Mitochondrial transfer from cancer-associated fibroblasts increases migration in aggressive breast cancer.

Cancer-associated fibroblasts (CAFs) have distinct roles within the tumor microenvironment, which can impact the mode and efficacy of tumor cell migration. CAFs are known to increase invasion of less-aggressive breast cancer cells through matrix remodeling and leader-follower dynamics. Here, we demonstrate that CAFs communicate with breast cancer cells through the formation of contact-dependent tunneling nanotubes (TNTs), which allow for the exchange of cargo between cell types. CAF mitochondria are an integral cargo component and are sufficient to increase the 3D migration of cancer cells. This cargo transfer results in an increase in mitochondrial ATP production in cancer cells, whereas it has a negligible impact on glycolytic ATP production. Manually increasing mitochondrial oxidative phosphorylation (OXPHOS) by providing extra substrates for OXPHOS fails to enhance cancer cell migration unless glycolysis is maintained at a constant level. Together, these data indicate that tumor-stromal cell crosstalk via TNTs and the associated metabolic symbiosis is a finely controlled mechanism by which tumor cells co-opt their microenvironment to promote cancer progression and may become a potential therapeutic target.

Engineering fiber anisotropy within natural collagen hydrogels.

It is well known that biophysical properties of the extracellular matrix (ECM), including stiffness, porosity, composition, and fiber alignment (anisotropy), play a crucial role in controlling cell behavior in vivo. Type I collagen (collagen I) is a ubiquitous structural component in the ECM and has become a popular hydrogel material that can be tuned to replicate the mechanical properties found in vivo. In this review article, we describe popular methods to create 2-D and 3-D collagen I hydrogels with anisotropic fiber architectures. We focus on methods that can be readily translated from engineering and materials science laboratories to the life-science community with the overall goal of helping to increase the physiological relevance of cell culture assays.

Ultrathin Dual-Scale Nano- and Microporous Membranes for Vascular Transmigration Models.

Selective cellular transmigration across the microvascular endothelium regulates innate and adaptive immune responses, stem cell localization, and cancer cell metastasis. Integration of traditional microporous membranes into microfluidic vascular models permits the rapid assay of transmigration events but suffers from poor reproduction of the cell permeable basement membrane. Current microporous membranes in these systems have large nonporous regions between micropores that inhibit cell communication and nutrient exchange on the basolateral surface reducing their physiological relevance. Here, the use of 100 nm thick continuously nanoporous silicon nitride membranes as a base substrate for lithographic fabrication of 3 µm pores is presented, resulting in a highly porous (≈30%), dual-scale nano- and microporous membrane for use in an improved vascular transmigration model. Ultrathin membranes are patterned using a precision laser writer for cost-effective, rapid micropore design iterations. The optically transparent dual-scale membranes enable complete observation of leukocyte egress across a variety of pore densities. A maximal density of ≈14 micropores per cell is discovered beyond which cell-substrate interactions are compromised giving rise to endothelial cell losses under flow. Addition of a subluminal extracellular matrix rescues cell adhesion, allowing for the creation of shear-primed endothelial barrier models on nearly 30% continuously porous substrates.

Extended live-tracking and quantitative characterization of wound healing and cell migration with SiR-Hoechst.

Cell migration is essential to many life processes, including immune response, tissue repair, and cancer progression. A reliable quantitative characterization of the cell migration can therefore aid in the high throughput screening of drug efficacy in wound healing and cancer treatments. In this work, we report what we believe is the first use of SiR-Hoechst for extended live tracking and automated analysis of cell migration and wound healing. We showed through rigorous statistical comparisons that this far-red label does not affect migratory behavior. We observed excellent automated tracking of random cell migration, in which the motility parameters (speed, displacement, path length, directionality ratio, persistence time, and direction autocorrelation) obtained closely match those obtained from manual tracking. We also present an analysis framework to characterize the healing of a scratch wound from the perspective of single cells. The use of SiR-Hoechst is advantageous for the crowded environments in wound healing assays because as long as cell nuclei do not overlap, continuous tracking can be maintained even if there is cell-cell contact. In this paper, we report wound recovery based on the number of cells migrating into the wound over time, normalized by the initial cell count prior to the infliction of the wound. This normalized cell count approach is impervious to operator bias during the arbitration of wound edges and is also robust against variability that arises due to differences in the cell density of different samples. Additional wound healing characteristics were also defined based on the evolution of cell speed and directionality during healing. Not unexpected, the wound healing cells exhibited much higher tendency to maintain the same migratory direction in comparison to the randomly migrating cells. The use of SiR-Hoechst thus greatly simplified the automation of single cell and whole population analysis with high spatial and temporal resolution over extended periods of time.

