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The Golgi apparatus comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region of most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi apparatus with intermediate filaments. In this study we show that the Golgi is in close physical proximity to vimentin intermediate filaments (IFs) in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with IFs. Although loss of vimentin and/or GORAB does not have major effects upon Golgi morphology at steady-state, the Golgi undergoes more rapid disassembly upon chemical disruption with the drug brefeldin A, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin causes reduced Golgi ribbon integrity when cells are cultured on high stiffness hydrogels, which is exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi apparatus, and suggest a role for GORAB in this process.

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localisation of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

This study investigated the effects of terminal sterilization of polyvinyl alcohol (PVA) biomaterials using clinically translatable techniques, specifically ethylene oxide (EtO) and gamma (γ) irradiation. While a few studies have reported the possibility of sterilizing PVA with γ-radiation, the use of EtO sterilization of PVA requires additional study. PVA solutions were chemically crosslinked with trisodium trimetaphosphate and sodium hydroxide. The three experimental groups included untreated control, EtO, and γ-irradiation, which were tested for the degree of swelling and water content, and mechanical properties such as radial compliance, longitudinal tensile, minimum bend radius, burst pressure, and suture retention strength. In addition, samples were characterized with scanning electron microscopy, differential scanning calorimetry, X-ray photoelectron spectroscopy, and water contact angle measurements. Cell attachment was assessed using the endothelial cell line EA.hy926, and the sterilized PVA cytotoxicity was studied with a live/dead stain. Platelet and fibrin accumulation was measured using an ex vivo shunt baboon model. Finally, the immune responses of PVA implants were analyzed after a 21-day subcutaneous implantation in rats and a 30-day implantation in baboon. EtO sterilization reduced the PVA graft wall thickness, its degree of swelling, and water content compared with both γ-irradiated and untreated PVA. Moreover, EtO sterilization significantly reduced the radial compliance and increased Young's modulus. EtO did not change PVA hydrophilicity, while γ-irradiation increased the water contact angle of the PVA. Consequently, endothelial cell attachment on the EtO-sterilized PVA showed similar results to the untreated PVA, while cell attachment significantly improved on the γ-irradiated PVA. When exposing the PVA grafts to circulating whole blood, fibrin accumulation of EtO-sterilized PVA was found to be significantly lower than γ-irradiated PVA. The immune responses of γ-irradiated PVA, EtO-treated PVA, and untreated PVA were compared. Implanted EtO-treated PVA showed the least MAC387 reaction. The terminal sterilization methods in this study changed PVA hydrogel properties; nevertheless, based on the characterizations performed, both sterilization methods were suitable for sterilizing PVA. We concluded that EtO can be used as an alternative method to sterilize PVA hydrogel material. IMPACT STATEMENT: Polyvinyl alcohol (PVA) hydrogels have been used for a variety of tissue replacements, including neural, cardiac, meniscal, cartilage, muscle, pancreatic, and ocular applications. In addition, PVA can be made into a tubular shape and used as a small-diameter vascular graft. Ethylene oxide (EtO) is one of the Food and Drug Administration-approved methods for sterilization, but its effect on PVA has not been studied extensively. The outcome of this study provides the effects of EtO and γ-irradiation of PVA grafts on both the material properties and the in vivo responses, particularly for vascular applications. Knowledge of these effects may ultimately improve the success rate of PVA vascular grafts.

The addition of nanoscale distortion to ordered nanotopographies consistently determines an osteogenic fate in stem cells. Although disordered and ordered nanopit arrays have identical surface areas, array symmetry has opposite effects on cell fate. We aimed to understand how cells sense disorder at the nanoscale. We observed effects in the early formation of cell and focal adhesions that controlled long-term cell fate. Disordered nanopits consistently yielded larger focal adhesions at a faster rate, prompting us to investigate this at the molecular scale. Super-resolution microscopy revealed that the nanopits did not act as nucleation points, as previously thought. Rather, nanopit arrays altered the plasma membrane and acted as barriers that changed molecular diffusion. The local areas corralled by four nanopits were the smallest structures that exerted diverging effects between ordered and disordered arrays. Heterogeneity in the local area on disordered arrays increased the proportion of fastest and slowest diffusing molecules. This resulted in higher quantity, more frequent formation and clustered arrangement of nascent adhesions, i.e., the modular units on which focal adhesions are built. This work presents a new pathway to exploit nanoscale sensing to dictate cell fate.

