Osteoarthritis (OA) affects millions of aging adults. In healthy joints, articular cartilage provides a cushioned surface that allows bones to glide over each other. When OA develops, degradation of the cartilage increases, as does production of cytokines and matrix-degrading enzymes. Development of OA therapeutics has been difficult because of the complexity of the joint. We have developed a novel, serum-free, three-dimensional culture system for human osteoarthritic articular chondrocytes (HOACs) obtained from patients who have undergone total knee arthroplasty. In this study, we optimize our HOAC cultures by altering the culture size, tissue harvest sites, and the number of cultures pooled to measure clinically-relevant endpoints. We have combined cells isolated from both the sides of greatest and least pathology and have scaled down the cultures to 22mm plates (plating density 1.8x106 cells/0.5 ml) to increase the number of cultures obtained from each patient. HOAC media fractions were collected on day 2 and 5 and the alginate-associated matrix collected on day 5 of culture. Intact and degraded collagens were measured by immunoassay. Both collagens type I and type II, intact and degraded, were detectable within the scale-down culture system, and pooling three cultures together provided measurable material. In summary, our optimization techniques have provided more cultures per patient, thus enhancing the use of HOACs as a screening system for potential OA therapeutics.