Impact of serum-free media on the expansion and functionality of CD19.CAR T-cells

Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) T-cells. In order to overcome a lot-to-lot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serum-free media [Prime-XV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ T-Cell Expansion Xeno-Free Medium, Takara Bio™ (TB) and TCM GMP-Prototype, CellGenix™ (CG)] to the standard CAR T-cell medium containing FBS (RCF). After 12 days of CD19.CAR T-cell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR T-cells was evaluated using intracellular staining, a chromium release assay and a long-term co-culture assay. Expansion and viability did not differ between the CAR T-cells generated in serum-free media compared to the standard FBS-containing medium. The CG CAR T-cells had a statistically significant higher frequency of IFNγ(+) and IFNγ(+)TNF-α(+) CAR T-cells than the CAR T-cells cultured with FBS (22.5 vs. 7.6%, P=0.0194; 15.3 vs. 6.2%, P=0.0399, respectively) as detected by intracellular cytokine staining. The CAR T-cells generated with serum-free media exhibited a higher cytotoxicity than the CAR T-cells cultured with FBS in the evaluation by chromium release assay [CG vs. RCF (P=0.0182), FF vs. RCF (P=0.0482) and TB vs. RCF (P=0.0482)]. Phenotyping on day 12 of CAR T-cell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyte-activation gene 3 and T-cell immunoglobulin and mucin-domain containing-3. The CAR T-cells cultured in FF had a higher percentage of central memory CAR T-cells (40.0 vs. 14.3%, P=0.0470) than the CAR T-cells cultured with FBS, whereas the CAR T-cells in FF (6.2 vs. 24.2%, P=0.0029) and CG (11.0% vs. 24.2%, P=0.0468) had a lower frequency of naïve CAR T-cells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serum-free media. Given the advantages of freedom from bovine material and consistent quality, serum-free media hold promise for the future development of the field of GMP manufacturing of CAR T-cells.