Abstract
White-spotted charr (Salvelinus leucomaenis, S. I.) is an anadromous cold water-adapted fish, distributed in the Far East. We have previously reported the complete mitochondrial DNA sequences of white-spotted chars (S. l. imbrius and S. l. pluvius) in Japan. In general, fish hepatocytes are useful for cellular and biochemical studies of fish. In this study, we isolated hepatocytes from the liver of white-spotted charr and used basic methods, such as enzyme digestion and low centrifugation, to analyze the molecular mechanisms involved in specific cellular responses. The isolated hepatocytes could be cultured at 5–20 °C but not 37 °C. The morphology of hepatocytes was altered in a temperature-dependent manner. The properties of hepatocyte were similar to those of living fish. Moreover, the proliferation rate and damage of isolated hepatocytes depended on the concentration of fetal bovine serum in the culture medium. Taken together, this study demonstrates that this simple method for isolation and culture of hepatocytes from white-spotted charr may be useful for other biochemical and cellular studies.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
References
Arai Y, Yokoyama C, Nagase K, Suwa M, Ogawa Y, Iuchi K, Hisatomi H (2019) Complete mitochondrial DNA sequences of two endemic subspecies, Salvelinus leucomaenis imbrius and Salvelinus leucomaenis pluvius (Salmonid, White spotted charr) in Japan. Mitochondrial DNA Part B 4:1524–1525. https://doi.org/10.1080/23802359.2019.1601517
Birnbaum MJ, Schultz J, Fain JN (1976) Hormone-stimulated glycogenolysis in isolated goldfish hepatocytes. Am J Physiol 231:191–197. https://doi.org/10.1152/ajplegacy.1976.231.1.191
Bols N, Dayeh V, Lee L, Schirmer K (2005) Use of fish cell lines in the toxicology and ecotoxicology of fish. Piscine cell lines in environmental toxicology, vol 6. Biochemistry and molecular biology of fishes. Elsevier, New York, pp 43–84. https://doi.org/10.1016/s1873-0140(05)80005-0
Bowen WC, Michalopoulos AW, Orr A, Ding MQ, Stolz DB, Michalopoulos GK (2014) Development of a chemically defined medium and discovery of new mitogenic growth factors for mouse hepatocytes: mitogenic effects of FGF1/2 and PDGF. PLoS ONE 9:e95487. https://doi.org/10.1371/journal.pone.0095487
Braunbeck T, Segner H (2000) Isolation and cultivation of teleost hepatocytes. Springer, Berlin, pp 49–71. https://doi.org/10.1007/978-94-017-3345-8_6
Broutier L et al (2017) Human primary liver cancer-derived organoid cultures for disease modeling and drug screening. Nat Med 23:1424–1435. https://doi.org/10.1038/nm.4438
Busby ER, Cooper GA, Mommsen TP (2002) Novel role for prostaglandin E2 in fish hepatocytes: regulation of glucose metabolism. J Endocrinol 174:137–146. https://doi.org/10.1677/joe.0.1740137
Egami N (1955) Effect of estrogen and androgen on the weight and structure of the liver of the fish, Oryzias latipes. Annot Zool Jpn 28:79–85
Hayashi S (2002) Regulation of synthesis and secretion of the lipoprotein by cultured eel hepatocytes. Fish Sci 68:1213–1216. https://doi.org/10.2331/fishsci.68.sup2_1213
Hayashi S, Kumagai A (2008) Studies on eel liver functions using perfused liver and primary cultured hepatocytes. Aqua-BioSci Monogr. https://doi.org/10.5047/absm.2008.00102.0001
Hewitt NJ et al (2007) Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev 39:159–234. https://doi.org/10.1080/03602530601093489
Hosoya K (2013) Salmonidae. Fishes of Japan with pictorial keys to the species, 3rd edn. Tokai University Press, Tokai
Ishii K (1971) Morphological changes in the liver of the Kokanee, Oncorhynchus nerka, accompanied with sexual maturation. Bull Fac Fish Hokkaido Univ 22:215–220
