Ecotoxicology

, Volume 21, Issue 8, pp 2419–2429 | Cite as

Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes

  • Matthew G. Baron
  • Wendy M. Purcell
  • Simon K. Jackson
  • Stewart F. Owen
  • Awadhesh N. Jha
Technical Note

Abstract

The use of fish primary cells and cell lines offer an in vitro alternative for assessment of chemical toxicity and the evaluation of environmental samples in ecotoxicology. However, their uses are not without limitations such as short culture periods and loss of functionality, particularly with primary tissue. While three-dimensional (spheroid) technology is now established for in vitro mammalian toxicity studies, to date it has not been considered for environmental applications in a model aquatic species. In this study we report development of a reproducible six-well plate, gyratory-mediated method for rainbow trout (Oncorhynchus mykiss) hepatocyte spheroid culture and compare their functional and biochemical status with two-dimensional (2D) monolayer hepatocytes. Primary liver spheroid formation was divided into two stages, immature (1–5 days) and mature (≥6 days) according to size, shape and changes in functional and biochemical parameters (protein, glucose, albumin and lactate dehydrogenase). Mature spheroids retained the morphological characteristics (smooth outer surface, tight cell–cell contacts) previously described for mammalian spheroids as demonstrated by light and scanning electron microscopy. Glucose production and albumin synthesis were significantly higher in mature spheroids when compared to conventional 2D monolayer cultures (P < 0.01) and increased as spheroids matured (P < 0.01). Basal lactate dehydrogenase (LDH) leakage significantly decreased during spheroid formation and was significantly lower than 2D cultures (P < 0.01). It is therefore suggested that mature spheroids can maintain a high degree of functional, biochemical and morphological status over-time in culture that is superior to conventional 2D models and can provide realistic organotypic responses in vitro. Trout spheroids that take ~6–8 days to reach maturity would be suitable for use in acute toxicological tests and since it is possible to culture individual spheroids for over a month, there is potential for this work to lead towards in vitro bioaccumulation alternatives and to conduct high throughput screens of chronic exposure. This is an important step forward for developing alternative in vitro tools in future fish ecotoxicological studies.

Keywords

Spheroid Monolayer Rainbow trout Hepatocyte Functionality Morphology In vitro toxicology 

