmacher, U. Noninvasive in vivo analysis of the human hepatic microcirculation using orthogonal polarization spectral imaging. Transplantation75, 756–761 (2003).

    Article  Google Scholar 

  15. 15.

    Chumo, B., Muluneh, M. & Issadore, D. Laser micromachined hybrid open/paper microfluidic chips. Biomicrofluidics7, 64109 (2013).

    CAS  Article  Google Scholar 

  16. 16.

    Prodanov, L., Jindal, R., Bale, S.S., Hegde, M., McCarty, W.J., Golberg, I., Bhushan, A., Yarmush, M.L. & Usta, O.B. Long-term maintenance of a microfluidic 3D human liver sinusoid: maintenance of a microfluidic 3D human liver sinusoid. Biotechnol. Bioeng.113, 241–246 (2016).

    CAS  Article  Google Scholar 

  17. 17.

    Wang, Y., Wang, H., Deng, P., Chen, W., Guo, Y., Tao, T. & Qin, J. In situ differentiation and generation of functional liver organoids from human iPSCs in a 3D perfusable chip system. Lab Chip18, 3606–3616 (2018).

    CAS  Article  Google Scholar 

  18. 18.

    Du, C., Narayanan, K., Leong, M.F. & Wan, A.C.A. Induced pluripotent stem cell-derived hepatocytes and endothelial cells in multi-component hydrogel fibers for liver tissue engineering. Biomaterials35, 6006–6014 (2014).

    CAS  Article  Google Scholar 

  19. 19.

    Dash, A., Simmers, M.B., Deering, T.G., Berry, D.J., Feaver, R.E., Hastings, N.E., Pruett, T.L., LeCluyse, E.L., Blackman, B.R. & Wamhoff, B.R. Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro. Am. J. Physiol.: Cell Physiol.304, C1053–C1063 (2013).

    CAS  Article  Google Scholar 

  20. 20.

    Fluconazole (Oral Route) Proper Use - Mayo Clinic. Available at: https://www.mayoclinic.org/drugs-supplements/fluconazole-oral-route/proper-use/drg-20071428. (Accessed: 21st March 2020)

  21. 21.

    Fluconazole Dosage Guide with Precautions. Available at: https://www.drugs.com/dosage/fluconazole.html. (Accessed: 21st March 2020)

  22. 22.

    Shulman, M. & Nahmias, Y. Long-term culture and coculture of primary rat and human hepatocytes. Methods Mol. Biol.945, 287–302 (2013).

    Article  Google Scholar 

  23. 23.

    Bokhari, M., Carnachan, R.J., Cameron, N.R. & Przyborski, S.A. Culture of HepG2 liver cells on three-dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge. J. Anat.211, 567–576 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Donato, M.T., Tolosa, L. & Gómez-Lechón, M.J. Culture and functional characterization of human hepatoma HepG2 cells. in Protocols in In Vitro Hepatocyte Research (eds. Vinken, M. & Rogiers, V.) 77–93 (Springer New York, 2015).

    Google Scholar 

  25. 25.

    Bhise, N.S., Manoharan, V., Massa, S., Tamayol, A., Ghaderi, M., Miscuglio, M., Lang, Q., Zhang, Y.S., Shin, S.R., Calzone, G., Annabi, N., Shupe, T.D., Bishop, C.E., Atala, A., Dokmeci, M.R. & Khademhosseini, A. A liver-on-a-chip platform with bioprinted hepatic spheroids. Biofabrication8, 014101 (2016).

    Article  Google Scholar 

  26. 26.

    Sison-Young, R.L., Lauschke, V.M., Johann, E., Alexandre, E., Antherieu, S., Aerts, H., Gerets, H.H.J., Labbe, G., Hoët, D., Dorau, M., Schofield, C.A., Lovatt, C.A., Holder, J.C., Stahl, S.H., Richert, L., Kitteringham, N.R., Jones, R.P., Elmasry, M., Weaver, R.J., Hewitt, P.G., Ingelman-Sundberg, M., Goldring, C.E. & Park, B.K. A multicenter assessment of single-cell models aligned to standard measures of cell health for prediction of acute hepatotoxicity. Arch. Toxicol.91, 1385–1400 (2017).

    CAS  Article  Google Scholar 

  27. 27.

    Wang, Y., Kim, M.H., Shirahama, H., Lee, J.H., Ng, S.S., Glenn, J.S. & Cho, N.-J. ECM proteins in a microporous scaffold influence hepatocyte morphology, function, and gene expression. Sci. Rep.6, 37427 (2016).

    CAS  Article  Google Scholar 

  28. 28.

    Hung, P.J., Lee, P.J., Sabounchi, P., Lin, R. & Lee, L.P. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays. Biotechnol. Bioeng.89, 1–8 (2005).

    CAS  Article  Google Scholar 

  29. 29.

    Trefts, E., Gannon, M. & Wasserman, D.H. The liver. Curr. Biol.27, R1147–R1151 (2017).

    CAS  Article  Google Scholar 

  30. 30.

    Pyzik, M., Rath, T., Kuo, T.T., Win, S., Baker, K., Hubbard, J.J., Grenha, R., Gandhi, A., Krämer, T.D., Mezo, A.R., Taylor, Z.S., McDonnell, K., Nienaber, V., Andersen, J.T., Mizoguchi, A., Blumberg, L., Purohit, S., Jones, S.D., Christianson, G., Lencer, W.I., Sandlie, I., Kaplowitz, N., Roopenian, D.C. & Blumberg, R.S. Hepatic FcRn regulates albumin homeostasis and susceptibility to liver injury. Proc. Natl. Acad. Sci. U. S. A.114, E2862–E2871 (2017).

    CAS  Article  Google Scholar 

  31. 31.

    Teo, Y.L., Ho, H.K. & Chan, A. Metabolism-related pharmacokinetic drug-drug interactions with tyrosine kinase inhibitors: current understanding, challenges and recommendations. Br. J. Clin. Pharmacol.79, 241–253 (2015).

    CAS  Article  Google Scholar 

  32. 32.

    Phenacetin. Available at: https://www.drugbank.ca/drugs/DB03783. (Accessed: 21st March 2020)

  33. 33.

    Fluconazole. Available at: https://www.drugbank.ca/drugs/DB00196. (Accessed: 21st March 2020)

  34. 34.

    Beckwitt, C.H., Clark, A.M., Wheeler, S., Taylor, D.L., Stolz, D.B., Griffith, L. & Wells, A. Liver ‘organ on a chip’. Exp. Cell Res.363, 15–25 (2018).

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by the pilot interdisciplinary grant from the BIO5 Institute at the University of Arizona and cardiovascular biomedical engineering training grant from U.S. National Institutes of Health, grant number T32HL00 7955. K.K. acknowledges the scholarship from the Development and Promotion of Science and Technology Talents Project (DPST) of Thailand. P.A. acknowledges the scholarship from One District One Scholarship (ODOS) of Thailand.

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Correspondence to Jeong-Yeol Yoon.

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Kaarj, K., Ngo, J., Loera, C. et al. Simple Paper-based Liver Cell Model for Drug Screening. BioChip J 14, 218–229 (2020). https://doi.org/10.1007/s13206-020-4211-6

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Keywords

  • Drug toxicity
  • Paper microfluidics
  • Organ-on-a-chip
  • Rat hepatocyte
  • HepG2