Iuchi K, Oya K, Hosoya K, Sasaki K, Sakurada Y, Nakano T, Hisatomi H (2020) Different morphologies of human embryonic kidney 293T cells in various types of culture dishes. Cytotechnology 72:131–140. https://doi.org/10.1007/s10616-019-00363-w
Kobayashi H (1953) Effect of estrone upon the structure, weight and fat content of the liver in the fishi, Misgurnus anguillicaudatus. Annot Zool Jpn 26:213–216
LeCluyse E, Bullock P, Madan A, Carroll K, Parkinson A (1999) Influence of extracellular matrix overlay and medium formulation on the induction of cytochrome P-450 2B enzymes in primary cultures of rat hepatocytes. Drug Metab Dispos 27:909–915
Lee LE et al (1993) Development and characterization of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity. Cell Biol Toxicol 9:279–294. https://doi.org/10.1007/bf00755606
Nakabo T (2000) Fishes of Japan with pictorial keys to the species, Japanese edition I. Tokai Univ. Press, Tokyo
Neerland ED, Bytingsvik J, Nikiforov VA, Evenset A, Krokje A (2019) DNA Double-Strand Breaks in Arctic Char (Salvelinus alpinus) from Bjornoya in the Norwegian Arctic. Environ Toxicol Chem 38:2405–2413. https://doi.org/10.1002/etc.4546
Nomura M (1963) Studies on reproduction of rainbow trout, Salmo gairdneri, with special reference to egg taking. V. Development of gonads and size of fish spawned firstly. Nippon Suisan Gakkaishi 29:976–984
Oguro C (1956) Some observations on the effect of estrogen upon the liver of the three-spined stickleback, Gasterosteus aculeatus aculeatus L. Environ Health Perspect 29:19–22
Ostrander GK et al (1995) Long-term primary culture of epithelial cells from rainbow trout Oncorhynchus mykiss) liver. In Vitro Cell Dev Biol Anim 31:367–378. https://doi.org/10.1007/bf02634286
Pesonen M, Teivainen P, Lundstrom J, Jakobsson E, Norrgren L (2000) Biochemical responses of fish sac fry and a primary cell culture of fish hepatocytes exposed to polychlorinated naphthalenes. Arch Environ Contam Toxicol 38:52–58. https://doi.org/10.1007/s002449910007
Segner H (1998) Isolation and primary culture of teleost hepatocytes. Compar Biochem Physiol Part A 120:71–81. https://doi.org/10.1016/s1095-6433(98)10012-0
Takami T, Kitano F, Nakano S (1997) High Water Temperature Influences on Foraging Responses and Thermal Deaths of Dolly Varden < i>Salvelinus malma </i > and White-spotted Charr < i>S. leucomaenis </i > in a Laboratory Fisheries science 63:6–8 https://doi.org/10.2331/fishsci.63.6
Takegawa Y, Kawaguchi Y, Mitsuhashi H, Taniguchi Y (2017) Implementing species distribution models to predict the impact of global warming on current and future potential habitats of white-spotted char Salvelinus leucomaenis for effective conservation planning in Japan. Jpn J Conserv Ecol 22:121–134
Yanhong F, Chenghua H, Guofang L, Haibin Z (2008) Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes. Cytotechnology 58:85–92. https://doi.org/10.1007/s10616-008-9169-5
Acknowledgements
We thank Mr. Yuji Tasaki, Ms. Rika Imaizumi and Mr. Tetsushi Seki for experimental support. We thank Prof. Naomi Kamimura for scientific advice.
Author information
Authors and Affiliations
Contributions
KI, YA, NS, CY, TS and HH conceived and designed the experiments. KI, YA, and KS performed the experiments and contributed the data analysis. KI, YA and TS wrote the manuscript. All authors contributed critically to drafts and gave final approval for submitting.
Corresponding author
Ethics declarations
Competing financial interests
The all authors declare no conflicts of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Iuchi, K., Arai, Y., Sasaki, K. et al. A simple method for isolation and culture of primary hepatocytes from Salvelinus leucomaenis (White-spotted Charr). Cytotechnology 72, 731–739 (2020). https://doi.org/10.1007/s10616-020-00415-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-020-00415-6