Cells respond in complex ways to their environment, making it challenging to predict a direct relationship between the two. A key problem is the lack of informative representations of parameters that translate directly into biological function. Here we present a platform to relate the effects of cell morphology to gene expression induced by nanotopography. This platform utilizes the ‘morphome’, a multivariate dataset of cell morphology parameters. We create a Bayesian linear regression model that uses the morphome to robustly predict changes in bone, cartilage, muscle and fibrous gene expression induced by nanotopography. Furthermore, through this model we effectively predict nanotopography-induced gene expression from a complex co-culture microenvironment. The information from the morphome uncovers previously unknown effects of nanotopography on altering cell–cell interaction and osteogenic gene expression at the single cell level. The predictive relationship between morphology and gene expression arising from cell-material interaction shows promise for exploration of new topographies., The surface nanotopography of biomaterials direct cell behavior, but screening for desired effects is inefficient. Here, the authors introduce a platform that enables prediction of nanotopography-induced gene expression changes from changes in cell morphology, including in co-culture environments.

3D printers suffer from the inverse relationship between throughput and minimum feature size; with smaller features inducing a cubic increase in print time. Here we introduce Nano-3DP, a hybrid process that combines digital light projection 3D printing with nanoscale-relief patterning. The tool enables large volume (cm3) prints with nanoscale details at a truly rapid rate (~120 mm/hour). 40 nm features, half the size of the finest printed details to date, are produced across a scalable print volume. We address the intrinsic issues of throughput and pixel induced surface inhomogeneity. To demonstrate the unique potential realized by this printing method across different areas of science optical lenses, injection molding tools and bio-implants originally acquired by x-ray CT are produced with functional nanoscale surface details. Notably, in vitro bone cell analysis delivered a profound 4.5-fold increase in osteogenesis purely through the inclusion of nanoscale features on the printed surfaces.

The endothelial lining of blood vessels is severely affected in type II diabetes. Yet, there is still a paucity on the use of diabetic endothelial cells for study and assessment of implantable devices targeting vascular disease. This critically impairs our ability to determine appropriate topographical cues to be included in implantable devices that can be used to maintain or improve endothelial cell function in vivo. Here, the functional responses of healthy and diabetic human coronary arterial endothelial cells were studied and observed to differ depending on topography. Gratings (2 μm) maintained normal endothelial functions such as adhesiveness, angiogenic capacity and cell-cell junction formation, and reduced immunogenicity of healthy cells. However, a significant and consistent effect was not observed in diabetic cells. Instead, diabetic endothelial cells cultured on the perpendicularly aligned multi-scale hierarchical gratings (250 nm gratings on 2 μm gratings) drastically reduced the uptake of oxidized low-density lipoprotein, decreased immune activation, and accelerated cell migration. Concave microlens (1.8 μm diameter) topography was additionally observed to overwhelmingly deteriorate diabetic endothelial cell function. The results of this study support a new paradigm and approach in the design and testing of implantable devices and biomedical interventions for diabetic patients.

Pancreatic ductal adenocarcinoma is one of the most invasive and metastatic cancers and has a dismal 5-year survival rate. We show that N-WASP drives pancreatic cancer metastasis, with roles in both chemotaxis and matrix remodeling. lysophosphatidic acid, a signaling lipid abundant in blood and ascites fluid, is both a mitogen and chemoattractant for cancer cells. Pancreatic cancer cells break lysophosphatidic acid down as they respond to it, setting up a self-generated gradient driving tumor egress. N-WASP-depleted cells do not recognize lysophosphatidic acid gradients, leading to altered RhoA activation, decreased contractility and traction forces, and reduced metastasis. We describe a signaling loop whereby N-WASP and the endocytic adapter SNX18 promote lysophosphatidic acid-induced RhoA-mediated contractility and force generation by controlling lysophosphatidic acid receptor recycling and preventing degradation. This chemotactic loop drives collagen remodeling, tumor invasion, and metastasis and could be an important target against pancreatic cancer spread.

