CELL FORCE MEASUREMENT USING MEMS-BASED MICROPILLAR ARRAYS

Focal contacts act as mechanosensors allowing cells to respond to their biomechanical environment. Force transmission through newly formed contact sites is a highly dynamic process requiring a stable link between the intracellular cytoskeleton and the extracellular environment. To simultaneously investigate cellular traction forces in several individual maturing adhesion sites within the same cell, we established a custom-built multiple trap optical tweezers setup. Beads functionalized with fibronectin or RGD-peptides were placed onto the apical surface of a cell and trapped with a maximum force of 160 pN. Cells form adhesion contacts around the beads as demonstrated by vinculin accumulation and start to apply traction forces after 30 seconds. Force transmission was found to strongly depend on bead size, surface density of integrin ligands and bead location on the cell surface. Highest traction forces were measured for beads positioned on the leading edge. For mouse embryonic fibroblasts, traction forces acting on single beads are in the range of 80 pN after 5 minutes. If two beads were positioned parallel to the leading edge and with a center-to-center distance less than 10 µm, traction forces acting on single beads were reduced by 40%. This indicates a spatial and temporal coordination of force development in closely related adhesion sites. We also used our setup to compare traction forces, retrograde transport velocities, and migration velocities between two cell lines (mouse melanoma and fibroblasts) and primary chick fibroblasts. We find that maximal force development differs considerably between the three cell types with the primary cells being the strongest. In addition, we observe a linear relation between force and retrograde transport velocity: a high retrograde transport velocity is associated with strong cellular traction forces. In contrast, migration velocity is inversely related to traction forces and retrograde transport velocity.

Silicon is the primary material of integrated circuit (IC) manufacturing in microelectronic industry. It has high thermal conductivity and superior thermomechanical properties compatible to most semiconductors. These characteristics make it an ideal material for fabricating micro/mini heat pipes and their wick structures. In this article, silicon wick structures, composed of cylindrical pillars 320 μm in height and 30–100 μm in diameter, are developed for studies of phase change capability. Fabrication of the silicon wick structures utilizes the standard microelectromechanical systems (MEMS) approach, which allows the precise definition on the wick dimensions, as well as the heated wick area. On these bases, experimental characterizations of temperature variations versus input heat fluxes, associated with simultaneous visualization on the liquid transport and the dryout, are performed to investigate the wick dimensional effects on the maximum phase change capability. On the wick structure with the pillar diameter/pores of 100 μm and a heated wick area of 2 mm × 2 mm, the phase change reached a maximum heat flux of 1130 W/cm2. Despite of the liquid bottom-feed approach, interactions between liquid and vapor phases enables the heated wick structure absorb liquid from its surrounding wick area, including from its top side with a longer liquid transport path. In contrast, a wick structure with fine pillars (10 μm in diameter) inhibited the generation of nucleate boiling. Evaporation on the meniscus interface becomes the major phase change mechanism. A large heated wick area (4 mm × 4 mm) increases the viscous loss in transporting liquid to wet the entire wick, advancing the dryout at 135 W/cm2. Mass transfer analysis, as well as discussion of the experimental results, indicates that a dimensional ratio r/l (pillar diameter/characteristic length of the heated wick area) is a key parameter in determining the maximum phase change capability. A low r/l ratio enhances heat and mass transport capability, as well as heat transfer coefficient.

High-throughput fabrication of graphically encoded hydrogel microparticles is achieved by combining flow contact lithography in a multichannel microfluidic device and a high capacity 25 mm LED UV source. Production rates of chemically homogeneous particles are improved by two orders of magnitude. Additionally, the custom-built contact lithography instrument provides an affordable solution for patterning complex microstructures on surfaces.

