ß-glycerophosphate, not low magnitude fluid shear stress, increases osteocytogenesis in the osteoblast-to-osteocyte cell line IDG-SW3.

AIM: As osteoblasts deposit a mineralized collagen network, a subpopulation of these cells differentiates into osteocytes. Biochemical and mechanical stimuli, particularly fluid shear stress (FSS), are thought to regulate this, but their relative influence remains unclear. Here, we assess both biochemical and mechanical stimuli on long-term bone formation and osteocytogenesis using the osteoblast-osteocyte cell line IDG-SW3. METHODS: Due to the relative novelty and uncommon culture conditions of IDG-SW3 versus other osteoblast-lineage cell lines, effects of temperature and media formulation on matrix deposition and osteocytogenesis were initially characterized. Subsequently, the relative influence of biochemical (ß-glycerophosphate (ßGP) and ascorbic acid 2-phosphate (AA2P)) and mechanical stimulation on osteocytogenesis was compared, with intermittent application of low magnitude FSS generated by see-saw rocker. RESULTS: ßGP and AA2P supplementation were required for mineralization and osteocytogenesis, with 33°C cultures retaining a more osteoblastic phenotype and 37°C cultures undergoing significantly higher osteocytogenesis. ßGP concentration positively correlated with calcium deposition, whilst AA2P stimulated alkaline phosphatase (ALP) activity and collagen deposition. We demonstrate that increasing ßGP concentration also significantly enhances osteocytogenesis as quantified by the expression of green fluorescent protein linked to Dmp1. Intermittent FSS (~0.06 Pa) rocker had no effect on osteocytogenesis and matrix deposition. CONCLUSIONS: This work demonstrates the suitability and ease with which IDG-SW3 can be utilized in osteocytogenesis studies. IDG-SW3 mineralization was only mediated through biochemical stimuli with no detectable effect of low magnitude FSS. Osteocytogenesis of IDG-SW3 primarily occurred in mineralized areas, further demonstrating the role mineralization of the bone extracellular matrix has in osteocyte differentiation.

Membrane-based microfluidic systems for medical and biological applications.

Microfluidic devices with integrated membranes that enable control of mass transport in constrained environments have shown considerable growth over the last decade. Membranes are a key component in several industrial processes such as chemical, pharmaceutical, biotechnological, food, and metallurgy separation processes as well as waste management applications, allowing for modular and compact systems. Moreover, the miniaturization of a process through microfluidic devices leads to process intensification together with reagents, waste and cost reduction, and energy and space savings. The combination of membrane technology and microfluidic devices allows therefore magnification of their respective advantages, providing more valuable solutions not only for industrial processes but also for reproducing biological processes. This review focuses on membrane-based microfluidic devices for biomedical science with an emphasis on microfluidic artificial organs and organs-on-chip. We provide the basic concepts of membrane technology and the laws governing mass transport. The role of the membrane in biomedical microfluidic devices, along with the required properties, available materials, and current challenges are summarized. We believe that the present review may be a starting point and a resource for researchers who aim to replicate a biological phenomenon on-chip by applying membrane technology, for moving forward the biomedical applications.

Assessing friction and damage of cell monolayers on soft substrates in vitro.

In the area of surgical applications, understanding the interaction between medical device materials and tissue is important since this interaction may cause complications. The interaction often consists of a cell monolayer touching the medical device that can be mimicked in vitro. Prominent examples of this are contact lenses, where epithelial cells interact with the contact lens, or stents and catheters, which are in contact with endothelial cells. To investigate those interactions, in previous studies, expensive microtribometers were used to avoid pressures in the contact area far beyond physiologically relevant levels. Here, we aim to present a new methodology that is cost- and time-efficient, more accessible than those used previously and allows for the application of more realistic pressures, while permitting a quantification of the damage caused to the monolayer. For this, a soft polydimethylsiloxane is employed that better mimics the mechanical properties of blood vessels than materials used in other studies. Furthermore, a technique to account for misalignments within the experiment set-up is presented. This is carried out using the raw spatial and force data recorded by the tribometer and adjusting for misalignments. The methodology is demonstrated using an endothelial cell (human umbilical vein endothelial cells) monolayer.

Corrigendum: 3D organ-on-a-chip: the convergence of microphysiological systems and organoids.

[This corrects the article DOI: 10.3389/fcell.2022.1043117.].

Corrigendum: Challenge of material haemocompatibility for microfluidic blood-contacting applications.

[This corrects the article DOI: 10.3389/fbioe.2023.1249753.].

Challenge of material haemocompatibility for microfluidic blood-contacting applications.

