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Ensuring experimental reproducibility and standardization of hepatocyte spheroid culture by evidence-based microplate choice

Establishing the appropriate culturing conditions for each cell type is vital to allow for experimental reproducibility across laboratories worldwide. Next to controlling key features of nutrient and growth factor supply as well as temperature and atmospheric conditions, choice of culturing ware, more specifically culturing surface, is decisive for successful cell growth.

In 2D cell culture, adhesion to the culturing surface can be enabled by well-established introduction of hydrophilic groups to the culturing plastic (learn more about our 2D cell culture options on or even by coating the culturing surface with extracellular matrix proteins. 
To prevent cell adhesion to the culturing surface thus promoting formation of three-dimensional multicellular structures, e.g. spheroids, pre-treated/pre-coated commercially available culturing ware currently presents as the option promising the most consistent and reproducible experimental results. 
By developing meaningful models for early prediction of hepatotoxicity and pharmacokinetic properties using canine or human primary hepatocytes, Prof. Dr. Lauschke's research group therefore aims to contribute to method standardization while diminishing need for animal testing. [1]

Liver spheroids have been established to maintain physiologically relevant phenotypes and functions upon long-term culture qualifying them for both high-throughput pharmacological and toxicological applications but also chronic disease modelling demanding repeated drug exposure. [2-9]

Commercially available pre-coated/pre-treated 3D cell culture plates present a rapid and easy method for spheroid formation. However, available plates differ in both plate geometry and coating - factors influencing cell viability, molecular phenotypes but also compound availability due to the coating's compound binding capacity. [10,11]

BIOFLOAT™ cell culture plates showed excellent performance for the crucial quality feature of single spheroid formation per well with single primary human hepatocyte (PHH) spheroids in ≥ 95 % of wells. PHH spheroids cultured with BIOFLOAT™ presented with ≥ 90 cell viability while preserving expression of key hepatic genes related to drug metabolism, ethanol catabolism, bile acid synthesis, and differentiation.

Upon treatment of PHH spheroids with prototypic hepatotoxic compounds, PHH spheroids BIOFLOAT™ clearly reacted at lowest compound concentrations showing compound availability was not reduced by the plate's coating.

To reduce the need for animal testing within preclinical drug testing, primary canine hepatocyte (PCH) spheroids can be used for previously established assays. For generation of physiologically relevant and functional PCH spheroids for acute and chronic toxicity tests, microplate choice is key with BIOFLOAT™ enabling successful PCH spheroid culture and thus, possibly, prospectively allowing extension of the preclinical method repertoire.

Taken together, these results emphasize the suitability of BIOFLOAT™ for hepatocyte culture in preclinical pharmacological and toxicological compound testing in comparison to various commercially available options. 



Interview with Prof Lauschke on the use of the BIOFLOAT™ plate



[1] Xing C, Kemas A, Mickols E, Klein K, Artursson P, Lauschke VM. The choice of ultra-low attachment plates impacts primary human and primary canine hepatocyte spheroid formation, phenotypes, and function. Biotechnol J. 2024 Feb;19(2):e2300587. doi: 10.1002/biot.202300587. PMID: 38403411.

[2] Bell CC, Lauschke VM, Vorrink SU, Palmgren H, Duffin R, Andersson TB, Ingelman-Sundberg M. Transcriptional, Functional, and Mechanistic Comparisons of Stem Cell-Derived Hepatocytes, HepaRG Cells, and Three-Dimensional Human Hepatocyte Spheroids as Predictive In Vitro Systems for Drug-Induced Liver Injury. Drug Metab Dispos. 2017 Apr;45(4):419-429. doi: 10.1124/dmd.116.074369. Epub 2017 Jan 30. PMID: 28137721; PMCID: PMC5363699.

[3] Vorrink SU, Ullah S, Schmidt S, Nandania J, Velagapudi V, Beck O, Ingelman-Sundberg M, Lauschke VM. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics. FASEB J. 2017 Jun;31(6):2696-2708. doi: 10.1096/fj.201601375R. Epub 2017 Mar 6. PMID: 28264975; PMCID: PMC5434660.

[4] Messner S, Fredriksson L, Lauschke VM, Roessger K, Escher C, Bober M, Kelm JM, Ingelman-Sundberg M, Moritz W. Transcriptomic, Proteomic, and Functional Long-Term Characterization of Multicellular Three-Dimensional Human Liver Microtissues. Appl In Vitro Toxicol. 2018 Mar 1;4(1):1-12. doi: 10.1089/aivt.2017.0022. PMID: 32953943; PMCID: PMC7500040.

[5] Vorrink SU, Zhou Y, Ingelman-Sundberg M, Lauschke VM. Prediction of Drug-Induced Hepatotoxicity Using Long-Term Stable Primary Hepatic 3D Spheroid Cultures in Chemically Defined Conditions. Toxicol Sci. 2018 Jun 1;163(2):655-665. doi: 10.1093/toxsci/kfy058. PMID: 29590495; PMCID: PMC5974779.

[6] Proctor WR, Foster AJ, Vogt J, Summers C, Middleton B, Pilling MA, Shienson D, Kijanska M, Ströbel S, Kelm JM, Morgan P, Messner S, Williams D. Utility of spherical human liver microtissues for prediction of clinical drug-induced liver injury. Arch Toxicol. 2017 Aug;91(8):2849-2863. doi: 10.1007/s00204-017-2002-1. Epub 2017 Jun 13. PMID: 28612260; PMCID: PMC5515971.

[7] Riede J, Wollmann BM, Molden E, Ingelman-Sundberg M. Primary human hepatocyte spheroids as an in vitro tool for investigating drug compounds with low clearance. Drug Metab Dispos. 2021 Jun 1:DMD-AR-2020-000340. doi: 10.1124/dmd.120.000340. Epub ahead of print. PMID: 34074732.

[8] Kanebratt KP, Janefeldt A, Vilén L, Vildhede A, Samuelsson K, Milton L, Björkbom A, Persson M, Leandersson C, Andersson TB, Hilgendorf C. Primary Human Hepatocyte Spheroid Model as a 3D In Vitro Platform for Metabolism Studies. J Pharm Sci. 2021 Jan;110(1):422-431. doi: 10.1016/j.xphs.2020.10.043. Epub 2020 Oct 26. PMID: 33122050.

[9] Preiss LC, Lauschke VM, Georgi K, Petersson C. Multi-Well Array Culture of Primary Human Hepatocyte Spheroids for Clearance Extrapolation of Slowly Metabolized Compounds. AAPS J. 2022 Mar 11;24(2):41. doi: 10.1208/s12248-022-00689-y. PMID: 35277751.

[10] Costa EC, de Melo-Diogo D, Moreira AF, Carvalho MP, Correia IJ. Spheroids Formation on Non-Adhesive Surfaces by Liquid Overlay Technique: Considerations and Practical Approaches. Biotechnol J. 2018 Jan;13(1). doi: 10.1002/biot.201700417. Epub 2017 Nov 15. PMID: 29058365.

[11] Palmgrén JJ, Mönkkönen J, Korjamo T, Hassinen A, Auriola S. Drug adsorption to plastic containers and retention of drugs in cultured cells under in vitro conditions. Eur J Pharm Biopharm. 2006 Nov;64(3):369-78. doi: 10.1016/j.ejpb.2006.06.005. Epub 2006 Jun 29. PMID: 16905298.