References

  1. Babich H, Borenfreund H (1986) In vitro cytotoxicity of metals to bluegill (BF-2) cells. Arch Environ Contam Toxicol 15:31–37CrossRefGoogle Scholar
  2. Babich H, Borenfreund H (1987) In vitro cytotoxicity of organic pollutants to bluegill sunfish (BF-2) cells. Environ Res 42:229–237CrossRefGoogle Scholar
  3. Bains OS, Kennedy CJ (2004) Energetic costs of pyrene metabolism in isolated hepatocytes of rainbow trout, Oncorhynchus mykiss. Aquat Toxicol 67:217–226CrossRefGoogle Scholar
  4. Bornschein J, Kidd M, Malfertheiner MV, Drozdov I, Pfragner R, Modlin IM (2008) Analysis of cytotoxic effects of chemotherapeutic agents on lung and small intestinal neuroendocrine cell lines. J Cancer Mol 4(2):47–54Google Scholar
  5. Castano A, Vega M, Blazquez T, Tarazona JV (1994) Biological alternatives to chemical identification for the ecotoxicological assessment of industrial effluents: the RTG-2 in vitro cytotoxicity test. Environ Toxicol Chem 13:1607–1611CrossRefGoogle Scholar
  6. Cravedi JP, Paris A, Monod G, Devaux A, Flouriot G, Valotaire Y (1996) Maintenance of cytochrome P450 content and phase I and phase II enzyme activities in trout hepatocytes cultured as spheroidal aggregates. Comp Biochem Physiol C 113(2):241–246Google Scholar
  7. Dowling K, Mothersill C (2001) The further development of rainbow trout primary epithelial cell cultures as a diagnostic tool in ecotoxicology risk assessment. Aquat Toxicol 53:279–290CrossRefGoogle Scholar
  8. Dunn JC, Yarmush ML, Koebe HG, Tompkins RG (1989) Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB J 3:174–177Google Scholar
  9. EMA (2006) European Medicine Agency. Committee for Medicinal Products for Human Use (CHMP): guideline on the environmental risk assessment of medicinal products for human use. http://www.emea.europa.eu/pdfs/human/swp/444700en.pdf Accessed 08 Feb 2012
  10. Flouriot G, Vaillant C, Salbert G, Pelissero C, Guiraud JM, Valotaire Y (1993) Monolayer and aggregate cultures of rainbow trout hepatocytes: long-term and stable liver-specific expression in aggregates. J Cell Sci 105(2):407–416Google Scholar
  11. Flouriot G, Monod G, Valotaire Y, Devaux A, Cravedi JP (1995) Xenobiotic metabolizing enzyme activities in aggregate culture of rainbow trout hepatocytes. Mar Environ Res 39(1–4):293–297CrossRefGoogle Scholar
  12. French CJ, Mommsen TP, Hochachka PW (1981) Amino acid utilisation in isolated hepatocytes from rainbow trout. Eur J Biochem 113:311–317CrossRefGoogle Scholar
  13. Garmanchuk LV, Perepelitsyna EM, Sydorenko MV, Ostapchenko LI (2010) Formation of multicellular aggregates under different conditions of microenvironment. Cytol Genet 44(1):19–22CrossRefGoogle Scholar
  14. Hamilton GA, Westmoreland C, George E (2001) Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers. In Vitro Cell Dev Biol 37:656–667CrossRefGoogle Scholar
  15. Hansen LK, Hisao CC, Friend JR, Wu FJ, Bridge GA, Remmel RP, Cerra FB, Hu WS (1998) Enhanced morphology and function in hepatocyte spheroids: a model of tissue self-assembly. Tissue Eng 41:65–74CrossRefGoogle Scholar
  16. Jha AN (2004) Genotoxicological studies in aquatic organisms: an overview. Mutat Res Fundam Mol Mech Mutagen 552:1–17CrossRefGoogle Scholar
  17. Juillerate M, Marceau N, Coeytaux S, Sierra F, Kolodziejczyk E, Guigoz Y (1997) Expression of organ-specific structures and functions in long-term cultures of aggregates from adult rat liver cells. Toxicol In Vitro 11:57–69CrossRefGoogle Scholar
  18. Katayama S, Tateno C, Asahara T, Katsutoshi Y (2001) Size-dependent in vivo growth potential of adult rat hepatocytes. Am J Pathol 158(1):97–105CrossRefGoogle Scholar
  19. Kessler MA, Wolfbeis OF (1992) Laser-induced fluorometric determination of albumin using longwave absorbing molecular probes. Anal Biochem 200(2):254–259CrossRefGoogle Scholar
  20. Kim JB (2005) Three-dimensional tissue culture models in cancer biology. Semin Cancer Biol 15(5):365–377CrossRefGoogle Scholar
