a-novel-method-for-localizing-reporter-fluorescent-beads-near-cell

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

modification-colliculo-thalamocortical-mouse-brain-slice

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

University of Illinois at Urbana-Champaign

University of Illinois Urbana-Champaign

We present a method of measuring force response of a single living cell, attached to a substrate, in situ, by a functionalized microelectromechanical systems sensor that applies local deformation on the cell. The sensor is a single crystal silicon microcantilever beam with prescribed shape and geometry, and is coated by a thin layer of fibronectin. It is brought in contact with a cell to form adhesion cites, and is then moved by a piezoactuator to deform the cell locally. The force is transmitted from the adhesion site(s) on the cantilever to the sites on the substrate through the cytoskeleton. The interaction force between the cell and the cantilever is measured from the deformation of the cantilever and its spring constant, which can be obtained by several independent means. The force and the cell deformation can be 10 s of nano-Newtons and micrometers, respectively. We demonstrate the method using two families of force sensors with spring constants of 18 and 0.4 nN/microm. Several cells, endothelial and fibroblast, are deformed by tens of micrometers until the adhesion sites failed. Their force-deformation response shows strong linearity. Several possible mechanisms are discussed to explain the linear response.

Functionalized biomicroelectromechanical systems sensors for force response study at local adhesion sites of single living cells on substrates.

We report the loading and unloading force response of single living adherent fibroblasts due to large lateral indentation obtained by a two-component microelectromechanical systems force sensor. Strong hysteretic force response is observed for all the tested cells. For the loading process, the force response is linear (often with small initial non-linearity) to a deformation scale comparable to the undeformed cell size, followed by plastic yielding. In situ visualization of actin fibers by tagging with green fluorescent protein indicates that during the indentation process, actin network possibly decomposes irreversibly at discrete locations where well-defined circular actin agglomerates appear all over the cell, which explains the irreversibility of the force response. Similar agglomeration is observed when the cell is compressed laterally by a micro plate. The distribution pattern of the agglomerates strongly correlates with the arrangement of the actin fibers of the pre-indented cell. The size of the agglomerates increases with time as t(alpha), initially with alpha=2-3 followed by alpha=0.5-1. The higher growth rate suggests influx of actin into the agglomerates. The slower rate suggests a diffusive spreading, but the diffusion constant is two orders of magnitude lower than that of an actin monomer through the cytoplasm. Actin agglomeration has previously been observed due to biochemical treatment, gamma-radiation, and ischemic injury, and has been identified as a precursor to cell death. We believe this is the first evidence of actin agglomeration due to mechanical indentation/compression. The study demonstrates that living cells may initiate similar functionalities in response to dissimilar mechanical and biochemical stimuli.

Force response and actin remodeling (agglomeration) in fibroblasts due to lateral indentation.

In nanocrystalline metals, lack of intragranular dislocation sources leads to plastic deformation mechanisms that substantially differ from those in coarse-grained metals. However, irrespective of grain size, plastic deformation is considered irrecoverable. We show experimentally that plastically deformed nanocrystalline aluminum and gold films with grain sizes of 65 nanometers and 50 nanometers, respectively, recovered a substantial fraction (50 to 100%) of plastic strain after unloading. This recovery was time dependent and was expedited at higher temperatures. Furthermore, the stress-strain characteristics during the next loading remained almost unchanged when strain recovery was complete. These observations in two dissimilar face-centered cubic metals suggest that strain recovery might be characteristic of other metals with similar grain sizes and crystalline packing.

Plastic deformation recovery in freestanding nanocrystalline aluminum and gold thin films.

Several experiments have shown that mechanical forces significantly influence the initiation, growth, and retraction of neurites of cultured neurons. A similar role has long been suggested for mechanical forces in vivo, but this hypothesis has remained unverified due to the paucity of in vivo studies of neuronal mechanical behavior. In this study, we used high-resolution micromechanical force sensors to study the mechanical response of motor neurons in live Drosophila embryos. Our experiments showed that Drosophila neurons maintained a rest tension (1-13 nN) and behaved like viscoelastic solids (i.e., with a linear force-deformation response followed by force relaxation to steady state) in response to sustained stretching. More importantly, when the tension was suddenly diminished by a release of the externally applied force, the neurons contracted and actively generated force to restore tension, sometimes to a value close to their rest tension. In addition, axons that were slackened by displacing the neuromuscular junction contracted and became taut in 10-30 min. These observations are remarkably similar to results from in vitro studies and suggest that mechanical tension may also strongly influence neuronal behavior in vivo.