Fabrication techniques enabling ultrathin nanostructured membranes for separations.

The fabrication of nanostructured materials is an area of continuous improvement and innovative techniques that fulfill the demand of many fields of research and development. The continuously decreasing size of the smallest patternable feature has expanded the catalog of methods enabling the fabrication of nanostructured materials. Several of these nanofabrication techniques have sprouted from applications requiring nanoporous membranes such as molecular separations, cell culture, and plasmonics. This review summarizes methods that successfully produce through-pores in ultrathin films exhibiting an approximate pore size to thickness ratio of one, which has been shown to be beneficial due to high permeability and improved separation potential. The material reviewed includes large-area, parallel, and affordable approaches such as self-organizing polymers, nanosphere lithography, anodization, nanoimprint lithography as well as others such as solid phase crystallization and nanosphere lens lithography. The aim of this review is to provide a set of inexpensive fabrication techniques to produce nanostructured materials exhibiting pores ranging from 10 to 350 nm and a pore size to thickness ratio close to one. The fabrication methods described in this work have reported the successful manufacture of nanoporous membranes exhibiting the ideal characteristics to improve selectivity and permeability when applied as separation media in ultrafiltration.

High-performance, low-voltage electroosmotic pumps with molecularly thin silicon nanomembranes.

We have developed electroosmotic pumps (EOPs) fabricated from 15-nm-thick porous nanocrystalline silicon (pnc-Si) membranes. Ultrathin pnc-Si membranes enable high electroosmotic flow per unit voltage. We demonstrate that electroosmosis theory compares well with the observed pnc-Si flow rates. We attribute the high flow rates to high electrical fields present across the 15-nm span of the membrane. Surface modifications, such as plasma oxidation or silanization, can influence the electroosmotic flow rates through pnc-Si membranes by alteration of the zeta potential of the material. A prototype EOP that uses pnc-Si membranes and Ag/AgCl electrodes was shown to pump microliter per minute-range flow through a 0.5-mm-diameter capillary tubing with as low as 250 mV of applied voltage. This silicon-based platform enables straightforward integration of low-voltage, on-chip EOPs into portable microfluidic devices with low back pressures.

Influence of silicon dioxide capping layers on pore characteristics in nanocrystalline silicon membranes.

Porous nanocrystalline silicon (pnc-Si) membranes are a new class of membrane material with promising applications in biological separations. Pores are formed in a silicon film sandwiched between nm thick silicon dioxide layers during rapid thermal annealing. Controlling pore size is critical in the size-dependent separation applications. In this work, we systematically studied the influence of the silicon dioxide capping layers on pnc-Si membranes. Even a single nm thick top oxide layer is enough to switch from agglomeration to pore formation after annealing. Both the pore size and porosity increase with the thickness of the top oxide, but quickly reach a plateau after 10 nm of oxide. The bottom oxide layer acts as a barrier layer to prevent the a-Si film from undergoing homo-epitaxial growth during annealing. Both the pore size and porosity decrease as the thickness of the bottom oxide layer increases to 100 nm. The decrease of the pore size and porosity is correlated with the increased roughness of the bottom oxide layer, which hinders nanocrystal nucleation and nanopore formation.

Highly porous silicon membranes fabricated from silicon nitride/silicon stacks.

Nanopore formation in silicon films has previously been demonstrated using rapid thermal crystallization of ultrathin (15 nm) amorphous Si films sandwiched between nm-thick SiO2 layers. In this work, the silicon dioxide barrier layers are replaced with silicon nitride, resulting in nanoporous silicon films with unprecedented pore density and novel morphology. Four different thin film stack systems including silicon nitride/silicon/silicon nitride (NSN), silicon dioxide/silicon/silicon nitride (OSN), silicon nitride/silicon/silicon dioxide (NSO), and silicon dioxide/silicon/silicon dioxide (OSO) are tested under different annealing temperatures. Generally the pore size, pore density, and porosity positively correlate with the annealing temperature for all four systems. The NSN system yields substantially higher porosity and pore density than the OSO system, with the OSN and NSO stack characteristics fallings between these extremes. The higher porosity of the Si membrane in the NSN stack is primarily due to the pore formation enhancement in the Si film. It is hypothesized that this could result from the interfacial energy difference between the silicon/silicon nitride and silicon/silicon dioxide, which influences the Si crystallization process.

Empirical and Computational Evaluation of Hemolysis in a Microfluidic Extracorporeal Membrane Oxygenator Prototype.

