Research Article
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High throughput production and characterization of primary rat hepatocyte spheroids

Year 2024, Volume: 52 Issue: 3, 139 - 146, 01.07.2024
https://doi.org/10.15671/hjbc.1394326

Abstract

Developing in vitro models to explore cell biology and physiology holds significant importance in biotechnology, cancer research, drug discovery, toxicity testing, and the emerging fields of tissue engineering and regenerative medicine. The conventional two-dimensional (2D) approaches of mammalian cell culture (2D) have limitations in replicating all of the mechanical and biochemical signals in vivo. Culturing cells as spheroids provides a three-dimensional environment that more accurately mimics physiological conditions compared to 2D culture. In this study, we established a method for high-throughput spheroid formation using primary rat hepatocytes (PRHs). Using a high throughput platform, we investigated the effect of varying concentrations of cell culture media supplements on spheroid formation. Additionally, we assessed different cell seeding densities and characterized their properties for 7 days. The average diameter and circularity of PRH spheroids remained stable on days 2, 4, and 7, regardless of the initial seeding density. Moreover, PRH spheroids demonstrated high viability (> 90 %) for up to 7 days across all seeding cell densities. Our results demonstrated that this technique enables straightforward, large scale, consistent, and repeatable spheroid manufacturing and presents an alternative approach for future applications.

References

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  • L. De Moor, I. Merovci, S. Baetens, J. Verstraeten, P. Kowalska, D.V. Krysko, W.H. De Vos, H. Declercq, High-throughput fabrication of vascularized spheroids for bioprinting, Biofabrication, 10 (2018) 035009-035019.
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  • D. Jayme, T. Watanabe, T. Shimada, Basal medium development for serum-free culture: a historical perspective, Cytotechnology, 23 (1997) 95-101.
  • J. A. Kyffin, C.R. Cox, J. Leedale, H.E. Colley, C. Murdoch, P. Mistry, S.D. Webb, P. Sharma, Preparation of primary rat hepatocyte spheroids utilizing the liquid‐overlay technique, Curr. Opin. Toxicol., 81 (2019) 82-99.
  • D. Jayme, D. Epstein, D. Conrad, Fetal bovine serum alternatives, Nature, 334 (1988) 547-548.
  • J.A. Kyffin, P. Sharma, J. Leedale, H.E. Colley, C. Murdoch, A.L. Harding, P. Mistry, S.D. Webb, Characterization of a functional rat hepatocyte spheroid model, Toxicol. In Vitro, 55 (2019) 160-172.
  • D.F. Hendriks, L. Fredriksson Puigvert, S. Messner, W. Mortiz, M. Ingelman-Sundberg, Hepatic 3D spheroid models for the detection and study of compounds with cholestatic liability, Sci. Rep., 6 (2016) 35434-35434.
  • W.R. Proctor, A.J. Foster, J. Vogt, C. Summers, B. Middleton, M.A. Pilling, D. Shienson, M. Kijanska, S. Ströbel, J.M. Kelm, Utility of spherical human liver microtissues for prediction of clinical drug-induced liver injury, Arch. Toxicol., 91 (2017) 2849-2863.
  • S. Messner, I. Agarkova, W. Moritz, J. Kelm, Multi-cell type human liver microtissues for hepatotoxicity testing, Arch. Toxicol., 87 (2013) 209-213.
  • D. Kratschmar, S. Messner, W. Moritz, A. Odermatt, Characterization of a rat multi-cell type 3D-liver microtissue system, Tissue Sci. Eng., 4 (2013) 2-10.
Year 2024, Volume: 52 Issue: 3, 139 - 146, 01.07.2024
https://doi.org/10.15671/hjbc.1394326

