Department of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, D‑69120 Heidelberg, Germany.
Bosch Health Campus, D‑70376 Stuttgart, Germany.
Int J Mol Med. 2023 Jul;52(1). doi: 10.3892/ijmm.2023.5261. Epub 2023 Jun 2.
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 IFNγTNF‑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.
胎牛血清（FBS）或人血清广泛用于嵌合抗原受体（CAR）T 细胞的生产。为了克服批次间的不一致性，使用不含动物成分的化学成分确定的培养基将是非常理想的。本研究比较了三种无血清培养基[Prime-XV™T 细胞 CDM、Fujifilm™（FF）、LymphoONE™T 细胞扩展无动物成分培养基、Takara Bio™（TB）和 TCM GMP-原型、CellGenix™（CG）]与含有 FBS（RCF）的标准 CAR T 细胞培养基。在 CD19.CAR T 细胞培养 12 天后，使用流式细胞术评估扩增、活力、转导效率和表型。通过细胞内染色、铬释放测定和长期共培养测定评估 CAR T 细胞的功能。与标准含 FBS 的培养基相比，无血清培养基中生成的 CAR T 细胞的扩增和活力没有差异。通过细胞内细胞因子染色检测，CG CAR T 细胞的 IFNγ和 IFNγTNF-αCAR T 细胞频率明显高于用 FBS 培养的 CAR T 细胞（22.5%比 7.6%，P=0.0194；15.3%比 6.2%，P=0.0399）。通过铬释放测定评估，无血清培养基中生成的 CAR T 细胞的细胞毒性高于用 FBS 培养的 CAR T 细胞[CG 比 RCF（P=0.0182）、FF 比 RCF（P=0.0482）和 TB 比 RCF（P=0.0482）]。CAR T 细胞生产第 12 天的表型分析未显示衰竭标志物、程序性细胞死亡蛋白 1、淋巴细胞激活基因 3 和 T 细胞免疫球蛋白和粘蛋白结构域 3 的表达有显著差异。与用 FBS 培养的 CAR T 细胞相比，FF 培养的 CAR T 细胞中中央记忆 CAR T 细胞的百分比更高（40.0%比 14.3%，P=0.0470），而 FF（6.2%比 24.2%，P=0.0029）和 CG（11.0%比 24.2%，P=0.0468）中的 CAR T 细胞幼稚 CAR T 细胞的频率较低。总的来说，本研究表明，一般来说，CAR T 细胞的功能和扩增在无血清培养基中得以维持。鉴于无牛材料和一致质量的优势，无血清培养基有望为 CAR T 细胞的 GMP 生产领域的未来发展做出贡献。
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