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. 2023 Oct 24;13:18160. doi: 10.1038/s41598-023-45197-x

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Experimental workflow, expansion rate, viability, and phenotyping of primary T cells during an 8-day culture process comparing two different media protocols. (A) Lab-scale process of mRNA-based CAR-T cell generation using primary T cells spanning separation (day -X), activation (day 0), and expansion (day 1–8). (B,C) Comparison of T cell expansion and viability during the 8-day culture process using protocols A (blue) and B (green). N = 4 independent experiments were performed. Values are displayed as the mean ± SEM. (D) Microscopic analysis of T cell morphology on day 7 after activation. One representative image each is displayed showing larger, more heterogeneous cell shapes in protocol A (blue) and a homogeneous, round-shaped cell population by using protocol B (green). Scale bar shows 200 μm. (E,F) Flow cytometric analysis of T cells during the 8-day culture process using protocols A and B. The percentage of marker expression is plotted against time in days. Analysis of T cell subsets (E) and activation status (F) revealed differences for both protocols tested. N = 4 independent experiments were performed. Values are displayed as the mean ± SEM.