Abstract

References

  • B.M. Leung, S.C. Lesher-Perez, T. Matsuoka, C. Moraes, S. Takayama, Media additives to promote spheroid circularity and compactness in hanging drop platform, Biomater. Sci., 3 (2015) 336-344.
  • M.C. Decarli, R. Amaral, D.P. Dos Santos, L.B. Tofani, E. Katayama, R.A. Rezende, J.V.L. da Silva, K. Swiech, C.A.T. Suazo, C. Mota, Cell spheroids as a versatile research platform: Formation mechanisms, high throughput production, characterization and applications, Biofabrication, 13 (2021) 032002-322039.
  • E. Fennema, N. Rivron, J. Rouwkema, C. van Blitterswijk, J. De Boer, Spheroid culture as a tool for creating 3D complex tissues, Trends Biotechnol, 31 (2013) 108-115.
  • G.L. Thomas, V. Mironov, A. Nagy-Mehez, J.C. Mombach, Dynamics of cell aggregates fusion: Experiments and simulations, Phys. A: Stat. Mech. Appl., 395 (2014) 247-254.
  • S. Raghavan, M.R. Ward, K.R. Rowley, R.M. Wold, S. Takayama, R.J. Buckanovich, G. Mehta, Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays, Gynecol. Oncol., 138 (2015) 181-189.
  • S. Breslin, L. O’Driscoll, Three-dimensional cell culture: the missing link in drug discovery, Drug Discov., Today 18 (2013) 240-249.
  • V.E. Santo, M.F. Estrada, S.P. Rebelo, S. Abreu, I. Silva, C. Pinto, S.C. Veloso, A.T. Serra, E. Boghaert, P.M. Alves, Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models, J. Biotechnol., 221 (2016) 118-129.
  • T. Boland, V. Mironov, A. Gutowska, E.A. Roth, R.R. Markwald, Cell and organ printing 2: Fusion of cell aggregates in three‐dimensional gels, Anat Rec A Discov Mol Cell Evol Biol., 272 (2003) 497-502.
  • S. Swaminathan, Q. Hamid, W. Sun, A.M. Clyne, Bioprinting of 3D breast epithelial spheroids for human cancer models, Biofabrication, 11 (2019) 025014.
  • G. Benton, I. Arnaoutova, J. George, H.K. Kleinman, J. Koblinski, Matrigel: from discovery and ECM mimicry to assays and models for cancer research, Adv. Drug Deliv., Rev. 79 (2014) 3-18.
  • H.K. Kleinman, G.R. Martin, Matrigel: basement membrane matrix with biological activity, Semin. Cancer Biol, 15 (2005) 378-386.
  • M.V. Wiles, G. Keller, Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture, Dev., 111 (1991) 259-267.
  • R.L. Amaral, M. Miranda, P.D. Marca to, K. Swiech, Comparative analysis of 3D bladder tumor spheroids obtained by forced floating and hanging drop methods for drug screening, Front. Physiol., 8 (2017) 605-621.
  • S. Bauer, C. Wennberg Huldt, K.P. Kanebratt, I. Durieux, D. Gunne, S. Andersson, L. Ewart, W.G. Haynes, I. Maschmeyer, A. Winter, Functional coupling of human pancreatic islets and liver spheroids on-a-chip: Towards a novel human ex vivo type 2 diabetes model, Sci. Rep., 7 (2017) 14620-14631.
  • M. Rogers, T. Sobolik, D.K. Schaffer, P.C. Samson, A.C. Johnson, P. Owens, S.G. Codreanu, S.D. Sherrod, J.A. McLean, J.P. Wikswo, Engineered microfluidic bioreactor for examining the three-dimensional breast tumor microenvironment, Biomicrofluidics, 12 (2018) 032102-034113.
  • K. Moshksayan, N. Kashaninejad, M.E. Warkiani, J.G. Lock, H. Moghadas, B. Firoozabadi, M.S. Saidi, N.-T. Nguyen, Spheroids-on-a-chip: Recent advances and design considerations in microfluidic platforms for spheroid formation and culture, Sens. Actuators B Chem., 263 (2018) 151-176.
  • A.N. Mehesz, J. Brown, Z. Hajdu, W. Beaver, J. Da Silva, R. Visconti, R. Markwald, V. Mironov, Scalable robotic biofabrication of tissue spheroids, Biofabrication, 3 (2011) 025002-0250011.
  • L. De Moor, I. Merovci, S. Baetens, J. Verstraeten, P. Kowalska, D.V. Krysko, W.H. De Vos, H. Declercq, High-throughput fabrication of vascularized spheroids for bioprinting, Biofabrication, 10 (2018) 035009-035019.
  • G. Gstraunthaler, T. Lindl, J. van der Valk, A plea to reduce or replace fetal bovine serum in cell culture media, Cytotechnology, 65 (2013) 791-793.
  • D. Jayme, T. Watanabe, T. Shimada, Basal medium development for serum-free culture: a historical perspective, Cytotechnology, 23 (1997) 95-101.
  • J. A. Kyffin, C.R. Cox, J. Leedale, H.E. Colley, C. Murdoch, P. Mistry, S.D. Webb, P. Sharma, Preparation of primary rat hepatocyte spheroids utilizing the liquid‐overlay technique, Curr. Opin. Toxicol., 81 (2019) 82-99.
  • D. Jayme, D. Epstein, D. Conrad, Fetal bovine serum alternatives, Nature, 334 (1988) 547-548.
  • J.A. Kyffin, P. Sharma, J. Leedale, H.E. Colley, C. Murdoch, A.L. Harding, P. Mistry, S.D. Webb, Characterization of a functional rat hepatocyte spheroid model, Toxicol. In Vitro, 55 (2019) 160-172.
  • D.F. Hendriks, L. Fredriksson Puigvert, S. Messner, W. Mortiz, M. Ingelman-Sundberg, Hepatic 3D spheroid models for the detection and study of compounds with cholestatic liability, Sci. Rep., 6 (2016) 35434-35434.
  • W.R. Proctor, A.J. Foster, J. Vogt, C. Summers, B. Middleton, M.A. Pilling, D. Shienson, M. Kijanska, S. Ströbel, J.M. Kelm, Utility of spherical human liver microtissues for prediction of clinical drug-induced liver injury, Arch. Toxicol., 91 (2017) 2849-2863.
  • S. Messner, I. Agarkova, W. Moritz, J. Kelm, Multi-cell type human liver microtissues for hepatotoxicity testing, Arch. Toxicol., 87 (2013) 209-213.
  • D. Kratschmar, S. Messner, W. Moritz, A. Odermatt, Characterization of a rat multi-cell type 3D-liver microtissue system, Tissue Sci. Eng., 4 (2013) 2-10.
There are 27 citations in total.