High-throughput measurement of cellular traction forces at the nanoscale remains a significant challenge in mechanobiology, limiting our understanding of how cells interact with their microenvironment. Here, we present a novel technique for fabricating protein nanopatterns in standard multiwell microplate formats (96/384-wells), enabling the high-throughput quantification of cellular forces using DNA tension gauge tethers (TGTs) amplified by CRISPR-Cas12a. Our method employs sparse colloidal lithography to create nanopatterned surfaces with feature sizes ranging from sub 100 to 800 nm on transparent, planar, and fully PEGylated substrates. These surfaces allow for the orthogonal immobilization of two different proteins or biomolecules using click-chemistry, providing precise spatial control over cellular signaling cues. We demonstrate the robustness and versatility of this platform through imaging techniques, including total internal reflection fluorescence microscopy, confocal laser scanning microscopy, and high-throughput imaging. Applying this technology, we measured the early stage mechanical forces exerted by 3T3 fibroblasts across different nanoscale features, detecting forces ranging from 12 to 56 pN. By integrating the Mechano-Cas12a Assisted Tension Sensor (MCATS) system, we achieved rapid and high-throughput quantification of cellular traction forces, analyzing over 2 million cells within minutes. Our findings reveal that nanoscale clustering of integrin ligands significantly influences the mechanical responses of cells. This platform offers a powerful tool for mechanobiology research, facilitating the study of cellular forces and mechanotransduction pathways in a high-throughput manner compatible with standard cell culture systems.

Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell-cell and cell-matrix interactions. The maximal cell-cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell-matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.

Precise determination of cellular traction forces has important significance in assessing cellular mechanical characteristic on micro/nano scale. Elastic substrate method is a useful way to study cellular traction forces, in which the cells are cultured on elastic gel substrate marked by randomly embedding fluorescent microbeads and they exert mechanical forces on the film to cause deformation of the substrate material. In this paper, we investigate the acquisition of deformation field induced by single cardiac myocyte by using digital image correlation (DIC) technique. In view of the fact that cellular force restoration is essentially an ill-posed inverse problem, which implies that the force reconstruction is susceptible to the input displacement noise, we develop a novel optimal filtering scheme in two-dimensional Fourier space to restrain displacement noise amplification. Experiments of traction force recovery for a real cardiac myocyte indicate the optimal scheme in combination with the DIC method enables us to reconstruct cellular traction fields with high accuracy.

3-D culture has been shown to provide cells with a more physiologically authentic environment than traditional 2-D (planar) culture [1, 2]. 3-D cues allow cells to exhibit more realistic functions and behaviors, e.g., adhesion, spreading, migration, metabolic activity, and differentiation. Knowledge of changes in cell morphology, mechanics, and mobility in response to geometrical cues and topological stimuli is important for understanding normal and pathological cell development [3]. Microfabrication provides unique in vitro approaches to recapitulating in vivo conditions due to the ability to precisely control the cellular microenvironment [4, 5]. Microwell arrays have emerged as robust alternatives to traditional 2D cell culture substrates as they are relatively simple and compatible with existing laboratory techniques and instrumentation [6, 7]. In particular, microwells have been adopted as a biomimetic approach to modeling the unique micro-architecture of the epithelial lining of the gastrointestinal (GI) tract [8–10]. The inner (lumen-facing) surface of the intestine has a convoluted topography consisting of finger-like projections (villi) with deep well-like invaginations (crypts) between them. The dimensions of villi and crypts are on the order of hundreds of microns (100–700 μm in height and 50–250 μm in diameter) [11]. While microwells have proven important in the development of physiologically realistic in vitro models of human intestine, existing methods of ensuring their surface is suitable for cell culture are lacking. Sometimes it is desirable to selectively seed cells within microwells and confine or restrict them to the microwells in which they are seeded. Existing methods of patterning microwells for cell attachment either lack selectivity, meaning cells can adhere and migrate anywhere on the microwell array, i.e., inside microwells or outside of them, or necessitate sophisticated techniques such as micro-contact printing, which requires precise alignment and control to selectively pattern the bottoms of microwells for cell attachment [12, 13].

