Most drugs are metabolized in the liver, which can lead to their activation or inactivation with a change in the parent compound pharmacology, as well as liver damage by active metabolites. Preclinical animal studies of drug safety do not always predict its effect on humans due to species specificity. Thus, for the rapid drug screening, and especially prodrugs, an in vitro system is required that allows predicting xenobiotic cytotoxicity with consideration of their metabolism in liver cells. The use of a microfluidic chip (BioClinicum) made it possible to cultivate a 2D culture of human HaCaT keratinocytes with spheroids of human hepatoma HepaRG cells. After incubation in a specially selected universal serum-free medium containing 3.8 mM cyclophosphamide, pronounced death of HaCaT cells was observed in comparison with culturing in the absence of liver cells.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Aleksandrova AV, Pulkova NP, Gerasimenko TN, Anisimov NY, Tonevitskaya SA, Sakharov DA. Mathematical and experimental model of oxygen diffusion for HepaRG cell spheroids. Bull. Exp. Biol. Med. 2016;160(6):857-860. doi: https://doi.org/10.1007/s10517-016-3326-1
Alexandrova AV, Pul’kova NV, Sakharov DA. Complex approach to xenobiotics hepatotoxicity testing using a microfluidic system. Bull. Exp. Biol. Med. 2016;161(1):50- 53. doi: https://doi.org/10.1007/s10517-016-3342-1
Bao J, Fisher JE, Lillegard JB, Wang W, Amiot B, Yu Y, Dietz AB, Nahmias Y, Nyberg SL. Serum-free medium and mesenchymal stromal cells enhance functionality and stabilize integrity of rat hepatocyte spheroids. Cell Transplant. 2013;22(2):299-308. doi: https://doi.org/10.3727/096368912X656054
de Jonge ME, Huitema AD, Rodenhuis S, Beijnen JH. Clinical pharmacokinetics of cyclophosphamide. Clin. Pharmacokinet. 2005;44(11):1135-1164. doi: https://doi.org/10.2165/00003088-200544110-00003
Deyrieux AF., Wilson VG. In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line. Cytotechnology. 2007;54(2):77-83. doi: https://doi.org/10.1007/s10616-007-9076-1
Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I, Guyomard C, Lucas J, Trepo C, Guguen-Guillouzo C. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl Acad. Sci. USA. 2002;99(24):15655-15660. doi: https://doi.org/10.1073/pnas.232137699
Guichard A, Humbert P, Tissot M, Muret P, Courderot- Masuyer C, Viennet C. Effects of topical corticosteroids on cell proliferation, cell cycle progression and apoptosis: in vitro comparison on HaCaT. Int. J. Pharm. 2015;479(2):422-429. doi: https://doi.org/10.1016/j.ijpharm.2014.12.066
Hintze JM, Tchoukalova YD, Sista R, Shah MK, Zhang N, Lott DG. Development of xeno-free epithelial differentiation media for adherent, non-expanded adipose stromal vascular cell cultures. Biochem. Biophys. Res. Commun. 2018;503(4):3128-3133. doi: https://doi.org/10.1016/j.bbrc.2018.08.104
ICCVAM test method evaluation report. In vitro cytotoxicity test methods for estimating starting doses for acute oral systemic toxicity testing. NIH Publication No. 07-4519, November 2006.