Automatic profiling of cell morphology is a powerful tool for inferring cell function. However, this technique retains a high barrier to entry. In particular, configuring image processing parameters for optimal cell profiling is susceptible to cognitive biases and dependent on user experience. Here, we use interactive machine learning to identify the optimum cell profiling configuration that maximises quality of the cell profiling outcome. The process is guided by the user, from whom a rating of the quality of a cell profiling configuration is obtained. We use Bayesian optimisation, an established machine learning algorithm, to learn from this information and automatically recommend the next configuration to examine with the aim to maximize the quality of the processing or analysis. Compared to existing interactive machine learning tools that require domain expertise for per-class or per-pixel annotations, we rely on users explicit assessment of output quality of the cell profiling task at hand. We validated our interactive approach against the standard human trial-and-error scheme to optimise an object segmentation task using the standard software CellProfiler. Our toolkit enabled rapid optimisation of an object segmentation pipeline, increasing the quality of object segmentation over a pipeline optimised through trial-and-error. Users also attested to the ease of use and reduced cognitive load enabled by our machine learning strategy over the standard approach. We envision that our interactive machine learning approach can enhance the quality and efficiency of pipeline optimisation to democratise image-based cell profiling.

Biophysical cues robustly direct cell responses and are thus important tools for in vitro and translational biomedical applications. High throughput platforms exploring substrates with varying physical properties are therefore valuable. However, currently existing platforms are limited in throughput, the biomaterials used, the capability to segregate between different cues and the assessment of dynamic responses. Here we present a multiwell array (3 × 8) made of a substrate engineered to present topography or rigidity cues welded to a bottomless plate with a 96-well format. Both the patterns on the engineered substrate and the well plate format can be easily customized, permitting systematic and efficient screening of biophysical cues. To demonstrate the broad range of possible biophysical cues examinable, we designed and tested three multiwell arrays to influence cardiomyocyte, chondrocyte and osteoblast function. Using the multiwell array, we were able to measure different cell functionalities using analytical modalities such as live microscopy, qPCR and immunofluorescence. We observed that grooves (5 μm in size) induced less variation in contractile function of cardiomyocytes. Compared to unpatterned plastic, nanopillars with 127 nm height, 100 nm diameter and 300 nm pitch enhanced matrix deposition, chondrogenic gene expression and chondrogenic maintenance. High aspect ratio pillars with an elastic shear modulus of 16 kPa mimicking the matrix found in early stages of bone development improved osteogenic gene expression compared to stiff plastic. We envisage that our bespoke multiwell array will accelerate the discovery of relevant biophysical cues through improved throughput and variety.

Cells respond in complex ways to topographies, making it challenging to identify a direct relationship between surface topography and cell response. A key problem is the lack of informative representations of topographical parameters that translate directly into biological properties. Here, we present a platform to relate the effects of nanotopography on morphology to function. This platform utilizes the ‘morphome’, a multivariate dataset containing single cell measures of focal adhesions, the cytoskeleton, and chromatin. We demonstrate that nanotopography-induced changes in cell phenotype (both morphological and functional) are uniquely encoded by the morphome. The morphome was used to create a Bayesian linear regression model that robustly predicted changes in bone, cartilage, muscle and fibrous gene expression induced by nanotopography. Furthermore, the morphome effectively predicted nanotopography-induced gene expression within a complex co-culture microenvironment. The spatial, morphological and functional resolution of the morphome uncovered previously unknown effects of nanotopography on selectively altering cell-cell interaction and osteogenesis at the single cell level. Thus, the morphome confers the ability to quantify phenotype arising from cell-material interactions. Our new platform shows promise for rapidly assessing novel surface-patterned biomaterials for tissue regeneration and enables cell function-oriented exploration of new topographies.