Conventional in vitro physiological models, relying on animal studies and 2D/3D cell cultures, are fundamentally limited by interspecies biological discrepancies, ethical constraints, or inadequate replication of human physiology. Organ-on-a-chip (OoC) technology overcomes these challenges through emulating organ-specific microphysiological systems. The transformative power of this innovation lies in multi-channel microfluidic chips. These chips facilitate the formation of 3D cellular organizations and tissue interfaces via integrated porous membranes, micropillar arrays, or perfusable vascular microchannels, simultaneously allowing for precise and dynamic modulation of chemical, biological, and physical factors. Rapid technological evolution has yielded functional models of lung alveoli, the blood-brain barrier, cardiac tissues, etc., thereby advancing drug testing and disease modeling accuracy. This review systematically examines the development of OoC technology through the lens of multi-channel microfluidics by focusing on four pivotal domains: (1) the biomimetic design overview for OoCs, (2) fabrication methods including soft lithography and 3D printing, (3) applications in pathophysiological investigations, preclinical drug evaluation, and toxicological assessment, and (4) current challenges and perspectives in structural design, materials and fabrication, biological applications, and other development directions. This review is intended to provide a reference for the technological iteration and interdisciplinary application of multi-channel microfluidic chip systems.

The influence of low-energy helium plasma on bubble formation in micro-engineered tungsten

Cellular traction forces are known to play an important role in the interactions between cells and their substrates. Although several studies have estimated traction forces using microfabrication techniques, little is known of how intracellular structures contribute to traction forces. In this study, microfabricated substrates with different arrays of microposts were used to estimate traction forces of smooth muscle cells, particularly exploring the contribution of cytoskeletal structures to traction forces. Smooth muscle cells isolated from bovine aortas were transfected with fluorescent protein conjugates, and then plated on an elastomer substrate with arrays of microposts (3 mum in diameter, 10 mum in height, 6 or 10 mum in spacing). The total area of the microposts arrays was designed to be 30 mum times 30 mum to control cell area. A BrdU incorporation assay revealed that cell proliferation significantly decreased when the spacing of microposts was increased from 6 to 10 mum. A significant increase in traction forces from 15.2 plusmn 1.4 to 22.4 plusmn 1.8 nN was also found by increasing the spacing of the microposts, although less organization of stress fibers was observed. This result indicates that, for the 10 mun spacing, actin-myosin interactions may be more activated, producing higher forces per microposts. Moreover, when microtubules were disrupted, using 10 mug/ml nocodazole, a 30% increase in traction forces was observed in cells plated on both sets of microposts. Therefore, this study proposes that the contraction forces of stress fibers are mainly involved in cellular traction forces, and that microtubules, directly or indirectly, contribute to the contraction forces of stress fibers

Roots of stunted broccoli plants (Brassica oleracea) from the Palo Verde Valley, CA, were observed at various stages of decay. A species of Pythium with large spiny oogonia was microscopically observed and consistently isolated from decayed roots. Isolates produced spherical, intercalary sporangia (average 34.5 μm in diameter) and aplerotic oospores (average 37.3 μm in diameter) in oogonia (average 47.4 μm in diameter) bearing numerous conical spines (average 8.5 μm in height and 5.5 μm basal width) with blunt apices. A representative broccoli isolate (P1) had a 99% internal transcribed spacer (ITS) sequence similarity with Pythium jasmonium (nom. inval., GenBank AF216654.1), a species which has not been formally described. Three other accessions in GenBank also carry the specific epithet of P. jasmonium and were originally isolated from diseased plants in the family Brassicaceae. In our study, these isolates were pathogenic on broccoli and morphologically similar to P1. P1 was pathogenic to 10 cultivated and 12 wild plants in the family Brassicaceae but not to 18 species of cultivated plants belonging to nine other plant families. Mycelial growth of our isolates occurred between <10 and 35°C, with an optimum of 25°C (maximum growth rate 25 mm/day). Our broccoli isolates are related to other species in subclade B of clade J. Members of subclade B include P. mastophorum, P. uncinulatum, P. buismaniae, P. polymastum, and P. megalacanthum. However, the broccoli isolates, in addition to all those in GenBank that carry the specific epithet of P. jasmonium, possess unique ITS, β-tubulin, and cox1 sequences that are sufficiently different from other species in subclade B to justify status as a new species. We propose that isolates previously designated as P. jasmonium (nom. inval.) be renamed as P. brassicum sp. nov. based on their apparent plant family-specific pathogenicity.