Microfluidic devices promise to overcome the limitations of conventional hemodialysis and oxygenation technologies by incorporating novel membranes with ultra-high permeability into portable devices with low blood volume. However, the characteristically small dimensions of these devices contribute to both non-physiologic shear that could damage blood components and laminar flow that inhibits transport. While many studies have been performed to empirically and computationally study hemolysis in medical devices, such as valves and blood pumps, little is known about blood damage in microfluidic devices. In this study, four variants of a representative microfluidic membrane-based oxygenator and two controls (positive and negative) are introduced, and computational models are used to predict hemolysis. The simulations were performed in ANSYS Fluent for nine shear stress-based parameter sets for the power law hemolysis model. We found that three of the nine tested parameters overpredict (5 to 10×) hemolysis compared to empirical experiments. However, three parameter sets demonstrated higher predictive accuracy for hemolysis values in devices characterized by low shear conditions, while another three parameter sets exhibited better performance for devices operating under higher shear conditions. Empirical testing of the devices in a recirculating loop revealed levels of hemolysis significantly lower (<2 ppm) than the hemolysis ranges observed in conventional oxygenators (>10 ppm). Evaluating the model's ability to predict hemolysis across diverse shearing conditions, both through empirical experiments and computational validation, will provide valuable insights for future micro ECMO device development by directly relating geometric and shear stress with hemolysis levels. We propose that, with an informed selection of hemolysis parameters based on the shear ranges of the test device, computational modeling can complement empirical testing in the development of novel high-flow blood-contacting microfluidic devices, allowing for a more efficient iterative design process. Furthermore, the low device-induced hemolysis measured in our study at physiologically relevant flow rates is promising for the future development of microfluidic oxygenators and dialyzers.

Use of the MicroSiM (µSiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier.

The microSiM (µSiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the µSiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between 'blood' and 'brain' chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the µSiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

Lineage-Specific Mesenchymal Stromal Cells Derived from Human iPSCs Showed Distinct Patterns in Transcriptomic Profile and Extracellular Vesicle Production.

Over the past decades, mesenchymal stromal cells (MSCs) have been extensively investigated as a potential therapeutic cell source for the treatment of various disorders. Differentiation of MSCs from human induced pluripotent stem cells (iMSCs) has provided a scalable approach for the biomanufacturing of MSCs and related biological products. Although iMSCs shared typical MSC markers and functions as primary MSCs (pMSCs), there is a lack of lineage specificity in many iMSC differentiation protocols. Here, a stepwise hiPSC-to-iMSC differentiation method is employed via intermediate cell stages of neural crest and cytotrophoblast to generate lineage-specific MSCs with varying differentiation efficiencies and gene expression. Through a comprehensive comparison between early developmental cell types (hiPSCs, neural crest, and cytotrophoblast), two lineage-specific iMSCs, and six source-specific pMSCs, are able to not only distinguish the transcriptomic differences between MSCs and early developmental cells, but also determine the transcriptomic similarities of iMSC subtypes to postnatal or perinatal pMSCs. Additionally, it is demonstrated that different iMSC subtypes and priming conditions affected EV production, exosomal protein expression, and cytokine cargo.

Pericytes Enrich the Basement Membrane and Reduce Neutrophil Transmigration in an In Vitro Model of Peripheral Inflammation at the Blood-Brain Barrier.

Sepsis is the most lethal and expensive condition treated in intensive care units. Sepsis survivors frequently suffer long-term cognitive impairment, which has been linked to the breakdown of the blood-brain barrier (BBB) during a sepsis-associated "cytokine storm". Because animal models poorly recapitulate sepsis pathophysiology, human models are needed to understand sepsis-associated brain injury and to develop novel therapeutic strategies. With the concurrent emergence of tissue chip technologies and the maturation of protocols for human induced pluripotent stem cell (hiPSC), we can now develop advanced in vitro models of the human BBB and immune system to understand the relationship between systemic inflammation and brain injury. Here, we present a BBB model of the primary barrier developed on the µSiM (microphysiological system enabled by an ultrathin silicon nanomembrane) tissue chip platform. The model features isogenically matched hiPSC-derived extended endothelial culture method brain microvascular endothelial cell-like cells (EECM-BMEC-like cells) and brain pericyte-like cells (BPLCs) in a back-to-back coculture separated by the ultrathin (100 nm) membrane. Both endothelial monocultures and cocultures with pericytes responded to sepsis-like stimuli, with increased small-molecule permeability, although no differences were detected between culture conditions. Conversely, BPLC coculture reduced the number of neutrophils that crossed the EECM-BMEC-like cell monolayer under sepsis-like stimulation. Interestingly, this barrier protection was not seen when the stimulus originated from the tissue side. Our studies are consistent with the reported role for pericytes in regulating leukocyte trafficking during sepsis but indicate that EECM-BMEC-like cells alone are sufficient to maintain the restrictive small-molecule permeability of the BBB.

