Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지
DOI QR코드

DOI QR Code

Evaluation of primary hepatocyte function using 2D or 3D culture method for primary rat hepatocytes

Rat Primary Hepatocyte의 2차원 배양과 3차원 배양에 따른 생리 활성능과 대사능에 관한 연구

  • Lim, Malgum (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Kim, Yeongji (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Shin, Yurianna (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Oh, Keon Bong (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Hwang, Seongsoo (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Kim, Youngim (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Hur, Tai-Young (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Ock, Sun A (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration)
  • 임맑음 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 김영지 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 신유리안나 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 오건봉 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 황성수 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 김영임 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 허태영 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 옥선아 (농촌진흥청 국립축산과학원 동물바이오공학과)
  • Received : 2016.08.10
  • Accepted : 2016.09.07
  • Published : 2016.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

There is a growing interest in the application of primary hepatocytes for treatment of liver diseases in humans and for drug development. Several studies have focused on long-term survival and di-differentiation blocking of primary hepatocytes in an in vitro culture system. Therefore, the present study also aimed to optimize an in vitro culture system using primary rat hepatocytes. Primary rat hepatocytes from 6-week-old male Crl:CD rats were isolated using a modified two-step collagenase perfusion. Healthy $3.5{\times}10^6$ primary rat hepatocytes were seeded into a 2 dimensional (2D) culture in a 25T culture flask coated with collagen type I or into a 3D culture in a 125-ml spinner flask for 7 days. Production of plasma protein (ALB and TF), apoptosis (BAX and BCL2), and CYP (CYP3A1) related genes were compared between the 2D and 3D culture systems. The 3D culture system had an advantage over the 2D system because of the relatively high expression of ALB and low expression of BAX in the 3D system. However, the level of CYP3A1 did not improve in the 3D culture with and without the presence of a dexamethasone inducer. Therefore, 3D culture has an advantage for albumin production and primary rat hepatocyte survivability, but a low expression of CYP3A1 indicated that primary rat hepatocytes require a high-density culture for stress reduction by continuous flow.

