Short paper Applicability of human osteoarthritic chondrocytes for in vitro efficacy testing of anti-TNFa drugs Sara Zigon-Branc a , Matja z Jeras b, c , Andrej Blejec d , Ariana Barli c a, * a Educell Cell Therapy Service Ltd., Prevale 9, 1236 Trzin, Slovenia b Faculty of Pharmacy, University of Ljubljana, Askerceva cesta 7, 1000 Ljubljana, Slovenia c Celica Biomedical Ltd., Tehnoloski Park 24, 1000 Ljubljana, Slovenia d National Institute of Biology, Vecna pot 101, 1000 Ljubljana, Slovenia article info Article history: Received 30 June 2016 Received in revised form 16 September 2016 Accepted 29 September 2016 Available online 20 October 2016 Keywords: Human osteoarthritic chondrocytes (OACs) TNF-a Gene expression In vitro drug testing abstract In vitro cell-based models are important tools for assessing efficacies of new leads in early phases of drug development. Human osteoarthritic chondrocytes (OACs), obtained from biomedical waste material, represent a valuable, relatively accessible cellular source that could be used for this purpose. By employing reverse transcription-polymerase chain reaction (qRT-PCR) we compared gene expression profiles of key anabolic, catabolic and inflammatory genes of freshly isolated vs. monolayer cultured OACs (passages P0-P2) and non-stimulated vs. tumor necrosis factor alpha (TNF-a) stimulated P2 OACs. After expansion of OACs in monolayer cultures, the expression of almost all analyzed genes significantly decreased. The subsequent addition of TNF-a to OACs at P2 significantly increased expressions of all catabolic and inflammatory genes, leaving the anabolic profile almost unchanged. TNF-a-treated OACs were later utilized for efficacy testing of anti-TNF-a drugs infliximab and etanercept and both signifi- cantly reduced the expressions of all catabolic and inflammatory genes tested. © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. 1. Introduction In vitro cell-based assays are important tools for determining functional properties of new drugs, thereby diminishing needs for animal testing and reducing drug development costs. Primary OACs can be obtained from discarded material at numerous knee and hip replacement surgeries and represent a potential cell source for in vitro testing of new anti-inflammatory drugs [1e4]. Although primary chondrocytes undergo dedifferentiation in monolayer cultures, their expression of genes coding for essential chondro- genic factors remains unchanged [5]. The inflammatory state of cells can be monitored at the mRNA level by employing qRT-PCR. TNF-a, as a pivotal inflammatory cytokine in OA, can be applied in vitro to induce the inflammatory state of normal primary chon- drocytes (NCs) [6e8]. Employing NCs and TNF-a, we have recently published a new analytical approach for in vitro assessment of neutralization efficacies of anti-TNF-a biologicals [8]. In the present study, we investigated the inflammatory gene expression profiles of OACs, both, freshly isolated from biopsies and harvested from their subsequent monolayer cultures (P0-P2), as well as the applicability of TNF-a untreated or pre-treated P2 OACs for in vitro efficacy testing of selected anti-TNF-a drugs. 2. Materials and methods 2.1. Chondrocyte acquisition, cultivation and stimulation with TNF-a in the absence or presence of selected anti-TNF-a drugs With approval of the National Medical Ethics Committee (Code 40/10/11), human articular chondrocytes were isolated from lateral and medial condyles of OA patients (3 women and 1 man; mean age 68, range 57e77) undergoing total knee replacement surgery due to high degeneration scores of 3 and/or 4, according to the Inter- national Cartilage Repair Society criteria. Small pieces of cartilage biopsies were digested in Dulbecco's Modified Eagle Medium: F-12 Nutrient Mixture (DMEM/F-12), containing 1 mg/mL collagenase II, 50 mg/mL gentamicin and 2 mg/mL Fungizone ® , at 37 C for up to 24 h. One portion of isolated OACs was stored as a biopsy sample, while the other was washed with Phosphate Buffer Saline (PBS), resuspended in DMEM/F-12 supplemented with 10% fetal bovine * Corresponding author. E-mail addresses: sara.zigon@educell.si (S. Zigon-Branc), Matjaz.Jeras@ffa.uni-lj. si (M. Jeras), Andrej.Blejec@nib.si (A. Blejec), ariana.barlic@educell.si (A. Barlic). Contents lists available at ScienceDirect Biologicals journal homepage: www.elsevier.com/locate/biologicals http://dx.doi.org/10.1016/j.biologicals.2016.09.013 1045-1056/© 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. Biologicals 45 (2017) 96e101