Drosophila neurons actively regulate axonal tension in vivo.

Forces generated by cells play a vital role in many cellular processes like cell spreading, motility, differentiation and apoptosis. Understanding the mechanics of single cells is essential to delineate the link between cellular force generation/sensing and function. MEMS sensors, because of their small size and fine force/displacement resolution, are ideal for force and displacement sensing at the single cell level. In addition, the amenability of MEMS sensors to batch fabrication methods allows the study of large cell populations simultaneously, leading to robust statistical studies. In this review, we discuss various microsystems used for studying cell mechanics and the insights on cell mechanical behavior that have resulted from their use. The advantages and limitations of these microsystems for biological studies are also outlined.

MEMS Sensors and Microsystems for Cell Mechanobiology.

Spatial patterning of proteins (extracellular matrix, ECM) for living cells on polyacrylamide (PA) hydrogels has been technically challenging due to the compliant nature of the hydrogels and their aqueous environment. Traditional micro-fabrication process is not applicable. Here we report a simple, novel and general method to pattern a variety of commonly used cell adhesion molecules, i.e. Fibronectin (FN), Laminin (LN) and Collagen I (CN), etc. on PA gels. The pattern is first printed on a hydrophilic glass using polydimethylsiloxane (PDMS) stamp and micro-contact printing (μCP). Pre-polymerization solution is applied on the patterned glass and is then sandwiched by a functionalized glass slide, which covalently binds to the gel. The hydrophilic glass slide is then peeled off from the gel when the protein patterns detach from the glass, but remain intact with the gel. The pattern is thus transferred to the gel. The mechanism of pattern transfer is studied in light of interfacial mechanics. It is found that hydrophilic glass offers strong enough adhesion with ECM proteins such that a pattern can be printed, but weak enough adhesion such that they can be completely peeled off by the polymerized gel. This balance is essential for successful pattern transfer. As a demonstration, lines of FN, LN and CN with widths varying from 5-400 μm are patterned on PA gels. Normal fibroblasts (MKF) are cultured on the gel surfaces. The cell attachment and proliferation are confined within these patterns. The method avoids the use of any toxic chemistry often used to pattern different proteins on gel surfaces.

A Novel Technique for Micro-patterning Proteins and Cells on Polyacrylamide Gels.

Many microorganisms, including spermatozoa and forms of bacteria, oscillate or twist a hair-like flagella to swim. At this small scale, where locomotion is challenged by large viscous drag, organisms must generate time-irreversible deformations of their flagella to produce thrust. To date, there is no demonstration of a self propelled, synthetic flagellar swimmer operating at low Reynolds number. Here we report a microscale, biohybrid swimmer enabled by a unique fabrication process and a supporting slender-body hydrodynamics model. The swimmer consists of a polydimethylsiloxane filament with a short, rigid head and a long, slender tail on which cardiomyocytes are selectively cultured. The cardiomyocytes contract and deform the filament to propel the swimmer at 5-10 μm s(-1), consistent with model predictions. We then demonstrate a two-tailed swimmer swimming at 81 μm s(-1). This small-scale, elementary biohybrid swimmer can serve as a platform for more complex biological machines.

A self-propelled biohybrid swimmer at low Reynolds number.

Combining biological components, such as cells and tissues, with soft robotics can enable the fabrication of biological machines with the ability to sense, process signals, and produce force. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. Whereas cardiac cell-driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel "bio-bots" with an asymmetric physical design and powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot. Geometric design and material properties of the hydrogel bio-bots were optimized using stereolithographic 3D printing, and the effect of collagen I and fibrin extracellular matrix proteins and insulin-like growth factor 1 on the force production of engineered skeletal muscle was characterized. Electrical stimulation triggered contraction of cells in the muscle strip and net locomotion of the bio-bot with a maximum velocity of ∼ 156 μm s(-1), which is over 1.5 body lengths per min. Modeling and simulation were used to understand both the effect of different design parameters on the bio-bot and the mechanism of motion. This demonstration advances the goal of realizing forward-engineered integrated cellular machines and systems, which can have a myriad array of applications in drug screening, programmable tissue engineering, drug delivery, and biomimetic machine design.

Three-dimensionally printed biological machines powered by skeletal muscle.

