1 2 Merging bioreactor technology with 3D hepatocyte-fibroblast culturing approaches: 3 Improved in vitro models for toxicological applications 4 Sofia B. Leite a,b , Ana P. Teixeira a,b , Joana P. Miranda a,1 , Rui M. Tostões a,b , João J. Clemente a , 5 Marcos F. Sousa a , Manuel J.T. Carrondo a,b , Paula M. Alves a,b,⇑ 6 a Instituto de Biologia Experimental e Tecnológica (IBET), Apartado 12, 2781-901 Oeiras, Portugal 7 b Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL), Apartado 127, 2781-901 Oeiras, Portugal 8 10 article info 11 Article history: 12 Received 22 March 2010 13 Accepted 3 February 2011 14 Available online xxxx 15 Keywords: 16 In vitro model 17 3D co-culture 18 Bioreactor 19 Hepatocytes 20 Fibroblasts 21 Long-term toxicology studies 22 CYP induction 23 Oxygen concentration 24 25 abstract 26 During the last years an increasing number of in vitro models have been developed for drug screening and 27 toxicity testing. Primary cultures of hepatocytes are, by far, the model of choice for those high-through- 28 put studies but their spontaneous dedifferentiation after some time in culture hinders long-term studies. 29 Thus, novel cell culture systems allowing extended hepatocyte maintenance and more predictive long 30 term in vitro studies are required. 31 It has been shown that hepatocytes functionality can be improved and extended in time when cultured 32 as 3D-cell aggregates in environmental controlled stirred bioreactors. In this work, aiming at further 33 improving hepatocytes functionality in such 3D cellular structures, co-cultures with fibroblasts were per- 34 formed. An inoculum concentration of 1.2 Â 10 5 cell/mL and a 1:2 hepatocyte:mouse embryonic fibro- 35 blast ratio allowed to improve significantly the albumin secretion rate and ECOD (phase I) and UGT 36 (phase II) enzymatic activities in 3D co-cultures, as compared to the routinely used 2D hepatocyte mono- 37 cultures. Significant improvements were also observed in relation to 3D monocultures of hepatocytes. 38 Furthermore, hepatocytes were able to respond to the addition of beta-Naphtoflavone by increasing 39 ECOD activity showing CYP1A inducibility. The dependence of CYP activity on oxygen concentration 40 was also observed. In summary, the improved hepatocyte specific functions during long term incubation 41 of 3D co-cultures of hepatocytes with fibroblasts indicate that this system is a promising in vitro model 42 for long term toxicological studies. 43 Ó 2011 Published by Elsevier Ltd. 44 45 46 47 1. Introduction 48 Primary cultures of freshly isolated hepatocytes are currently 49 the in vitro model of choice for drug screening applications. When 50 cultured under well-defined and stringent conditions, these pri- 51 mary cells perform alike common hepatocyte cell lines (e.g. 52 HepG2) in terms of ability to induce P450 (CYP) activity upon addi- 53 tion of specific compounds; however, they have improved synthe- 54 sis and secretion rates of both albumin and urea as well as 55 improved capacity for phase I metabolism (Donato et al., 1994; 56 Viollon-Abadie et al., 2000; Gebhardt et al., 2003). 57 Hepatocyte cultures of human origin can be obtained from liver 58 biopsies. However, these cells are rather difficult to obtain and of- 59 ten their profile is highly dependent on the donor sources present- 60 ing a high batch-to-batch variability. Primary cultures of rat 61 hepatocytes are a good alternative often used by the pharmaceuti- 62 cal industry because: (i) they have higher metabolic responses 63 than common human cell lines and (ii) the inter-donor variability 64 can be minimized by selecting animals of the same sex, age and 65 similar feeding regimes (Coecke et al., 2000; O’Brien et al., 2004). 66 Moreover, the strategies developed to improve hepatocytes func- 67 tionalities can often be applied easily to primary human 68 hepatocytes. 69 Conventional 2D in vitro models of hepatocytes present the ma- 70 jor drawback of quick loss of expression of specific liver-functions 71 (O’Brien et al., 2004). Strategies to decrease or, ideally, overcome, 72 in vitro dedifferentiation of hepatocytes constitute a major goal in 73 drug development. Several reports on the effect of: (i) extracellular 0887-2333/$ - see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.tiv.2011.02.002 Abbreviations: 3D, three dimensional; 2D, two dimensional; ECM, extracellular matrix; HGF, hepatocyte growth factor; CYP, cytochrome P450; mef, mouse embryonic fibroblasts; hep, hepatocytes; pO 2 , partial pressure of oxygen; BNF, beta-Naphtoflavone; EGTA, ethylene glycol tetracetic acid; PBS, phosphate buffered saline; FBS, foetal bovine serum; MEM, minimum essential medium eagle’s; hff, human foreskin fibroblasts; ECOD, 7-ethoxycoumarin-o-deethylase; UGT, uridine diphosphate glucuronoltransferase; Lac/Glc, lactate formation to glucose consump- tion ratio. ⇑ Corresponding author at: ITQB-UNL/IBET, Apartado 12, 2781-901 Oeiras, Portugal. Tel.: +351 214469425. E-mail address: marques@itqb.unl.pt (P.M. Alves). 1 Present address: iMED, Av Prof Gama Pinto, 1649-003 Lisboa, Portugal. Toxicology in Vitro xxx (2011) xxx–xxx Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit TIV 2562 No. of Pages 8, Model 5G 15 February 2011 Please cite this article in press as: Leite, S.B., et al. Merging bioreactor technology with 3D hepatocyte-fibroblast culturing approaches: Improved in vitro models for toxicological applications. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.02.002