Chimeric antigen receptor (CAR-) T cells are designed to exploit the intrinsic cytotoxic function of T cells whilst manipulating specificity by expressing a nominal antigen-specific receptor containing a cytoplasmic activation domain. Such cells have recently revealed remarkable success in the clinic, with multiple studies reporting the ability to ameliorate CD19+ malignancies in particular. However, transitioning this technology to the treatment of solid tumours has posed many challenges, in particular the identity of appropriate target antigens and the need to penetrate the protective cancer stromal microenvironment. Accordingly, we developed second generation CAR-T constructs against an ovarian cancer antigen TAG-72 using lentiviral transduction of T cells from donated healthy blood. Resultant CAR+ve T cells were isolated by flow cytometry and maintained in basal media supplemented with IL-2, IL-7, IL-15 or IL-21 ± serum supplements for 24-48h. Cell activation status was determined by flow cytometry examining expression levels of CD137 and HLA-DR relative to baseline. Function was monitored in vitro using the real-time xCELLigence platform where both TAG-72hi and TAG-72low/neg target ovarian cancer cell lines were exposed to CAR-T for at least 20h. Changes in cell impedance were monitored throughout, where a reduction is indicative of target cell death. Parallel studies were performed with non-transduced T cells from the same donors. High levels of IL-2 resulted in T cell hyper-stimulation and potent, indiscriminate elimination of target cells induced by both CAR-T and non-transduced T cells. This was ameliorated in part by the reduction of IL-2 in the culture system. In contrast, addition of IL-7 alone maintained low levels of activation of CAR-T cells, as determined by comparable levels of HLA-DR/CD137 expression to baseline levels. This translated to complete elimination of TAG-72hi but not TAG-72low/neg cells in vitro, suggesting antigen-specific killing; non-transduced “resting” T cells showed no killing. Through sensitive, label-free, real-time cell monitoring we have been able to identify a collection of culture conditions which augment CAR-T function in vitro. These studies highlight the importance of the production process in the ability to achieve the fine balance between highly antigen-specific but potent cytotoxicity CAR-T cells for the eradication of cancer.