(603f) Hydrogel-Based Cell Culture System for Scalable Expansion of Human Primary T Cells

Introduction: Adoptive immunotherapy refers to the transfer of immune cells (e.g. T lymphocytes) with antitumor activity into a patient to mediate tumor regression. Basic, translational and clinical studies have shown adoptive immunotherapy is highly effective for treating many cancers, such as melanoma, cervical cancer, lymphoma, leukemia. However, the cost for manufacturing T cells with current cell culturing technologies is extremely high. For instance, one dose of a recently approved engineered T cells for treating children and young adults with B-cell acute lymphoblastic leukemia costs $475,000. Thus novel cell culturing technologies that can significantly reduce the manufacturing cost and increase the manufacturing capacity is highly wanted for the widespread application of adoptive immunotherapy.

Materials and Methods: A micro-extruder was designed and made for processing alginate hydrogel tubes. To process the alginate hydrogel tubes, a solution containing single T cells, T cell activators (e.g. anti-CD3/CD28/CD2 antibodies) and 2% hyaluronic acid polymer, and a solution containing 1.5% alginate polymer was pumped into the central channel and side channel of the micro-extruder, respectively, to form a coaxial core-shell flow that was extruded into a CaCl₂ buffer. The shell alginate flow was instantly crosslinked by Ca²⁺ ions to form an alginate hydrogel tube. Subsequently, the CaCl₂ buffer was replaced by the T cell culture medium and cells were grown in the tubes. To collect or passage cells, the alginate hydrogel tubes could be dissolved with the cell-compatible ethylenediaminetetraacetic acid (EDTA) solution (0.5 mM, 5 minutes at room temperature) to release the micro cell masses that could be dissociated into single cells by gently mechanical pipetting for the following analysis or passage.

Results: Here, we report a novel cell culture technology for expanding primary human T cells for adoptive immunotherapy. T cells are suspended and cultured in microscale alginate hydrogel tubes (AlgTubes) that are suspended in the cell culture medium in a culture vessel. The hydrogel tubes protect cells from hydrodynamic stresses and confine the cell mass less than 400 μm (in radial diameter) to ensure efficient mass transport, creating a cell-friendly microenvironment for growing T cells. This system is simple, scalable, highly efficient, defined, cost-effective and compatible with the current Good Manufacturing Practices. Under optimized culture conditions, the AlgTubes enable culturing T cells with high cell viability, low DNA damage, high growth rate (~320-fold expansion over 14 days), high purity (~98% CD3+) and high yield (~3.2x10⁸ cells/mL hydrogel), all offer considerable advantages compared to current T cell culturing approaches. This new culture technology can significantly reduce the culture volume, time and cost, while increase the production capacity to make the adoptive immunotherapy broadly available and affordable.

Conclusions: In summary, we believe the alginate hydrogel tubes will significantly advance the adoptive immunotherapy and will be of broad interest to individual laboratories, institutions, and biotechnology companies working on adoptive immunotherapy.