A variety of cell types exhibit phenotype changes in response to the mechanical stiffness of the substrate. Many cells excluding neurons display an increase in the spread area, actin stress fiber formation and larger focal adhesion complexes as substrate stiffness increases in a sparsely populated culture. Cell proliferation is also known to directly correlate with these phenotype changes/changes in substrate stiffness. Augmented spreading and proliferation on stiffer substrates require nuclear transcriptional regulator YAP (Yes associated protein) localization in the cell nucleus and is tightly coupled to larger traction force generation. In this study, we show that different types of fibroblasts can exhibit spread morphology, well defined actin stress fibers, and larger focal adhesions even on very soft collagen gels (modulus in hundreds of Pascals) as if they are on hard glass substrates (modulus in GPa, several orders of magnitude higher). Strikingly, we show, for the first time, that augmented spreading and other hard substrate cytoskeleton architectures on soft collagen gels are not correlated with the cell proliferation pattern and do not require YAP localization in the cell nucleus. Finally, we examine the response of human colon carcinoma (HCT-8) cells on soft collagen gels. Recent studies show that human colon carcinoma (HCT-8) cells form multicellular clusters by 2-3 days when cultured on soft polyacrylamide (PA) gels with a wide range of stiffness (0.5-50 kPa) and coated with an extracellular matrix, ECM (collagen monomer/fibronectin). These clusters show limited spreading/wetting on PA gels, form 3D structures at the edges, and eventually display a remarkable, dissociative metastasis like phenotype (MLP), i.e., epithelial to rounded morphological transition after a week of culture on PA gels only, but not on collagen monomer coated stiff polystyrene/glass where they exhibit enhanced wetting and form confluent monolayers. Here, we show that HCT-8 cell clusters also show augmented spreading/wetting on soft collagen gels and eventually form confluent monolayers as on rigid glass substrates and MLP is completely inhibited on soft collagen gels. Overall, these results suggest that cell-material interactions (soft collagen gels in this case) can induce cellular phenotype and cytoskeleton organization in a remarkably distinct manner compared to a classical synthetic polyacrylamide (PA) hydrogel cell culture model and may contribute in designing new functional biomaterials.

Reprogramming cellular phenotype by soft collagen gels.

Here, we present the development, characterization and quantification of a novel 1D/2D like polymeric platform for cell culture. The platform consists of a 2D surface anchoring a long (few millimeters) narrow filament (1D) with a single cell scale (micro scale) cross section. We plate C2C12 cells on the platform and characterize their migration, proliferation, and differentiation patterns in contrast to 2D culture. We find that the cells land on the 2D surface, and then migrate to the filament only when the 2D surface has become nearly confluent. Individual and isolated cells randomly approaching the filament always retract away towards the 2D surface. Once on the filament, their differentiation to myotubes is expedited compared to that on 2D substrate. The myotubes generate periodic twitching forces that deform the filament producing more than 17 μm displacement at the tip. Such flagellar motion can be used to develop autonomous micro scale bio-bots.

ArticleTotal : 2	Year
Publication title Cited by 47	2014
Publication title Cited by 8	2015

Article	Year
Functionalized biomicroelectromechanical systems sensors for force response study at local adhesion sites of single living cells on substrates. Annals of biomedical engineering\| PubMed ID: 12918910	2003
Force response and actin remodeling (agglomeration) in fibroblasts due to lateral indentation. Acta biomaterialia\| PubMed ID: 17049324	2007
Plastic deformation recovery in freestanding nanocrystalline aluminum and gold thin films. Science (New York, N.Y.)\| PubMed ID: 17395826	2007
Drosophila neurons actively regulate axonal tension in vivo. Biophysical journal\| PubMed ID: 21081068	2010
MEMS Sensors and Microsystems for Cell Mechanobiology. Journal of micromechanics and microengineering : structures, devices, and systems\| PubMed ID: 21886944	2011
A Novel Technique for Micro-patterning Proteins and Cells on Polyacrylamide Gels. Soft matter\| PubMed ID: 23002394	2012
A self-propelled biohybrid swimmer at low Reynolds number. Nature communications\| PubMed ID: 24435099	2014
Three-dimensionally printed biological machines powered by skeletal muscle. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24982152	2014
Reprogramming cellular phenotype by soft collagen gels. Soft matter\| PubMed ID: 25284029	2014
Cell culture on microfabricated one-dimensional polymeric structures for bio-actuator and bio-bot applications. Lab on a chip\| PubMed ID: 25712193	2015

M. Taher A. Saif

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