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. Here, we present an adaptable microfluidic electroporation platform with the capability for rapid, multiplexed optimization with 96-well plates. Once parameters are optimized using small volumes of cells, transfection can be seamlessly scaled to high-volume cell manufacturing without re-optimization. We demonstrate optimizing transfection of plasmid DNA to Jurkat cells, screening hundreds of different electrical waveforms of varying shapes at a speed of ~3 s per waveform using ~20 µL of cells per waveform. We selected an optimal set of transfection parameters using a low-volume flow cell. These parameters were then used in a separate high-volume flow cell where we obtained similar transfection performance by design. This demonstrates an alternative non-viral and economical transfection method for scaling to the volume required for producing a cell therapy without sacrificing performance. Importantly, this transfection method is disease-agnostic with broad applications beyond cell therapy.

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. Here, we present an adaptable microfluidic electroporation platform with the capability for rapid, multiplexed optimization with 96-well plates. Once parameters are optimized using small volumes of cells, transfection can be seamlessly scaled to high-volume cell manufacturing without re-optimization. We demonstrate optimizing transfection of plasmid DNA to Jurkat cells, screening hundreds of different electrical waveforms of varying shapes at a speed of ~3 s per waveform using ~ 20 µL of cells per waveform. We selected an optimal set of transfection parameters using a low-volume flow cell. These parameters were then used in a separate high-volume flow cell where we obtained similar transfection performance by design. This demonstrates an economical method for scaling to the volume required for producing a cell therapy without sacrificing performance.

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. We demonstrate delivery of plasmid DNA and mRNA to primary human T cells with high efficiency and viability, such as > 95% transfection efficiency for mRNA delivery with < 2% loss of cell viability compared to control cells. We present methods for scaling delivery that achieve an experimental throughput of 256 million cells/min. Finally, we demonstrate a therapeutically relevant modification of primary T cells using CRISPR/Cas9 to knockdown T cell receptor (TCR) expression. This study displays the capabilities of our system to address unmet needs for efficient, non-viral engineering of T cells for cell manufacturing.

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. We model the biophysical environment of healthy and cancerous prostate tissue using polyacrylamide gels of different stiffnesses. Significant increases in calcium steady-state concentration, vimentin expression, and aspect ratio, and decreases in E-cadherin expression were observed by increasing matrix stiffness and also after treatment with Yoda1, a chemical agonist of Piezo1. Overall, this study concludes that Piezo1-regulated calcium flux plays a role in prostate cancer cell metastatic potential by sensing changes in ECM stiffness and modulating EMT markers.

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. The stiffness interface results in an oscillatory pattern of neutrophil transendothelial migration, symmetrical about the interface and well-fit by a sinusoid function. 'Peaks' of the sinusoid were found to have increased cellular contractility and decreased barrier function relative to 'troughs' of the sinusoid. Pharmacological modulation of contractility was observed to break symmetry, altering the amplitude and wavelength of the sinusoid, indicating that contractility may regulate this effect. This work illuminates a novel biophysical phenomenon of the role of stiffness-mediated cell-matrix interactions on cell-cell interactions at a distance. Additionally, it provides insight into the range at which intimal matrix stiffness heterogeneities will impact endothelial barrier function and potentially contribute to atherogenesis.

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. We found that cells migrate faster when they are fully confined, contacting all four walls (top, bottom, and two sides) of a collagen microtrack, compared with cells that are partially confined, contacting less than four walls. When fully confined, cells exhibit fewer but larger vinculin-containing adhesions and create greater strains in the surrounding matrix directed toward the cell body. In contrast, partially confined cells develop a more elongated morphology with smaller but significantly more vinculin-containing adhesions and displace the surrounding matrix less than fully confined cells. The resulting effect of increasing cell contractility via Rho activation is dependent on the number of walls with which the cell is in contact. Although matrix strains increase in both fully and partially confined cells, cells that are partially confined increase speed, whereas those in full confinement decrease speed. Together, these results suggest that the degree of cell-ECM contact during confined migration is a key determinant of speed, morphology, and cell-generated substrate strains during motility, and these factors may work in tandem to facilitate metastatic cell migration.

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. Energetic costs, driven by the cellular work needed to generate force for matrix displacement, increase with increasing cell stiffness, matrix stiffness, and degree of spatial confinement, limiting migration. By assessing energetic costs between possible migration paths, we can predict the probability of migration choice. Our findings indicate that motility in confined spaces imposes high energetic demands on migrating cells, and cells migrate in the direction of least confinement to minimize energetic costs. Therefore, therapeutically targeting metabolism may limit cancer cell migration and metastasis.