The high mortality of pancreatic cancer demands that new therapeutic avenues be developed. The orally available small-molecule inhibitor AT13148 potently inhibits ROCK1 and ROCK2 kinases that regulate the actomyosin cytoskeleton. We previously reported that ROCK kinase expression increases with human and mouse pancreatic cancer progression and that conditional ROCK activation accelerates mortality in a genetically modified LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre; (KPC) mouse pancreatic cancer model. In this study, we show that treatment of KPC mouse and human TKCC5 patient-derived pancreatic tumor cells with AT13148, as well as the ROCK-selective inhibitors Y27632 and H1152, act comparably in blocking ROCK substrate phosphorylation. AT13148, Y27632, and H1152 induced morphologic changes and reduced cellular contractile force generation, motility on pliable discontinuous substrates, and three-dimensional collagen matrix invasion. AT13148 treatment reduced subcutaneous tumor growth and blocked invasion of healthy pancreatic tissue by KPC tumor cells in vivo without affecting proliferation, suggesting a role for local tissue invasion as a contributor to primary tumor growth. These results suggest that AT13148 has antitumor properties that may be beneficial in combination therapies or in the adjuvant setting to reduce pancreatic cancer cell invasion and slow primary tumor growth. AT13148 might also have the additional benefit of enabling tumor resection by maintaining separation between tumor and healthy tissue boundaries. Significance: Preclinical evaluation of a small-molecule ROCK inhibitor reveals significant effects on PDAC invasion and tumor growth, further validating ROCK kinases as viable therapeutic targets in pancreatic cancer. Cancer Res; 78(12); 3321–36. ©2018 AACR.

Pancreatic ductal adenocarcinoma is one of the most invasive and metastatic cancers and has a dismal 5-year survival rate. Here we show that N-WASP is required for the metastatic process, with roles in both chemotaxis, steering cells out of the tumour, and matrix remodelling, allowing them to escape. Lysophosphatidic acid, a signalling lipid abundant in blood and ascites fluid, is both a mitogen and chemoattractant for PDAC cells. We find they efficiently break LPA down as they respond to it, setting up a self-generated gradient that directs cells out of the tumour. N-WASP depleted cells are unable to respond to LPA gradients and show altered RhoA activation, leading to a loss of cell contractility and traction forces, and reduced metastasis in vitro and in vivo. N-WASP couples LPA receptor signalling to RhoA via the endocytic adapter SNX18, and promotes sensitivity by recycling the receptor back to the surface. Coordinated by N-WASP, the LPA-LPAR signalling loop promotes RhoA-mediated contractility and force generation. Perturbing this pathway chemically or by CRISPR deletion causes PDAC cells to lose their ability to invade through complex 3D environments or peritoneal explants, and remodel fibrillar collagen. We thus reveal N-WASP as a central controller of a chemotactic loop between PDAC cells and microenvironmental conditions that drives metastasis.

Interfacial polyelectrolyte (polyion) complexation (IPC) is a process whereby fibers and capsules are formed through interactions at the interface of oppositely charged polymers. Since its discovery in the late 1990s, the IPC fiber process, in particular, has been investigated for various applications such as tissue engineering, drug delivery, flexible electronics and biosensing. The advent of the IPC fiber and process has been supported by its unique mechanism of formation that makes it amenable to encapsulation and functionalization. In this first review on IPC fibers, we consolidate the current knowledge of the IPC process, mechanism of fiber formation and fiber physical properties, while documenting the various polycation–polyanion pairs and encapsulants that have been used to date. We review the rapidly accumulating literature on IPC fibers for tissue engineering, describing how they have been used to release protein factors in a sustained manner, made into random or spatially well-defined scaffolds and decorated with appropriate functionalities and extracellular matrices in order to tailor the microenvironment for cell growth and function.

