Massively-Parallelized, Deterministic Mechanoporation for Intracellular Delivery

Microfluidic intracellular delivery approaches based on plasma membrane poration have shown promise for addressing the limitations of conventional cellular engineering techniques in a wide range of applications in biology and medicine. However, the inherent stochasticity of the poration process in many of these approaches often results in a trade-off between delivery efficiency and cellular viability, thus potentially limiting their utility. Herein, we present a novel microfluidic device concept that mitigates this trade-off by providing opportunity for deterministic mechanoporation (DMP) of cells en masse. This is achieved by the impingement of each cell upon a single needle-like penetrator during aspiration-based capture, followed by diffusive influx of exogenous cargo through the resulting membrane pore, once the cells are released by reversal of flow. Massive parallelization enables high throughput operation, while single-site poration allows for delivery of small and large-molecule cargos in difficult-to-transfect cells with efficiencies and viabilities that exceed both conventional and emerging transfection techniques. As such, DMP shows promise for advancing cellular engineering practice in general and engineered cell product manufacturing in particular

Stirred Suspension Culture for Scalable Production and Differentiation of Human Pluripotent Stem Cells

The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges in part on the availability of a robust scalable culture system that can readily produce clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one of such promising platforms due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development in the past several years, and little is known beyond empirically optimized culture parameters. The goal of this study was to elucidate the impact of fluidic agitation on hPSCs in stirred suspension culture. In particular, we systematically investigated various agitation rates to characterize their impact on cell yield, viability, and maintenance of pluripotency. Additionally, we closely examined the distribution of cell aggregates and how the observed culture outcomes are attributed to their size distribution. Our results showed that moderate agitation maximized the propagation of hPSCs by controlling the cell aggregates below the critical size, beyond which the cells suffer from diffusion limitation, while limiting cell death caused by excessive fluidic forces. Furthermore, we observed that fluidic agitation could regulate not only cell aggregation, but also expression of some key signaling proteins in hPSCs. Upon discovering this mechanosensitive effector enabled a novel approach for linking expansion and cardiac differentiation to generate over 90% cardiomyocytes. In addition, these cardiomyocytes displayed highly organized sarcomere structure which suggests an improved maturation in their morphology. Altogether, results presented in this study indicate the new possibility of guiding stem cell fate determination by fluidic agitation in stirred suspension cultures

A multivariate, quantitative assay that disentangles key kinetic parameters of primary human T cell function in vitro.

Cell therapy is poised to play a larger role in medicine, most notably for immuno-oncology. Despite the recent success of CAR-T therapeutics in the treatment of blood tumors and the rapid progress toward improved versions of both CAR- and TCR-Ts, important analytical aspects of preclinical development and manufacturing of engineered T cells remain immature. One limiting factor is the absence of robust multivariate assays to disentangle key parameters related to function of engineered effector cells, especially in the peptide-MHC (pMHC) target realm, the natural ligand for TCRs. Here we describe an imaging-based primary T cell assay that addresses several of these limitations. To our knowledge, this assay is the first quantitative, high-content assay that separates the key functional parameters of time- and antigen-dependent T cell proliferation from cytotoxicity. We show that the assay sheds light on relevant biology of CAR- and TCR-T cells, including response kinetics and the influence of effector:target ratio