  21. Klaunig J, Ruch R, Goldblatt P (1985) Trout hepatocyte culture: isolation and primary culture. In Vitro Cell Dev Biol Plant 21(4):221–228. doi:10.1007/bf02620933 CrossRefGoogle Scholar
  22. Kocal T, Quinn BA, Smith IR, Ferguson HW, Hayes MA (1988) Use of trout serum to prepare primary attached monolayer cultures of hepatocytes from rainbow trout (Salmo gairdneri). In Vitro Cell Dev Biol 24(4):304–308CrossRefGoogle Scholar
  23. Lee LE, Clemons JH, Bechtel DG, Caldwell SJ, Han KB, Pasitschniak-Arts M, Mosser DD, Bols NC (1993) Development and characterisation of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity. Cell Biol Toxicol 9:279–294CrossRefGoogle Scholar
  24. Lin KH, Maeda S, Saito T (1995) Long-term maintenance of liver-specific functions in three-dimensional culture of adult rat hepatocytes with a porous gelatin sponge support. Biotechnol Appl Biochem 21:19–27Google Scholar
  25. Lipsky MM, Sheridan TR, Bennett BO, May EB (1986) Comparison of trout hepatocyte culture on different substrates. In Vitro Cell Dev Biol 22(6):360–362CrossRefGoogle Scholar
  26. Liu J, Kuznetsova LA, Edwards GO, Xu J, Ma M, Purcell WM, Jackson SK, Coakley WT (2007) Functional three-dimensional HepG2 aggregate cultures generated from an ultrasound trap: comparison with HepG2 spheroids. J Cell Biochem 102:1180–1189CrossRefGoogle Scholar
  27. Ma M, Xu J, Purcell WM (2003) Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: I. morphological maturation and kinetic changes of energy metabolism, albumin synthesis, and activities of some enzymes. J Cell Biochem 90:1166–1175CrossRefGoogle Scholar
  28. Mazzoleni G, Di Lorenzo D, Steimberg N (2009) Modelling tissues in 3D: the next future of pharmaco-toxicology and food research? Genes Nutr 4(1):13–22CrossRefGoogle Scholar
  29. Mommsen TP, Vijayan MM, Moon TW (1999) Cortisol in teleosts: dynamics, mechanisms of action, and metabolic regulation. Rev Fish Biol Fish 9:211–268CrossRefGoogle Scholar
  30. Morata P, Vargas AM, Pita ML, Sanchez-Medina F (1982) Hormonal effects on the liver glucose metabolism in rainbow trout (Salmo gairdneri). Comp Biochem Physiol B Comp Biochem 72B:543–545Google Scholar
  31. Nabb DL, Mingoia RT, Yang CH, Han X (2006) Comparison of basal level metabolic enzyme activities of freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and rat. Aquat Toxicol 80:52–59CrossRefGoogle Scholar
  32. Niwa T, Koid N, Tsuji T, Imaoka S, Ishibashi F, Funae Y, Katagiri M (1996) Cytochrome P450s of isolated rat hepatocytes in spheroids and monolayer cultures. Res Commun Chem Pathol Pharmacol 91:372–378Google Scholar
  33. Pampaloni F, Reynaud EG, Stelzer EH (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8:839–845CrossRefGoogle Scholar
  34. Pannevis MC, Houlihan DF (1992) The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss). J Comp Physiol B 162(5):393–400CrossRefGoogle Scholar
  35. Papis E, Davies SJ, Jha AN (2011) Relative sensitivity of fish and mammalian cells to the antibiotic, trimethoprim: cytotoxic and genotoxic responses as determined by neutral red retention, comet and micronucleus assays. Ecotoxicology 20:208–217CrossRefGoogle Scholar
  36. Pesonen M, Andersson T (1992) Toxic effects of bleached and unbleached paper mill effluents in primary cultures of rainbow trout hepatocytes. Ecotoxicol Environ Saf 24:63–71CrossRefGoogle Scholar
  37. Pesonen M, Goksoyr A, Andersson T (1992) Expression of P4501A1 in a primary culture of rainbow trout hepatocytes exposed to β-napthoflavone or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Arch Biochem Biophys 292(1):228–233CrossRefGoogle Scholar
  38. PPPR (2009) Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009. Legislation concerning the protection of plants products that replaced directives 79/117/EEC and 91/414/EEC on the 14th June 2011. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:309:0001:0050:EN:PDF Accessed 08 Feb 2012
