Mutation Research 758 (2013) 69–79 Contents lists available at ScienceDirect Mutation Research/Genetic Toxicology and Environmental Mutagenesis jou rn al h om ep age: www.elsevier.com/locate/gentox C om mu n i ty add ress: www.elsevier.com/locate/mutres Optimization of upcyte ® human hepatocytes for the in vitro micronucleus assay Astrid Nörenberg a , Stefan Heinz a , Katharina Scheller a,1 , Nicola J. Hewitt a , Joris Braspenning a,∗ , Michael Ott b a Medicyte GmbH, Heidelberg, Germany b MHH, Medizinische Hochschule Hannover, Germany a r t i c l e i n f o Article history: Received 8 April 2013 Received in revised form 5 September 2013 Accepted 28 September 2013 Available online 16 October 2013 Keywords: Upcyte ® hepatocyte Genotoxicity Micronucleus MN assay a b s t r a c t “Upcyte ® human hepatocytes” have the unique property of combining proliferation with the expression of drug metabolising activities. In our current study, we evaluated whether these cells would be suitable for early in vitro micronucleus (MN) tests. A treatment period of 96 h without a recovery period was most reliable for detecting MN formation in upcyte ® hepatocytes from Donor 740. The basal MN rate in upcyte ® hepatocytes varied considerably between donors (7–28%); therefore, modifications to the assay medium were tested to determine whether they could decrease inherent MN formation. Optimal medium supplements were 10 ng/ml oncostatin M for the pre-culture and recovery periods and 25 ng/ml epider- mal growth factor and 10 ng/ml oncostatin M for the treatment period. Using the optimised conditions and outcome criteria, the upcyte ® hepatocyte MN assay could correctly identify directly acting (e.g. mit- omycin C, etoposide) and metabolically activated genotoxins (e.g. benzo[a]pyrene, cyclophosphamide). “True negative” and “false positive” compounds were also correctly identified as negative. The basal %MN in upcyte ® hepatocytes from Donor 740 treated with DMSO, cyclophosphamide or MMC, was essentially unaffected by the growth stage ranging from population doublings of 14–61, suggesting that billions of cells could be produced from a single donor for standardised drug toxicity testing. In conclusion, we have established and optimised an in vitro MN test by using upcyte ® hepatocytes to correctly identify known direct and metabolically activated genotoxicants as well as “false positives” and true negative compounds. The almost unlimited supply of cells from a single donor and optimised test conditions increase reproducibility in early and more predictive in vitro MN tests. © 2013 Elsevier B.V. All rights reserved. 1. Introduction The in vitro micronucleus (MN) assay is used routinely as part of the assessment of compound safety. This assay detects clasto- gens (compounds which directly react with DNA) and aneugens (compounds which interact with the components of the mitotic and meiotic cell division cycle). This assay has a good sensitivity (∼80%); however, it also has a high “false positive” (FP) rate (i.e. a low specificity) of only ∼30% [1]. This problem is especially impor- tant for the cosmetics industry, since the 7th amendment to the Cosmetics Directive [2] bans the use animals for follow-up studies ∗ Corresponding author at: Medicyte GmbH, 69120 Heidelberg, Germany. Tel.: +49 6221 72925 30; fax: +49 6221 72925 31. E-mail addresses: a.noerenberg@medicyte.com (A. Nörenberg), s.heinz@medicyte.com (S. Heinz), k.scheller@medicyte.com (K. Scheller), n.hewitt@medicyte.com (N.J. Hewitt), j.braspenning@medicyte.com (J. Braspenning). 1 Current address: TRM, Translationszentrum fur Regenerative Medizin, Leipzig, Germany. when the current in vitro test battery gives a positive outcome. In the chemicals industry, the re-testing of chemicals (as per the EU chemicals legislation [3]) would result in vast numbers of animals being used to follow-up on positive results from in vitro assays. Therefore, a number of projects and workshops were initiated in order to reduce FP rates in in vitro assays [4–7]. The main factors thought to affect the high rate of FPs include the selection of the cell type [4] and measurement of cytotoxicity [5]. To address the selection of an appropriate cell type, we have investigated whether “upcyte ® hepatocytes” could be used in the MN assay as part of early genotoxicity studies, which may be car- ried out using flow cytometry to speed up the scoring of MN. These cells have the unique feature of exhibiting metabolic activities and proliferative capacity, both of which are required for this assay. The upcyte ® technology generates non-transformed proliferating liver cells from primary human hepatocytes [8]. The new upregu- lated cells (called “upcytes”) start to grow after transduction of the primary cells with a defined cocktail of lentiviral vectors. Human upcyte ® hepatocytes proliferate and still express differentiated marker proteins (-1-antitrypsin, cytokeratin-8 and -18, human 1383-5718/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2013.09.008