Microvascular surgery is becoming a prevalent surgical practice. Replantation surgeries, hand reconstruction, orthopedic and free tissue transfer procedures all rely on microvascular surgery for the repair of venous and arterial defects at the millimeter and sub-millimeter levels. Often, a vascular graft is required for the procedure as a means to bridge the gap between native arteries. While autologous vessels are desired for their bioactivity and non-thrombogenicity, the tedious harvest process, lack of availability and caliber or mechanical mismatch contribute to graft failure. Thus, there is a need for an off-the-shelf artificial vascular graft that has low thrombogenic properties and mechanical properties matching those of sub-millimeter vessels. Poly(vinyl alcohol) hydrogel (PVA) has excellent prospects as a vascular graft due to its bioinertness, low thrombogenicity, high water content and tunable mechanical properties. Here, we fabricated PVA grafts with sub-millimeter diameter and mechanical properties that closely approximated those of the rabbit femoral artery. In vitro platelet adhesion and microparticle release assay verified the low thrombogenicity of PVA. A stringent proof of concept in vivo test was performed by implanting PVA grafts in rabbit femoral artery with multi-level arterial occlusion. Laser Doppler measurements indicated the improved perfusion of the distal limb after implantation of the PVA grafts. Moreover, ultrasound Doppler and angiography verified that the sub-millimeter diameter PVA vascular grafts remained patent for two weeks without the aid of anti-coagulant or anti-thrombotics. Endothelial cells were observed in the luminal surface of one patent PVA graft. The advantageous non-thrombogenic and tunable mechanical properties of PVA that are retained even in sub-millimeter diameter dimensions support the application of this biomaterial for vascular replacement in microvascular surgery.

International audience; Poly(vinyl alcohol) hydrogel (PVA) is a widely used material for biomedical devices, yet there is a need to enhance its biological functionality for in vitro and in vivo vascular application. Significance of surface topography in modulating cellular behaviour is increasingly evident. However, hydrogel patterning remains challenging. Using a casting method, planar PVA were patterned with micro-sized features. To achieve higher patterning resolution, nanoimprint lithography with high pressure and temperature was used. In vitro experiment showed enhanced human endothelial cell (EC) density and adhesion on patterned PVA. Additional chemical modification via nitrogen gas plasma on patterned PVA further improved EC density and adhesion. Only EC monolayer grown on plasma modified PVA with 2 mm gratings and 1.8 mm concave lens exhibited expression of vascular endothelial cadherin, indicating EC functionality. Patterning of the luminal surface of tubular hydrogels is not widely explored. The study presents the first method for simultaneous tubular molding and luminal surface patterning of hydrogel. PVA graft with 2 mm gratings showed patency and endothelialization, while unpatterned grafts were occluded after 20 days in rat aorta. The reproducible, high yield and high-fidelity methods enable planar and tubular patterning of PVA and other hydrogels to be used for biomedical applications.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Hydrogels are highly preferred in soft tissue engineering because they recapitulate the hydrated extracellular matrix. Naturally derived polysaccharides, like pullulan and dextran, are attractive materials with which to form hydrophilic polymeric networks due to their non-immunogenic and non-antigenic properties. However, their inherent hydrophilicity prevents adherent cell growth. In this study, we modified pullulan–dextran scaffolds with interfacial polyelectrolyte complexation (IPC) fibers to improve their ability to support adherent cell growth. We showed that the pullulan–dextran–IPC fiber composite scaffold laden with extracellular matrix protein has improved cell adhesion and proliferation compared to the plain polysaccharide scaffold. We also demonstrated the zero-order release kinetics of the biologics bovine serum albumin and vascular endothelial growth factor (VEGF) incorporated in the composite scaffold. Lastly, we showed that the VEGF released from the composite scaffold retained its capacity to stimulate endothelial cell growth. The incorporation of IPC fibers in the pullulan–dextran hydrogel scaffold improved its functionality and biological activity, thus enhancing its potential in tissue engineering applications.