Dynamics of adhesion molecule domains on neutrophil membranes: surfing the dynamic cell topography.

Lateral organization and mobility of adhesion molecules play a significant role in determining the avidity with which cells can bind to target cells or surfaces. Recently, we have shown that the lateral mobility of the principal adhesion molecules on neutrophils is lower for rolling associated adhesion molecules (RAAMs: L-selectin and PSGL-1) than for ß2 integrins (LFA-1 and Mac-1). Here we report that all four adhesion molecules exhibit distinct punctate distributions that are mobile on the cell surface. Using uniform illumination image correlation microscopy, we measure the lateral mobility of these topologically distinct domains. For all four molecules, we find that diffusion coefficients calculated from domain mobility agree with measurements we made previously using fluorescence recovery after photobleaching. This agreement indicates that the transport of receptors on the surface of the resting neutrophil is dominated by the lateral movement of domains rather than individual molecules. The diffusion of pre-assembled integrin domains to zones of neutrophil/endothelial contact may provide a mechanism to facilitate high avidity adhesion during the earliest stages of firm arrest.

Charge- and size-based separation of macromolecules using ultrathin silicon membranes.

Commercial ultrafiltration and dialysis membranes have broad pore size distributions and are over 1,000 times thicker than the molecules they are designed to separate, leading to poor size cut-off properties, filtrate loss within the membranes, and low transport rates. Nanofabricated membranes have great potential in molecular separation applications by offering more precise structural control, yet transport is also limited by micrometre-scale thicknesses. This limitation can be addressed by a new class of ultrathin nanostructured membranes where the membrane is roughly as thick (approximately 10 nm) as the molecules being separated, but membrane fragility and complex fabrication have prevented the use of ultrathin membranes for molecular separations. Here we report the development of an ultrathin porous nanocrystalline silicon (pnc-Si) membrane using straightforward silicon fabrication techniques that provide control over average pore sizes from approximately 5 nm to 25 nm. Our pnc-Si membranes can retain proteins while permitting the transport of small molecules at rates an order of magnitude faster than existing materials, separate differently sized proteins under physiological conditions, and separate similarly sized molecules carrying different charges. Despite being only 15 nm thick, pnc-Si membranes that are free-standing over 40,000 microm2 can support a full atmosphere of differential pressure without plastic deformation or fracture. By providing efficient, low-loss macromolecule separations, pnc-Si membranes are expected to enable a variety of new devices, including membrane-based chromatography systems and both analytical and preparative microfluidic systems that require highly efficient separations.

Optimization of Parylene C and Parylene N thin films for use in cellular co-culture and tissue barrier models.

Parylene has been used widely used as a coating on medical devices. It has also been used to fabricate thin films and porous membranes upon which to grow cells. Porous membranes are integral components of in vitro tissue barrier and co-culture models, and their interaction with cells and tissues affects the performance and physiological relevance of these model systems. Parylene C and Parylene N are two biocompatible Parylene variants with potential for use in these models, but their effect on cellular behavior is not as well understood as more commonly used cell culture substrates, such as tissue culture treated polystyrene and glass. Here, we use a simple approach for benchtop oxygen plasma treatment and investigate the changes in cell spreading and extracellular matrix deposition as well as the physical and chemical changes in material surface properties. Our results support and build on previous findings of positive effects of plasma treatment on Parylene biocompatibility while showing a more pronounced improvement for Parylene C compared to Parylene N. We measured relatively minor changes in surface roughness following plasma treatments, but significant changes in oxygen concentration at the surface persisted for 7 days and was likely the dominant factor in improving cellular behavior. Overall, this study offers facile and relatively low-cost plasma treatment protocols that provide persistent improvements in cell-substrate interactions on Parylene that match and exceed tissue culture polystyrene.

Engineering cell-substrate interactions on porous membranes for microphysiological systems.

