Non-Destructive Approaches for Retrieving T-Cells from Fibrous Scaffolds for Therapeutic Applications

Flasks and plates have traditionally been used to culture cells required for cell-based therapies. Recent success of adoptive T-cell transfer therapy (ACT) for various pathological conditions warrants development of more physiologically relevant <i>ex vivo</i> cell culture platforms. Electrospun (Espun) scaffolds hold promise for culturing cells by mimicking features of extracellular matrix (ECM). However, unlike traditional 2D culture, recovering cells from these fibrous scaffolds is challenging and poses a critical roadblock in their development as cell culture platforms. We used electrospun matrices to culture Jurkat T-cells and observed that the cells remain entrapped in these matrices, facilitating their growth and clustering, which are the key phenomena for their activation and expansion, especially in the context of adoptive cell therapy. Yet, their retrieval using the pipette-aided gentle aspiration method proved difficult. This challenge was amplified with stimulating (anti-CD3 antibody-coated) substrates. Our study compared different recovery strategies using enzymatic agents (Accutase and TrypLE) and non-enzymatic manual flushing to determine the most effective method. A comparable cell yield was obtained and the viability of recovered cells was found to be unaffected for all the methods tested. However, the unstimulated substrate had a significantly higher cell recovery than its stimulated counterpart. Further investigation revealed that cells recovered from scaffolds after enzymatic treatment with Accutase had better proliferation and clustering ability when compared with those cultured on 2D substrates. The insights from this study may be critical in generating clinical-grade T-cells <i>ex vivo</i> for immunotherapeutic applications.