Joy MS, La M, Wang J, Bridges AS, Hu Y, Hogan SL, Frye RF, Blaisdell J, Goldstein JA, Dooley MA, Brouwer KL, Falk RJ. Cyclophosphamide and 4-hydroxycyclophosphamide pharmacokinetics in patients with glomerulonephritis secondary to lupus and small vessel vasculitis. Br. J. Clin. Pharmacol. 2012;74(3):445-455. doi: https://doi.org/10.1111/j.1365-2125.2012.04223.x
Kane BJ, Zinner MJ, Yarmush ML, Toner M. Liver-specific functional studies in a microfluidic array of primary mammalian hepatocytes. Anal. Chem. 2006;78(13):4291- 4298. doi: https://doi.org/10.1021/ac051856v
Kao JS, Fluhr JW, Man MQ, Fowler AJ, Hachem JP, Crumrine D, Ahn SK, Brown BE, Elias PM, Feingold KR. Short-term glucocorticoid treatment compromises both permeability barrier homeostasis and stratum corneum integrity: inhibition of epidermal lipid synthesis accounts for functional abnormalities. J. Invest. Dermatol. 2003;120(3):456-464. doi: https://doi.org/10.1046/j.1523-1747.2003.12053.x
Li AP, Uzgare A, LaForge YS. Definition of metabolismdependent xenobiotic toxicity with co-cultures of human hepatocytes and mouse 3T3 fibroblasts in the novel integrated discrete multiple organ co-culture (IdMOC) experimental system: results with model toxicants aflatoxin B1, cyclophosphamide and tamoxifen. Chem. Biol. Interact. 2012;199(1):1-8. doi: https://doi.org/10.1016/j.cbi.2012.05.003
Ma LD, Wang YT, Wang JR, Wu JL, Meng XS, Hu P, Mu X, Liang QL, Luo GA. Design and fabrication of a liver-on-a-chip platform for convenient, highly efficient, and safe in situ perfusion culture of 3D hepatic spheroids. Lab Chip. 2018;18(17):2547-2562. doi: https://doi.org/10.1039/c8lc00333e
Mandon M, Huet S, Dubreil E, Fessard V, Le Hégarat L. Three-dimensional HepaRG spheroids as a liver model to study human genotoxicity in vitro with the single cell gel electrophoresis assay. Sci. Rep. 2019;9(1):10548. doi: https://doi.org/10.1038/s41598-019-47114-7
Nelson LJ, Morgan K, Treskes P, Samuel K, Henderson CJ, LeBled C, Homer N, Grant MH, Hayes PC, Plevris JN. Human hepatic HepaRG cells maintain an organotypic phenotype with high intrinsic CYP450 activity/metabolism and significantly outperform standard HepG2/C3A cells for pharmaceutical and therapeutic applications. Basic Clin. Pharmacol. Toxicol. 2017;120(1):30-37. doi: https://doi.org/10.1111/bcpt.12631
Richardson J, Shah B, Bondarenko PV, Bhebe P, Zhang Z, Nicklaus M, Kombe MC. Metabolomics analysis of soy hydrolysates for the identification of productivity markers of mammalian cells for manufacturing therapeutic proteins. Biotechnol. Prog. 2015;31(2):522-531. doi: https://doi.org/10.1002/btpr.2050
Runge D, Runge DM, Jäger D, Lubecki KA, Beer Stolz D, Karathanasis S, Kietzmann T, Strom SC, Jungermann K, Fleig WE, Michalopoulos GK. Serum-free, long-term cultures of human hepatocytes: maintenance of cell morphology, transcription factors, and liver-specific functions. Biochem. Biophys. Res. Commun. 2000;269(1):46-53. doi: https://doi.org/10.1006/bbrc.2000.2215
Samatov TR, Shkurnikov MU, Tonevitskaya SA, Tonevitsky AG. Modelling the metastatic cascade by in vitro microfluidic platforms. Prog. Histochem. Cytochem. 2015;49(4):21-29. doi: https://doi.org/10.1016/j.proghi.2015.01.001
Semenova OV, Petrov VA, Gerasimenko TN, Aleksandrova AV, Burmistrova OA, Khutornenko AA, Osipyants AI, Poloznikov AA, Sakharov DA. Effect of circulation parameters on functional status of HepaRG spheroids cultured in microbioreactor. Bull. Exp. Biol. Med. 2016;161(3):425- 429. doi: https://doi.org/10.1007/s10517-016-3430-2
Tjin MS, Chua AWC, Tryggvason K. Chemically defined and xenogeneic-free culture method for human epidermal keratinocytes on laminin-based matrices. Nat. Protoc. 2020;15(2):694-711. doi: https://doi.org/10.1038/s41596-019-0270-3
van Tonder A, Joubert AM, Cromarty AD. Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC Res. Notes. 2015;8:47. doi: https://doi.org/10.1186/s13104-015-1000-8
Author information
Authors and Affiliations
Corresponding author
Additional information
Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 39-44, March, 2022
Rights and permissions
About this article
Cite this article
Pulkova, N.V., Zyrina, A.N., Mnafki, N.A. et al. Microfluidic Chip as a Tool for Effective In Vitro Evaluation of Cyclophosphamide Prodrug Toxicity. Bull Exp Biol Med 173, 146–150 (2022). https://doi.org/10.1007/s10517-022-05510-6
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10517-022-05510-6