The movement of collective cells is affected through changes in physical interactions of cells in response to external mechanical stimuli, including fluid flow. Most tissues are affected by fluid flow at the interstitial level, but few studies have investigated the physical effects in collective cells affected by a low flow rate. In this study, collective cell migration of Madin–Darby canine kidney (MDCK) epithelial cells was investigated under static or interstitial flow (0, 0.1, and 1 μL/min) using a traction microfluidic device. The optimization of calculation of cellular traction forces was first achieved by changing interrogation window size from the fluorescent bead images. Migration analysis of cell collectives patterned with a 700 μm circular shape reveals that cells under the slow flow (0.1 and 1 μL/min) showed the inhibitory migration by decreasing cell island size and cellular speed compared to that of static condition. Analysis of cellular forces shows that level of traction forces was lower in the slow flow condition (~20 Pa) compared to that of static condition (~50 Pa). Interestingly, the standard deviation of traction force of cells was dramatically decreased as the flow rate increased from 0 to 1 μL/min, which indicates that flow affects the distribution of cellular traction forces among cell collectives. Cellular tension was increased by 50% in the cells under the fluid flow rate of 1 μL/min. Treatment of calcium blocker increased the migratory speed of cells under the flow condition, whereas there is little change of cellular forces. In conclusion, it has been shown that the interstitial flow inhibited the collective movement of epithelial cells by decreasing and re-distributing cellular forces. These findings provide insights into the study of the effect of interstitial flow on cellular behavior, such as development, regeneration, and morphogenesis.

A 193-nm ArF excimer laser was used to induce the photodissociation of Si–O bonds of silicone rubber in order to fabricate a periodic micro/nano-suction cup silicone structure, approximately 1 μm in diameter and 2 μm in height at regular intervals of 2.5 μm. The laser was focused on Al-coated silicone rubber by each silica glass microsphere 2.5 μm in diameter, which covered the entire surface of the silicone rubber. The silicone rubber underneath each microsphere photochemically swelled after laser-ablating the coated Al to limit the diameter of the swelling. Simultaneously, the coated Al was able to adjust the focal point to the surface of the silicone rubber to form a hole approximately 500 nm in diameter, centered at the swollen silicone. The dependences of the thickness of the coated-Al and the laser pulse number are discussed, based on the observations of a scanning electron microscope (SEM) and an atomic force microscope (AFM). The superhydrophobic property of the fabricated micro/nano-suction cup structure was successfully found.

SUMMARYA new rhodymeniacean species, Chamaebotrys prolifera, is described from a shallow water habitat in Puerto Rico, representing the first occurrence of the genus in the Atlantic Ocean. Plants, to 5 cm across, are decumbent and comprised of compressed vesicles that are originally proliferously branched at the perimeter. Older vesicles become branched from their dorsal surfaces as well. Branches are septate at their origin and become irregularly shaped with age. Anastomoses between adjacent vesicles is common. Individual vesicles measure to 15 mm in broadest dimension. Vesicle walls consist of two layers of medullary cells and two layers of cortical cells. Tetrasporangia, which occur in diffuse nemathecia, are spherical, to 30 µm in diameter and are cut off terminally from an inner cortical cell. Cystocarps are hemispherical measuring to 800 µm in diameter and 350 µm in height. Spermatangia are apparently cut off randomly from outer cortical cells across the thallus surface. Molecular evidence confirms placement of Chamaebotrys within the Rhodymeniaceae.

In article number 1806380, Benjamin D. Almquist and co-workers describe a bioinspired nanotechnology platform that uses the cellular traction force as a trigger for controlling the release of growth factors. This method of release is straightforward to integrate with diverse biomaterial scaffolds, substrates, and fabrication methods, and enables novel capabilities like the selective activation of growth factors by different cell types.