Keywords

References

  1. Allen JW, Hassanein T and Bhatia SN. 2001. Adavances in bioartificial liver devices. Hepatology 34:447-455.
  2. Berthiaume F, Moghe PV, Toner M and Yarmush ML. 1996. Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration. FASEB J. 10:1471-1484. https://doi.org/10.1096/fasebj.10.13.8940293
  3. Chenxia H and Lanjuan L. 2015. In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration. Protein cell. 6:562-574. https://doi.org/10.1007/s13238-015-0180-2
  4. Dunn JC, Yaramush ML, Koebe HG and Tompkins RG. 1989. Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB J. 3:174-177. https://doi.org/10.1096/fasebj.3.2.2914628
  5. Giada Sebastiani and Ann P Walker. 2007. HFE gene in primary and secondary hepatic iron overload. World J Gastroenterol. 13:4673-4689. https://doi.org/10.3748/wjg.v13.i35.4673
  6. Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Bottger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CE, Gomez-Lechon MJ, Groothuis GM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Haussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhutter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, LeCluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EH, Stieger B, Stober R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM and Hengstler JG. 2013. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 87:1315-1530. https://doi.org/10.1007/s00204-013-1078-5
  7. Gomez-Lechon MJ, Lahoz A, Gombau L, Castell JV and Donato MT. 2010. Evaluation of Potential Hepatotoxicity Induced by Drugs. Curr. Pharm. Des. 16:1-15. https://doi.org/10.2174/138161210789941829
  8. Hu X, Xie P, Li W, Li Z and Shan H. 2016. Direct induction of hepatocyte-like cells from immortalized human bone marrow mesenchymal stem cells by overexpression of HNF4${\alpha}$. Biochem Biophys Res Commun. 478:791-797. https://doi.org/10.1016/j.bbrc.2016.08.026
  9. Kegel V, Deharde D, Pfeiffer E, Zeilinger K, Seehofer D and Damm G. 2016. Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells. J. Vis. Exp. 30:e53069.
  10. Knobeloch D, Ehnert S, Schyschka L, Buchler P, Schoenberg M, Kleeff J, Thasler WE, Nussler NC, Godoy P, Hengstler J and Nussler AK. 2012. Human hepatocytes: isolation, culture, and quality procedures. Methods Mol. Biol. 806:99-120.
  11. Koide N, Skaguchi K, Koide Y, Asano K, Kawaguchi M, Matsushima H. Takenami, T, Shinji, Mori M and Tsuji T. 1990. Formation of Multicellular Spheroids Composed of Adult Rat Hepatocytes in Dishes with Positively Charged Surfaces and under Other Nonadherent Environmants. Exp. Cell. Res 1860:227-235.
  12. Landry J, Bernier D, Ouellet C, Goyette R and Marceau N. 1985. Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities. J. Cell Biol. 1101:914-923.
  13. Lecluyse EL and Alexandre E. 2010. Isolation and culture of primary hepatocytes from resected human liver tissue. Methods Mol. Biol. 640:57-82.
  14. Lee DH, Yoon HH and Park JK. 2014. Hepatocyte culture technology and its application to bioartificial liver. 42:129-138.
  15. Lu C and Li AP. 2001. Species comparison in P450 induction: effects of dexamethasone, omeprazole, and rifampin on P450 isoforms 1A and 3A in primary cultured hepatocytes from man, Sprague-Dawley rat, minipig, and beagle dog. Chem Biol Interact. 134:271-281. https://doi.org/10.1016/S0009-2797(01)00162-4
  16. Maruyama M, Fujisawa M, Yokosuka M, Saito TR, Hayama S, Akimoto T and Hakamata Y. 2016. A new in vivo analysis model to detect sexually dimorphic rat liver cytochrome P450 gene expression dependent on growth hormone secretory patterns. Exp Anim. [Epub ahead of print]
  17. No da Y, Lee SA, Choi YY, Park D, Jang JY, Kim DS and Lee SH. 2012. Functional 3D human primary hepatocyte spheroids made by co-culturing hepatocytes from partial hepatectomy specimens and human adipose-derived stem cells. Plos one, 7:e50723. https://doi.org/10.1371/journal.pone.0050723
  18. Nyberg SL, Hibbs JR, Hardin JA, Germer JJ, Platt JL, Paya CV and Wiesner RH. 2000. Influence of human fulminant hepatic failure sera on endogenous retroviral expression in pig hepatocytes. Liver Transplant. 6:76-84.
  19. Rajan N, Habermehl J, Cote MF, Doillon CJ and Mantovani D. 2006. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc. 1:2753-2758.
  20. Schyschka L, Sanchez JJ, Wang Z, Burkhardt B, Muller-Vieira U, Zeilinger K, Bachmann A, Nadalin S, Damm G and Nussler AK. 2013. Hepatic 3D cultures but not 2D cultures preserve specific transporter activity for acetaminophen-induced hepatotoxicity. Arch Toxicol. 87:1581-1593. https://doi.org/10.1007/s00204-013-1080-y
  21. Selgan PO. 1976. Preparation of isolated rat liver cells. Methods Cell Biol. 13:29-83.
  22. Shulman M and Nahmias Y. 2013. Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes. Methods Mol. Biol. 915:287-302.
  23. Soldatow VY, Griffith LG, Rusyn I and LeCluyse EL. 2013. In vitro models for liver toxicity testing. Toxicol. Res. 2:23-39. https://doi.org/10.1039/C2TX20051A
  24. Wojcikowski J, Haduch A and Daniel WA. 2012. Effect of classic and atypical neuroleptics on cytochrome P450 3A (CYP3A1) in rat liver. Pharmacol Rep. 64:1411-1418. https://doi.org/10.1016/S1734-1140(12)70938-6
  25. Wu FJ, Friend JR, Hsiao CC, Zilliox MJ, Ko WJ, Cerra FB and Hu WS. 1996. Efficient assembly of rat hepatocyte spheroids for tissue engineering applications. Biotechnol Bioeng. 50:404-415. https://doi.org/10.1002/(SICI)1097-0290(19960520)50:4<404::AID-BIT7>3.0.CO;2-P
  26. Xiao-Lei Shi, Yimeng Gao, Yupeng Yan, Hucheng Ma, Lulu Sun, Pengyu Huang, Xuan Ni, Ludi Zhang, Xin Zhao, Haozhen Ren, Dan Hu, Yan Zhou, Feng Tian, Yuan Ji, Xin Cheng, Guoyu Pan, Yi-Tao Ding and Lijian Hui. 2016. Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes. Cell Research. 26:206-216. https://doi.org/10.1038/cr.2016.6
  27. Yang SM, Lee DH and Park JK. 2000. Effects of Degree of Cell-Cell Contact on Liver Specific Functions of Rat Primary Hepatocytes. Biotechnol. Bioprocess Eng. 5:99-105. https://doi.org/10.1007/BF02931879
  28. Zuber R, Anzenbacherova E and Anzenbacher P. 2002. Cytochromes P450 and experimental models of drug metabolism. J Cell Mol Med. 6:189-198. https://doi.org/10.1111/j.1582-4934.2002.tb00186.x