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.
• CFD simulations of a spinner flask and a rotary wall vessel were performed. • Increasing impeller speed reduced the Kolmogorov length scales. • The Kolmogorov length scales were strongly correlated with cell aggregate sizes. • The rotary wall vessel was designed to minimize shear stress distributions. • The rotary vessel reduced shear stress distribution by half than the spinner flask.
The
CEACAM5
gene product [carcinoembryonic antigen (CEA)] is an attractive target for colorectal cancer because of its high expression in virtually all colorectal tumors and limited expression in most healthy adult tissues. However, highly active CEA-directed investigational therapeutics have been reported to be toxic, causing severe colitis because CEA is expressed on normal gut epithelial cells. Here, we developed a strategy to address this toxicity problem: the Tmod dual-signal integrator. CEA Tmod cells use two receptors: a chimeric antigen receptor (CAR) activated by CEA and a leukocyte Ig-like receptor 1 (LIR-1)–based inhibitory receptor triggered by human leukocyte antigen (HLA)-A*02. CEA Tmod cells exploit instances of HLA heterozygous gene loss in tumors to protect the patient from on-target, off-tumor toxicity. CEA Tmod cells potently killed CEA-expressing tumor cells in vitro and in vivo. But in contrast to a traditional CEA-specific T cell receptor transgenic T cell, Tmod cells were highly selective for tumor cells even when mixed with HLA-A*02–expressing cells. These data support further development of the CEA Tmod construct as a therapeutic candidate for colorectal cancer.
The survival, growth, self-renewal, and differentiation of human pluripotent stem cells (hPSCs) are influenced by their microenvironment, or so-called "niche," consisting of particular chemical and physical cues. Previous studies on mesenchymal stem cells and other stem cells have collectively uncovered the importance of physical cues and have begun to shed light on how stem cells sense and process such cues. In an attempt to support similar progress in mechanobiology of hPSCs, we review mechanosensory machinery, which plays an important role in cell-extracellular matrix interactions, cell-cell interactions, and subsequent intracellular responses. In addition, we review recent studies on the mechanobiology of hPSCs, in which engineered micromechanical environments were used to investigate effects of specific physical cues. Identifying key physical cues and understanding their mechanism will ultimately help in harnessing the full potential of hPSCs for clinical applications.
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.
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