HUMAN PRIMARY RESTING T-CELL MANUFACTURING VIA MICROFLUIDIC TRANSFECTION PLATFORM

• Published in: Cytotherapy (Oxford, England) Vol. 26; no. 6; pp. S209 - S210
• Main Authors: Goff, A., Han, S., Zamarayeva, A., Sulchek, T., Ni, C.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.06.2024
• Subjects: Microfludic transfection; Resting T-cell manufacturing; T cell stemness; Resting T-cell manufacturing; Microfludic transfection; T cell stemness
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2024.03.419

Current workflows for T-cell manufacturing utilize fully activated T-cells via anti-CD3/CD28 co-stimulation, but T-cell differentiation in the final cell products may lead to shorter in vivo lifespans and lower anti-tumor efficacy. Hence, maintaining T cell stemness during the manufacturing process is valuable. However, engineering T cells with a high degree of stemness poses a challenge due to their quiescent state. In this study, we developed a workflow to process resting T cells and identified a cytokine-primed process and employed non-viral transfection using a microfluidic platform for resting T-cell manufacturing. Fresh human primary T cells were isolated and frozen until use. Cells were thawed and cultured in different conditions including medium, combination of interleukins, and duration of culture. Cells from individual conditions were processed using microfluidic transfection to evaluate transfection efficiency, cell viability, activation status, and differentiation. Processed cells were also tested in a secondary freeze-and-thaw process to confirm the capacity for future storage and expansion. Our results were consistent with general knowledge that resting T-cells are quiescent in the ex vivo culture. We successfully identified a cytokine-primed culture condition for these cells that 1) maintain cell survival; 2) preserve stemness; and 3) result in effective CRISPR/Cas9 knockouts and DNA transfection. This process was found to significantly promote the percentage of the Tscm subset, which was also found to be highly transfectable with potent self-renewal and proliferative ability. Transient transfection of such cells using mRNA or plasmid DNA resulted in efficiency of 60-80% and 5-12%, respectively. Importantly, cell viability remained comparable to the non-processed control (70-90%). Furthermore, CRISPR knockouts, tested at the TRAC, B2M, and CD52 loci, resulted in knockout efficiency ranged from 40-60% with similar viability to control. Strikingly, the transfected cells retained their expansion capacity even after second round of freeze-and-thaw cycle in response to activation. In sum, our cytokine-primed process prepared resting T cells for microfluidic transfection. This workflow preserves the Tscm subset in the engineered cells which allows for future expansion ex vivo or in vivo. This study enables many options for cell therapy including off-the-shelf allogeneic T-cell products or shortening the time frame for autologous T-cell manufacturing.