Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta). New in vitro testing systems are needed to evaluate efficacies of new anti-inflammatory biological drugs, ideally in a patient-specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti-inflammatory drugs, spheroids were exposed to TNF-alpha, IL-1 beta, or to supernatant containing secretome from activated macrophages (MCM). The anti-inflammatory efficacies of anti-TNF-alpha biologicals adalimumab, infliximab, and etanercept, and the anti-IL-1 beta agent anakinra were assessed in short-term microspheroid and long-term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF-alpha or IL-1 beta. The differences in potency of anti-TNF-alpha biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short-term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti-TNF-alpha drugs in personalized and preclinical studies. (C) 2018 American Institute of Chemical Engineers