Biological applications of microfluidics technology is beginning to expand beyond the original focus of diagnostics, analytics and organ-on-chip devices. There is a growing interest in the development of microfluidic devices for therapeutic treatments, such as extra-corporeal haemodialysis and oxygenation. However, the great potential in this area comes with great challenges. Haemocompatibility of materials has long been a concern for blood-contacting medical devices, and microfluidic devices are no exception. The small channel size, high surface area to volume ratio and dynamic conditions integral to microchannels contribute to the blood-material interactions. This review will begin by describing features of microfluidic technology with a focus on blood-contacting applications. Material haemocompatibility will be discussed in the context of interactions with blood components, from the initial absorption of plasma proteins to the activation of cells and factors, and the contribution of these interactions to the coagulation cascade and thrombogenesis. Reference will be made to the testing requirements for medical devices in contact with blood, set out by International Standards in ISO 10993-4. Finally, we will review the techniques for improving microfluidic channel haemocompatibility through material surface modifications-including bioactive and biopassive coatings-and future directions.

3D organ-on-a-chip: The convergence of microphysiological systems and organoids.

Medicine today faces the combined challenge of an increasing number of untreatable diseases and fewer drugs reaching the clinic. While pharmaceutical companies have increased the number of drugs in early development and entering phase I of clinical trials, fewer actually successfully pass phase III and launch into the market. In fact, only 1 out of every 9 drugs entering phase I will launch. In vitro preclinical tests are used to predict earlier and better the potential of new drugs and thus avoid expensive clinical trial phases. The most recent developments favor 3D cell culture and human stem cell biology. These 3D humanized models known as organoids better mimic the 3D tissue architecture and physiological cell behavior of healthy and disease models, but face critical issues in production such as small-scale batches, greater costs (when compared to monolayer cultures) and reproducibility. To become the gold standard and most relevant biological model for drug discovery and development, organoid technology needs to integrate biological culture processes with advanced microtechnologies, such as microphysiological systems based on microfluidics technology. Microphysiological systems, known as organ-on-a-chip, mimic physiological conditions better than conventional cell culture models since they can emulate perfusion, mechanical and other parameters crucial for tissue and organ physiology. In addition, they reduce labor cost and human error by supporting automated operation and reduce reagent use in miniaturized culture systems. There is thus a clear advantage in combining organoid culture with microsystems for drug development. The main objective of this review is to address the recent advances in organoids and microphysiological systems highlighting crucial technologies for reaching a synergistic strategy, including bioprinting.

Design and Evaluation of an Osteogenesis-on-a-Chip Microfluidic Device Incorporating 3D Cell Culture.

Microfluidic-based tissue-on-a-chip devices have generated significant research interest for biomedical applications, such as pharmaceutical development, as they can be used for small volume, high throughput studies on the effects of therapeutics on tissue-mimics. Tissue-on-a-chip devices are evolving from basic 2D cell cultures incorporated into microfluidic devices to complex 3D approaches, with modern designs aimed at recapitulating the dynamic and mechanical environment of the native tissue. Thus far, most tissue-on-a-chip research has concentrated on organs involved with drug uptake, metabolism and removal (e.g., lung, skin, liver, and kidney); however, models of the drug metabolite target organs will be essential to provide information on therapeutic efficacy. Here, we develop an osteogenesis-on-a-chip device that comprises a 3D environment and fluid shear stresses, both important features of bone. This inexpensive, easy-to-fabricate system based on a polymerized High Internal Phase Emulsion (polyHIPE) supports proliferation, differentiation and extracellular matrix production of human embryonic stem cell-derived mesenchymal progenitor cells (hES-MPs) over extended time periods (up to 21 days). Cells respond positively to both chemical and mechanical stimulation of osteogenesis, with an intermittent flow profile containing rest periods strongly promoting differentiation and matrix formation in comparison to static and continuous flow. Flow and shear stresses were modeled using computational fluid dynamics. Primary cilia were detectable on cells within the device channels demonstrating that this mechanosensory organelle is present in the complex 3D culture environment. In summary, this device aids the development of 'next-generation' tools for investigating novel therapeutics for bone in comparison with standard laboratory and animal testing.

Thermoplastic elastomer with advanced hydrophilization and bonding performances for rapid (30 s) and easy molding of microfluidic devices.