Details

Primary Language English
Subjects Cellular Interactions
Journal Section Articles
Authors

Ayşe Aslıhan Gökaltun 0000-0002-5880-0049

Publication Date July 1, 2024
Submission Date November 22, 2023
Acceptance Date December 13, 2023
Published in Issue Year 2024 Volume: 52 Issue: 3

Cite

APA Gökaltun, A. A. (2024). High throughput production and characterization of primary rat hepatocyte spheroids. Hacettepe Journal of Biology and Chemistry, 52(3), 139-146. https://doi.org/10.15671/hjbc.1394326
AMA Gökaltun AA. High throughput production and characterization of primary rat hepatocyte spheroids. HJBC. July 2024;52(3):139-146. doi:10.15671/hjbc.1394326
Chicago Gökaltun, Ayşe Aslıhan. “High Throughput Production and Characterization of Primary Rat Hepatocyte Spheroids”. Hacettepe Journal of Biology and Chemistry 52, no. 3 (July 2024): 139-46. https://doi.org/10.15671/hjbc.1394326.
EndNote Gökaltun AA (July 1, 2024) High throughput production and characterization of primary rat hepatocyte spheroids. Hacettepe Journal of Biology and Chemistry 52 3 139–146.
IEEE A. A. Gökaltun, “High throughput production and characterization of primary rat hepatocyte spheroids”, HJBC, vol. 52, no. 3, pp. 139–146, 2024, doi: 10.15671/hjbc.1394326.
ISNAD Gökaltun, Ayşe Aslıhan. “High Throughput Production and Characterization of Primary Rat Hepatocyte Spheroids”. Hacettepe Journal of Biology and Chemistry 52/3 (July 2024), 139-146. https://doi.org/10.15671/hjbc.1394326.
JAMA Gökaltun AA. High throughput production and characterization of primary rat hepatocyte spheroids. HJBC. 2024;52:139–146.
MLA Gökaltun, Ayşe Aslıhan. “High Throughput Production and Characterization of Primary Rat Hepatocyte Spheroids”. Hacettepe Journal of Biology and Chemistry, vol. 52, no. 3, 2024, pp. 139-46, doi:10.15671/hjbc.1394326.
Vancouver Gökaltun AA. High throughput production and characterization of primary rat hepatocyte spheroids. HJBC. 2024;52(3):139-46.

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