  39. Raisuddin S, Jha AN (2004) Relative sensitivity of fish and mammalian cells to sodium arsenate and arsenite as determined by alkaline single-cell gel electrophoresis and cytokinesis-block micronucleus assay. Environ Mol Mutagen 44:83–89CrossRefGoogle Scholar
  40. REACH (2009) Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006. Legislation concerning the registration, evaluation, authorisation and restriction of chemicals. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:309:0001:0050:EN:PDF Accessed 08 Feb 2012
  41. Reeves JF, Davie SJ, Dodd NJF, Jha AN (2008) Hydroxyl radicals (OH) are associated with titanium dioxide (TiO2) nanoparticel-induced cytotoxicity and oxidative DNA damage in fish cells. Mutat Res Fundam Mol Mech Mutagen 640:113–122CrossRefGoogle Scholar
  42. Scholtz S, Segner H (1999) Induction of CYP1A in primary cultures of rainbow trout (Oncorhynchus mykiss) liver cells: concentration-response relationships of four model substances. Ecotoxicol Environ Saf 43:252–260CrossRefGoogle Scholar
  43. Schrattenholz A, Klemm M (2006) How human embryonic stem cell research can impact in vitro drug screening technologies of the future. In: Marx U, Sandig V (eds) Drug Testing in vitro breakthroughs and trends in cell culture technology. Wiley-VCH, New York, pp 205–228CrossRefGoogle Scholar
  44. Segner H (1998) Isolation and primary culture of teleost hepatocytes. Comp Biochem Physiol A Mol Integr Physiol 120:71–81CrossRefGoogle Scholar
  45. Segner H, Cravedi JP (2001) Metabolic activity in primary cultures of fish hepatocytes. Altern Lab Anim 29:251–257Google Scholar
  46. Tong JZ, De Lagausie P, Furlan V, Crsteil T, Bernard O, Alvarez F (1992) Long-term culture of adult rat hepatocyte spheroids. Exp Cell Res 200:326–332CrossRefGoogle Scholar
  47. Vevers WF, Jha AN (2008) Genotoxic and cytotoxic potential of titanium dioxide (TiO2) nanoparticles on fish cells in vitro. Ecotoxicology 17:410–420CrossRefGoogle Scholar
  48. Walker TM, Rhodes PC, Westmoreland C (2000) The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures. Toxicol In Vitro 14:475–485CrossRefGoogle Scholar
  49. Weber JM, Shanghavi DS (2000) Regulation of glucose production in rainbow trout: role of epinephrine in vivo and in isolated hepatocytes. Am J Physiol Regul Integ Comp Physiol 278:956–963Google Scholar
  50. Xiao R, Yu HL, Zhao HF, Liang J, Feng JF, Wang W (2007) Developmental neurotoxicity role of cyclophosphamide on post-neural tube closure of rodents in vitro and in vivo. Int J Dev Neurosci 25:531–537CrossRefGoogle Scholar
  51. Xu J, Ma M, Purcell WM (2002) Optimising the enzymatic determination of galactose in the culture media of rat liver and HepG2 cell spheroids. Anal Biochem 311:179–181CrossRefGoogle Scholar
  52. Xu J, Ma M, Purcell WM (2003a) Characterisation of some cytotoxic endpoints using rat liver and HepG2 spheroids as in vitro models and their application in hepatotoxicity studies. I. Glucose metabolism and enzyme release as cytotoxic markers. Toxicol Appl Pharmacol 189(2):100–111CrossRefGoogle Scholar
  53. Xu J, Ma M, Purcell WM (2003b) Characterisation of some cytotoxic endpoints using rat liver and HepG2 spheroids as in vitro models and their application in hepatotoxicity studies. II. Spheroid cell spreading inhibition as a new cytotoxic marker. Toxicol Appl Pharmacol 189(2):112–119CrossRefGoogle Scholar
  54. Zubay G (1988) Biochemistry. Collier MacMillan Publishers, New YorkGoogle Scholar
  55. Zucco F, De Angelis I, Testai E, Stammati A (2004) Toxicology investigations with cell culture systems: 20 years after. Toxicol In Vitro 18(2):153–163CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Matthew G. Baron
    • 1
    • 2
  • Wendy M. Purcell
    • 1
  • Simon K. Jackson
    • 1
  • Stewart F. Owen
    • 2
  • Awadhesh N. Jha
    • 1
  1. 1.School of Biomedical and Biological SciencesPlymouth UniversityPlymouthUK
  2. 2.AstraZeneca Safety Health and EnvironmentBrixham Environmental LaboratoryBrixhamUK

Personalised recommendations