Electrospun poly(dimethyl siloxane)-based meshes as a platform for t cell expansion

Abstract

Event Abstract Back to Event Electrospun poly(dimethyl siloxane)-based meshes as a platform for t cell expansion Alex Dang1, Sarah E. De Leo1, Danielle R. Bogdanowicz1, Helen H. Lu1 and Lance C. Kam1 1 Columbia University, Biomedical Engineering, United States Introduction: Recent advances in the genetic manipulation of T cells have led to striking successes in immunotherapy of cancer. However, advances in cell production and manufacturing are needed to fully realize the power of this approach. The current gold standard for initiating clinical ex vivo expansion of T cell populations consists of rigid (GPa) polystyrene beads presenting activating antibodies to CD3 and CD28. Recent work by our lab demonstrated that improved human T cell stimulation can be achieved using softer poly(dimethyl siloxane) (PDMS) substrates (~30 kPa – 2 MPa)[1]m[2]. In this report, we leverage this mechanosensing property of T cells in a fiber-based mesh system to improve human T cell expansion on a clinically relevant scale. This format preserves the high surface area provided by beads while reducing the risks associated with incomplete removal of these structures from the final cell product. We herein examine the effect of composition, rigidity, fiber density, and pore size of electrospun polymer fibers on long-term (19 days) T cell expansion on clinically relevant scales. Materials and Methods: Fibrous meshes were synthesized via electrospinning of NuSil MED-4086 PDMS mixed with poly(ε-caprolactone) (PCL) in a 3:1 v/v dichloromethane (DCM) / N,N-dimethylformamide (DMF) solution at 10 – 12 kV onto a grounded collection electrode with a working distance between 10-12 cm. Polymer concentration was varied from 19 to 50% wt/wt. Meshes were coated with goat-anti-mouse IgG linkers followed by a 1:4 ratio of mouse IgG-anti-human CD3 (OKT3) to mouse IgG-anti-human CD28 (9.3) (2 h, 23 °C). Cells were subjected to a carboxy-fluorescein succinimidyl ester (CFSE) assay and seeded at 1 million cells / mL at either small-scale (1 mL) or clinical scale (15-20 mL) volumes. Cell proliferation studies were performed in vitro with human naïve CD4+/CD8+ T cells as previously described[1]. Assays for CD107b expression and IFNγ secretion were performed upon completion of blast phase and in vitro re-stimulation for 4 and 12 hours, respectively. Assays for memory markers CD45RO and CCR7 were performed following re-stimulation. Results and Discussion: Scanning electron microscopy (SEM) revealed tunable pore sizes and fiber densities as a function of polymer composition. Long-term culture for 17-19 days coupled with approximately 9.8 maximum doublings for PDMS/PCL meshes indicated a robust system for expansion of T cells. Expansions on experimental meshes yielded up to 3.2 and 4 times more cells compared to Dynabead® and pure PCL mesh controls, respectively (see fig. 1). Flow cytometry revealed higher levels of proliferation on experimental meshes than on the current gold standard and pure PCL controls. Assays for CD107b / CD45RO / CCR7 expression and IFNγ secretion showed PDMS/PCL mesh-expanded cell activation levels on par with or higher than cells expanded on the current gold standard (see fig. 2). Memory marker analysis revealed high levels of central memory T cells after re-stimulation (data not shown). Conclusion: We have shown that electrospun, antibody-functionalized PDMS/PCL fibrous meshes with microscale diameters and pore sizes can increase T cell proliferation. Cells produced yielded substantially higher population doublings and were shown to have equal or better levels of effector cell function than the current gold standard. This platform holds significant promise in the ex vivo expansion of T cells. National Institutes of Health (R01AI088377); Wallace H. Coulter Foundation; National Science Foundation GRFP; Columbia University Medical Center Flow Cytometry Core