One of the most important areas of research on microfluidic technologies focuses on the identification and characterisation of novel materials with enhanced properties and versatility. Here we present a fast, easy and inexpensive microstructuration method for the fabrication of novel, flexible, transparent and biocompatible microfluidic devices. Using a simple hot press, we demonstrate the rapid (30 s) production of various microfluidic prototypes embossed in a commercially available soft thermoplastic elastomer (sTPE). This styrenic block copolymer (BCP) material is as flexible as PDMS and as thermoformable as classical thermoplastics. It exhibits high fidelity of replication using SU-8 and epoxy master molds in a highly convenient low-isobar (0.4 bar) and iso-thermal process. Microfluidic devices can then be easily sealed using either a simple hot plate or even a room-temperature assembly, allowing them to sustain liquid pressures of 2 and 0.6 bar, respectively. The excellent sorption and biocompatibility properties of the microchips were validated via a standard rhodamine dye assay as well as a sensitive yeast cell-based assay. The morphology and composition of the surface area after plasma treatment for hydrophilization purposes are stable and show constant and homogenous distribution of block nanodomains (∼22° after 4 days). These domains, which are evenly distributed on the nanoscale, therefore account for the uniform and convenient surface of a "microfluidic scale device". To our knowledge, this is the first thermoplastic elastomer material that can be used for fast and reliable fabrication and assembly of microdevices while maintaining a high and stable hydrophilicity.

Microfluidic traction force microscopy to study mechanotransduction in angiogenesis.

The formation of new blood vessels from existing vasculature, angiogenesis, is driven by coordinated endothelial cell migration and matrix remodeling in response to local signals. Recently, a growing body of evidence has shown that mechanotransduction, along with chemotransduction, is a major regulator of angiogenesis. Mechanical signals, such as fluid shear stress and substrate mechanics, influence sprouting and network formation, but the mechanisms behind this relationship are still unclear. Here, we present cellular traction forces as possible effectors activated by mechanosensing to mediate matrix remodeling, and encourage the use of TFM to study mechanotransduction in angiogenesis. We also suggest that deciphering the response of EC to mechanical signals could reveal an optimal angiogenic mechanical environment, and provide insight into development, wound healing, the initiation and growth of tumors, and new strategies for tissue engineering.

In Vitro Bone Cell Models: Impact of Fluid Shear Stress on Bone Formation.

This review describes the role of bone cells and their surrounding matrix in maintaining bone strength through the process of bone remodeling. Subsequently, this work focusses on how bone formation is guided by mechanical forces and fluid shear stress in particular. It has been demonstrated that mechanical stimulation is an important regulator of bone metabolism. Shear stress generated by interstitial fluid flow in the lacunar-canalicular network influences maintenance and healing of bone tissue. Fluid flow is primarily caused by compressive loading of bone as a result of physical activity. Changes in loading, e.g., due to extended periods of bed rest or microgravity in space are associated with altered bone remodeling and formation in vivo. In vitro, it has been reported that bone cells respond to fluid shear stress by releasing osteogenic signaling factors, such as nitric oxide, and prostaglandins. This work focusses on the application of in vitro models to study the effects of fluid flow on bone cell signaling, collagen deposition, and matrix mineralization. Particular attention is given to in vitro set-ups, which allow long-term cell culture and the application of low fluid shear stress. In addition, this review explores what mechanisms influence the orientation of collagen fibers, which determine the anisotropic properties of bone. A better understanding of these mechanisms could facilitate the design of improved tissue-engineered bone implants or more effective bone disease models.

Traction Forces of Endothelial Cells under Slow Shear Flow.

Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeostasis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow regime in which endothelial cell mechanics is acutely responsive to shear stress.

Structural finite element analysis to explain cell mechanics variability.

The ability to model the mechanical responses of different cell types presents many opportunities to tissue engineering research to further identify changes from physiological conditions to disease. Using a previously validated finite element cell model we aim to show how variation of the material properties of the intracellular components affects cell response after compression and shearing. A parametric study was performed to understand the key mechanical features from different cell types, focussing on specific cytoskeleton components and prestress. Results show that actin cortex does not have a mechanical role in resisting shearing loading conditions. The sensitivity analysis predicted that cell force to compression and shearing is highly affected by changes in cortex thickness, cortex Young's modulus and rigidity of the remaining cytoplasm. Variation of prestress affects mainly the response of cells under shear loads and the model defines a relationship between cell force and prestress depending on the specific loading conditions, which is in good agreement with in vitro experiments. The results are used to make predictions that can relate mechanical properties with cell phenotype to be used as guidelines for individual cytoskeletal structures for future modelling efforts of the structure-function relationships of living cells.

A multi-structural single cell model of force-induced interactions of cytoskeletal components.