Microphysiological systems are now widely used to recapitulate physiological and pathological microenvironments in order to study and understand a variety of cellular processes as well as drug delivery and stem cell differentiation. Central to many of these systems are porous membranes that enable tissue barrier formation as well as compartmentalization while still facilitating small molecule diffusion, cellular transmigration and cell-cell communication. The role or impact of porous membranes on the cells cultured upon them has not been widely studied or reviewed. Although many chemical and physical substrate characteristics have been shown to be effective in controlling and directing cellular behavior, the influence of pore characteristics and the ability to engineer porous membranes to influence these responses is not fully understood. In this mini-review, we show that many studies point to a multiphasic cell-substrate response, where increasing pore sizes and pore-pore spacing generally leads to improved cell-substrate interactions. However, the smallest pores in the nano-scale sometimes promote the strongest cell-substrate interactions, while the very largest micron-scale pores hinder cell-substrate interactions. This synopsis provides an insight into the importance of membrane pores in controlling cellular responses, and may help with the design and utilization of porous membranes for induction of desired cell processes in the development of biomimetic platforms.

Disrupted Surfaces of Porous Membranes Reduce Nuclear YAP Localization and Enhance Adipogenesis through Morphological Changes.

The disrupted surface of porous membranes, commonly used in tissue-chip and cellular coculture systems, is known to weaken cell-substrate interactions. Here, we investigated whether disrupted surfaces of membranes with micron and submicron scale pores affect yes-associated protein (YAP) localization and differentiation of adipose-derived stem cells. We found that these substrates reduce YAP nuclear localization through decreased cell spreading, consistent with reduced cell-substrate interactions, and in turn enhance adipogenesis while decreasing osteogenesis.

Local extensional flows promote long-range fiber alignment in 3D collagen hydrogels.

Randomly oriented type I collagen (COL1) fibers in the extracellular matrix are reorganized by biophysical forces into aligned domains extending several millimeters and with varying degrees of fiber alignment. These aligned fibers can transmit traction forces, guide tumor cell migration, facilitate angiogenesis, and influence tissue morphogenesis. To create aligned COL1 domains in microfluidic cell culture models, shear flows have been used to align thin COL1 matrices (<50µm in height) in a microchannel. However, there has been limited investigation into the role of shear flows in aligning 3D hydrogels (>130µm). Here, we show that pure shear flows do not induce fiber alignment in 3D atelo COL1 hydrogels, but the simple addition of local extensional flow promotes alignment that is maintained across several millimeters, with a degree of alignment directly related to the extensional strain rate. We further advance experimental capabilities by addressing the practical challenge of accessing a 3D hydrogel formed within a microchannel by introducing a magnetically coupled modular platform that can be released to expose the microengineered hydrogel. We demonstrate the platform's capability to pattern cells and fabricate multi-layered COL1 matrices using layer-by-layer fabrication and specialized modules. Our approach provides an easy-to-use fabrication method to achieve advanced hydrogel microengineering capabilities that combine fiber alignment with biofabrication capabilities.

The Modular µSiM Reconfigured: Integration of Microfluidic Capabilities to Study In Vitro Barrier Tissue Models under Flow.

Microfluidic tissue barrier models have emerged to address the lack of physiological fluid flow in conventional "open-well" Transwell-like devices. However, microfluidic techniques have not achieved widespread usage in bioscience laboratories because they are not fully compatible with traditional experimental protocols. To advance barrier tissue research, there is a need for a platform that combines the key advantages of both conventional open-well and microfluidic systems. Here, a plug-and-play flow module is developed to introduce on-demand microfluidic flow capabilities to an open-well device that features a nanoporous membrane and live-cell imaging capabilities. The magnetic latching assembly of this design enables bi-directional reconfiguration and allows users to conduct an experiment in an open-well format with established protocols and then add or remove microfluidic capabilities as desired. This work also provides an experimentally-validated flow model to select flow conditions based on the experimental needs. As a proof-of-concept, flow-induced alignment of endothelial cells and the expression of shear-sensitive gene targets are demonstrated, and the different phases of neutrophil transmigration across a chemically stimulated endothelial monolayer under flow conditions are visualized. With these experimental capabilities, it is anticipated that both engineering and bioscience laboratories will adopt this reconfigurable design due to the compatibility with standard open-well protocols.

Pore size control of ultrathin silicon membranes by rapid thermal carbonization.

Rapid thermal carbonization in a dilute acetylene (C(2)H(2)) atmosphere has been used to chemically modify and precisely tune the pore size of ultrathin porous nanocrystalline silicon (pnc-Si). The magnitude of size reduction was controlled by varying the process temperature and time. Under certain conditions, the carbon coating displayed atomic ordering indicative of graphene layer formation conformal to the pore walls. Initial experiments show that carbonized membranes follow theoretical predictions for hydraulic permeability and retain the precise separation capabilities of untreated membranes.