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. In this study, we have generated aligned collagen matrices and characterized their pore sizes and mechanical properties at the micro- and macro-scale. Our results indicate that collagen alignment can alter pore-size of matrices depending on the polymerization temperature of the collagen. Furthermore, alignment does not affect the macro-scale stiffness but alters the micro-scale stiffness in a temperature independent manner. Overall, these results describe the manifestation of confounding variables that arise due to alignment and the importance of fully characterizing biomaterials at both micro- and macro-scales.

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. We found equivalent disruption of adherens junctions within monolayers on step-rigidity and uniformly stiff substrates relative to uniformly compliant substrates. Similarly, monolayers cultured on step-rigidity substrates exhibited equivalent percentages of leukocyte transmigration to monolayers on rigidity-matched, uniformly stiff substrates. Adherens junction tension and focal adhesion density, but not size, increased within monolayers on step-rigidity and uniformly stiff substrates compared to more compliant substrates suggesting that elevated tension is disrupting adherens junction integrity. Leukocyte transmigration frequency and time, focal adhesion size, and focal adhesion density did not differ between stiff and compliant sub-regions of step-rigidity substrates. Overall, our results suggest that endothelial monolayers exposed to mechanically heterogeneous substrates adopt the phenotype associated with the stiffer matrix, indicating that spatial heterogeneities in intimal stiffness observed with age could disrupt endothelial barrier integrity and contribute to atherogenesis.

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. Here, we first review the best characterized of these changes, including physiological intimal thickening throughout the arterial tree, fibronectin and collagen deposition, and collagen cross-linking. We then address the most common in vivo and in vitro models used to gain mechanistic insight into the consequences of intimal remodeling. Finally, we consider the impacts of intimal stiffening upon endothelial cell mechanotransduction with emphasis on the emerging impact of increased complexity in cellular traction forces and substrate rigidity upon endothelial barrier integrity.

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. Here we introduce the use of the genetically encoded fluorescent biomarkers, PercevalHR and pHRed, to quantitatively assess ATP, ADP, and pH levels in MDA-MB-231 metastatic cancer cells as a function of the local collagen microenvironment. We find that the use of the probe is an effective tool for exploring the thermodynamics of cancer cell migration and invasion. Specifically, we find that the ATP:ADP ratio increases in cells in denser matrices, where migration is impaired, and it decreases in cells in aligned collagen matrices, where migration is facilitated. When migration is pharmacologically inhibited, the ATP:ADP ratio decreases. Together, our data indicate that matrix architecture alters cellular energetics and that intracellular ATP:ADP ratio is related to the ability of cancer cells to effectively migrate.

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. Collagen fibrils can be aligned during or post-fabrication, cross-linked via numerous techniques, polymerized to create various fibril sizes and densities, and copolymerized into a wide array of composite scaffolds. Here, we review approaches that have been used to tune collagen to better recapitulate physiological environments for use in tissue engineering applications and studies of basic cell behavior. We discuss techniques to control fibril alignment, methods for cross-linking collagen constructs to modulate stiffness, and composite collagen constructs to better mimic physiological extracellular matrix.

Controlling collagen fiber microstructure in three-dimensional hydrogels using ultrasound.

Type I collagen is the primary fibrillar component of the extracellular matrix, and functional properties of collagen arise from variations in fiber structure. This study investigated the ability of ultrasound to control collagen microstructure during hydrogel fabrication. Under appropriate conditions, ultrasound exposure of type I collagen during polymerization altered fiber microstructure. Scanning electron microscopy and second-harmonic generation microscopy revealed decreased collagen fiber diameters in response to ultrasound compared to sham-exposed samples. Results of mechanistic investigations were consistent with a thermal mechanism for the effects of ultrasound on collagen fiber structure. To control collagen microstructure site-specifically, a high frequency, 8.3-MHz, ultrasound beam was directed within the center of a large collagen sample producing dense networks of short, thin collagen fibrils within the central core of the gel and longer, thicker fibers outside the beam area. Fibroblasts seeded onto these gels migrated rapidly into small, circularly arranged aggregates only within the beam area, and clustered fibroblasts remodeled the central, ultrasound-exposed collagen fibrils into dense sheets. These investigations demonstrate the capability of ultrasound to spatially pattern various collagen microstructures within an engineered tissue noninvasively, thus enhancing the level of complexity of extracellular matrix microenvironments and cellular functions achievable within three-dimensional engineered tissues.