Several computational models based on experimental techniques and theories have been proposed to describe cytoskeleton (CSK) mechanics. Tensegrity is a prominent model for force generation, but it cannot predict mechanics of individual CSK components, nor explain the discrepancies from the different single cell stimulating techniques studies combined with cytoskeleton-disruptors. A new numerical concept that defines a multi-structural 3D finite element (FE) model of a single-adherent cell is proposed to investigate the biophysical and biochemical differences of the mechanical role of each cytoskeleton component under loading. The model includes prestressed actin bundles and microtubule within cytoplasm and nucleus surrounded by the actin cortex. We performed numerical simulations of atomic force microscopy (AFM) experiments by subjecting the cell model to compressive loads. The numerical role of the CSK components was corroborated with AFM force measurements on U2OS-osteosarcoma cells and NIH-3T3 fibroblasts exposed to different cytoskeleton-disrupting drugs. Computational simulation showed that actin cortex and microtubules are the major components targeted in resisting compression. This is a new numerical tool that explains the specific role of the cortex and overcomes the difficulty of isolating this component from other networks in vitro. This illustrates that a combination of cytoskeletal structures with their own properties is necessary for a complete description of cellular mechanics.

The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells.

We have shown that the microtopography (mT) underlying colon cancer changes as a tumor de-differentiates. We distinguish the well-differentiated mT based on the increasing number of "pits" and poorly differentiated mT on the basis of increasing number of "posts." We investigated Rho A as a mechanosensing protein using mT features derived from those observed in the ECM of colon cancer. We evaluated Rho A activity in less-tumorogenic (Caco-2 E) and more tumorigenic (SW620) colon cancer cell-lines on microfabricated pits and posts at 2.5 mum diameter and 200 nm depth/height. In Caco-2 E cells, we observed a decrease in Rho A activity as well as in the ratio of G/F actin on surfaces with either pits or posts but despite this low activity, knockdown of Rho A led to a significant decrease in confined motility suggesting that while Rho A activity is reduced on these surfaces it still plays an important role in controlling cellular response to barriers. In SW620 cells, we observed that Rho A activity was greatest in cells plated on a post microtopography which led to increased cell motility, and an increase in actin cytoskeletal turnover.

Chamber and microfluidic probe for microperfusion of organotypic brain slices.

Microfluidic systems are increasingly being used for the culture and study of dissociated cells because they require only minute amounts of materials while enabling drug screening and chemotaxis studies down to the single cell level. However, the culture of organized tissue, such as brain slices, has been more difficult to adapt to microfluidic devices. Here, we present a microfluidic system, comprising (i) a perfusion chamber for the culture of organotypic slices that is compatible with high resolution imaging on inverted microscopes, and (ii) a novel transparent microfluidic probe (MFP) for the localized microperfusion of the brain tissue. The MFP is made in poly(dimethylsiloxane), features six micrometre-scale apertures and can be assembled within a few hours in a standard laboratory. Each aperture can indiscriminately be used either for the injection or aspiration of solutions, giving rise to many possible combinations. The MFP was successfully used for the perfusion of a small number of cells in a brain slice with concurrent confocal fluorescence imaging of the perfused dye and sub-cellular structures within the tissue.

The microfluidic probe: operation and use for localized surface processing.

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. In this video we present the microfluidic probe (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

Nitric oxide cytoskeletal-induced alterations reverse the endothelial progenitor cell migratory defect associated with diabetes.

Stromal-derived factor-1 (SDF-1) is a critical chemokine for endothelial progenitor cell (EPC) recruitment to areas of ischemia, allowing these cells to participate in compensatory angiogenesis. The SDF-1 receptor, CXCR4, is expressed in developing blood vessels as well as on CD34+ EPCs. We describe that picomolar and nanomolar concentrations of SDF-1 differentially influence neovascularization, inducing CD34+ cell migration and EPC tube formation. CD34+ cells isolated from diabetic patients demonstrate a marked defect in migration to SDF-1. This defect is associated, in some but not all patients, with a cell surface activity of CD26/dipeptidyl peptidase IV, an enzyme that inactivates SDF-1. Diabetic CD34+ cells also do not migrate in response to vascular endothelial growth factor and are structurally rigid. However, incubating CD34+ cells with a nitric oxide (NO) donor corrects this migration defect and corrects the cell deformability. In addition, exogenous NO alters vasodilator-stimulated phosphoprotein and mammalian-enabled distribution in EPCs. These data support a common downstream cytoskeletal alteration in diabetic CD34+ cells that is independent of growth factor receptor activation and is correctable with exogenous NO. This inability of diabetic EPCs to respond to SDF-1 may contribute to aberrant tissue vascularization and endothelial repair in diabetic patients.