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Cartilage defects represent an increasing pathology among active individuals that affects the ability to contribute to sports and daily life. Cell therapy, such as autologous chondrocyte implantation (ACI), is a widespread option to treat larger cartilage defects still lacking standardization of in vitro cell culture parameters. We hypothesize that mRNA expression of cytokines and proteases before and after ACI is influenced by in vitro parameters: cell-passage, cell-density and membrane-holding time. Knee joint articular chondrocytes, harvested from rabbits (n = 60), were cultured/processed under varying conditions: after three different cell-passages (P1, P3, and P5), cells were seeded on 3D collagen matrices (approximately 25mm3) at three different densities (2 x 10 5/matrix, 1 x 10 6/matrix, and 3 x 10 6/matrix) combined with two different membrane-holding times (5 h and two weeks) prior autologous transplantation. Those combinations resulted in 18 different in vivo experimental groups. Two defects/knee/animal were created in the trochlear groove (defect dimension: radius 4 mm x 2 mm). Four identical cell-seeded matrices (CSM) were assembled and grouped in two pairs: One pair giving pre-operative in vitro data (CSM-i), the other pair was implanted in vivo and harvested 12 weeks post-implantation (CSM-e). CSMs were analyzed for TNF-alpha, IL-1beta, MMP-1, and MMP-3 via qPCR. CSM-i showed higher expression of IL-1beta, MMP-1, and MMP-3 compared to CSM-e. TNF-alpha expression was higher in CSM-e. Linearity between CSM-i and CSM-e values was found, except for TNF-alpha. IL-1beta expression was higher in CSM-i at higher passage and longer membrane-holding time. IL-1beta expression decreased with prolonged membrane-holding time in CSM-e. For TNF-alpha, the reverse was true. Lower cell-passages and lower membrane-holding time resulted in stronger TNF-alpha expression. Prolonged membrane-holding time resulted in increased MMP levels among CSM-i and CSM-e. Cellular densi