A multiplexed microfluidic continuous-flow electroporation system for efficient cell transfection.

Cellular therapies have the potential to advance treatment for a broad array of diseases but rely on viruses for genetic reprogramming. The time and cost required to produce viruses has created a bottleneck that constricts development of and access to cellular therapies. Electroporation is a non-viral alternative for genetic reprogramming that bypasses these bottlenecks, but current electroporation technology suffers from low throughput, tedious optimization, and difficulty scaling to large-scale cell manufacturing.

A multiplexed microfluidic continuous-flow electroporation system for efficient cell transfection.

Cellular therapies have the potential to advance treatment for a broad array of diseases but rely on viruses for genetic reprogramming. The time and cost required to produce viruses has created a bottleneck that constricts development of and access to cellular therapies. Electroporation is a non-viral approach for genetic reprogramming that bypasses these bottlenecks, but current electroporation technology suffers from low throughput, tedious optimization, and difficulty scaling to large-scale cell manufacturing.

Scalable continuous-flow electroporation platform enabling T cell transfection for cellular therapy manufacturing.

Viral vectors represent a bottleneck in the manufacturing of cellular therapies. Electroporation has emerged as an approach for non-viral transfection of primary cells, but standard cuvette-based approaches suffer from low throughput, difficult optimization, and incompatibility with large-scale cell manufacturing. Here, we present a novel electroporation platform capable of rapid and reproducible electroporation that can efficiently transfect small volumes of cells for research and process optimization and scale to volumes required for applications in cellular therapy.

Matrix stiffness induces epithelial-to-mesenchymal transition via Piezo1-regulated calcium flux in prostate cancer cells.

Cells utilize calcium channels as one of the main signaling mechanisms to sense changes in the extracellular space and convert these changes to intracellular signals. Calcium regulates several key signaling networks, such as the induction of EMT. The current study expands on the understanding of how EMT is controlled via the mechanosensitive calcium channel Piezo1 in cancerous cells, which senses changes in the extracellular matrix stiffness.

A discrete interface in matrix stiffness creates an oscillatory pattern of endothelial monolayer disruption.

Intimal stiffening upregulates endothelial cell contractility, disrupting barrier integrity; however, intimal stiffening is non-uniform. The impact of local changes in intimal stiffness on proximal and distal cell-cell interactions is unknown. To investigate the range at which matrix stiffness heterogeneities impact neighboring endothelial cells within a monolayer, we built a micropillar system with adjacent regions of stiff and compliant matrix.

Author Correction: Energetic costs regulated by cell mechanics and confinement are predictive of migration path during decision-making.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Extent of Cell Confinement in Microtracks Affects Speed and Results in Differential Matrix Strains.

During metastasis, cancer cells navigate through a spatially heterogeneous extracellular matrix (ECM). Physical properties of ECM, including the degree of confinement, influence cell migration behavior. Here, utilizing in vitro three-dimensional collagen microtracks, we demonstrate that cell-ECM interactions, specifically the degree of spatial confinement, regulate migratory behavior.

Energetic costs regulated by cell mechanics and confinement are predictive of migration path during decision-making.

Cell migration during the invasion-metastasis cascade requires cancer cells to navigate a spatially complex microenvironment that presents directional choices to migrating cells. Here, we investigate cellular energetics during migration decision-making in confined spaces. Theoretical and experimental data show that energetic costs for migration through confined spaces are mediated by a balance between cell and matrix compliance as well as the degree of spatial confinement to direct decision-making.

Fiber alignment drives changes in architectural and mechanical features in collagen matrices.

Aligned collagen architecture is a characteristic feature of the tumor extracellular matrix (ECM) and has been shown to facilitate cancer metastasis using 3D in vitro models. Additional features of the ECM, such as pore size and stiffness, have also been shown to influence cellular behavior and are implicated in cancer progression. While there are several methods to produce aligned matrices to study the effect on cell behavior in vitro, it is unclear how the alignment itself may alter these other important features of the matrix.

Substrate stiffness heterogeneities disrupt endothelial barrier integrity in a micropillar model of heterogeneous vascular stiffening.

Intimal stiffening has been linked with increased vascular permeability and leukocyte transmigration, hallmarks of atherosclerosis. However, recent evidence indicates age-related intimal stiffening is not uniform but rather characterized by increased point-to-point heterogeneity in subendothelial matrix stiffness, the impact of which is much less understood. To investigate the impact of spatially heterogeneous matrix rigidity on endothelial monolayer integrity, we develop a micropillar model to introduce closely-spaced, step-changes in substrate rigidity and compare endothelial monolayer phenotype to rigidity-matched, uniformly stiff and compliant substrates.

The Role of Age-Related Intimal Remodeling and Stiffening in Atherosclerosis.

Age-related vascular stiffening is closely associated with cardiovascular risk. The clinical measure of arterial stiffness, pulse wave velocity, reflects bulk structural changes in the media observed with age, but does not reflect intimal remodeling that also drives atherosclerosis. Endothelial barrier integrity is disrupted during early atherogenesis and is regulated by the mechanics and composition of the underlying intima, which undergoes significant atherogenic remodeling in response to age and hemodynamics.

Regulation of ATP utilization during metastatic cell migration by collagen architecture.

Cell migration in a three-dimensional matrix requires that cells either remodel the surrounding matrix fibers and/or squeeze between the fibers to move. Matrix degradation, matrix remodeling, and changes in cell shape each require cells to expend energy. While significant research has been performed to understand the cellular and molecular mechanisms guiding metastatic migration, less is known about cellular energy regulation and utilization during three-dimensional cancer cell migration.

Manipulation of in vitro collagen matrix architecture for scaffolds of improved physiological relevance.

Type I collagen is a versatile biomaterial that is widely used in medical applications due to its weak antigenicity, robust biocompatibility, and its ability to be modified for a wide array of applications. As such, collagen has become a major component of many tissue engineering scaffolds, drug delivery platforms, and substrates for in vitro cell culture. In these applications, collagen constructs are fabricated to recapitulate a diverse set of conditions.