A practical review on the measurement tools for cellular adhesion force

Cell cell and cell matrix adhesions are fundamental in all multicellular organisms. They play a key role in cellular growth, differentiation, pattern formation and migration. Cell-cell adhesion is substantial in the immune response, pathogen host interactions, and tumor development. The success of tissue engineering and stem cell implantations strongly depends on the fine control of live cell adhesion on the surface of natural or biomimetic scaffolds. Therefore, the quantitative and precise measurement of the adhesion strength of living cells is critical, not only in basic research but in modern technologies, too. Several techniques have been developed or are under development to quantify cell adhesion. All of them have their pros and cons, which has to be carefully considered before the experiments and interpretation of the recorded data. Current review provides a guide to choose the appropriate technique to answer a specific biological question or to complete a biomedical test by measuring cell adhesion

Magnetic manipulation and multimodal imaging for single cell direct mechanosensing

The study of internal mechanics of single cells is paramount to understand mechanisms of mechanoregulation. External loading and cell-mediated force generation result in changes in cell shape, rheology, and the deformation of subcellular structures such as the nucleus. Moreover, alterations in the processes that regulate these responses have been further correlated to specific pathologies. Cellular deformation is often studied through application of forces in the environment of the cell, relying on strain and stress transfer through focal adhesions and the cytoskeletal system. However, the transfer of these external forces to internal mechanics can introduce uncertainties in the interpretation of subcellular responses. Our group has focused on minimally-invasive techniques for the study of internal mechanical perturbation and mechanobiology measures. We have been particularly interested in multimodal imaging methods that combine and leverage nano-scale spatial localization, visualization, biophysical and physico-chemical analysis features to reveal information that cannot be attained by any single method alone. We recently fabricated novel atomic force microscopy (AFM) cantilevers, functionalized to generate small, highly-localized magnetic fields, for the controlled force application and sensing of single cells. In combination with AFM and fluorescence microscopy detection capabilities, this technique enables the selective stimulation and monitoring of cells injected with superparamagnetic microbeads. Though the targeted magnetic force application, we are able to apply various waveforms to direct the microdisplacements of the injected beads to allow insight into the structural architecture of the cell. Coupling this with AFM techniques further yields insight into internal and external mechanics over time. This technique can be extended to include studies of intranuclear strain dynamics through fluorescent labeling of specific cellular targets and image post-processing algorithms such as hyperelastic warping. Furthermore, the ability to alter the culture environment (e.g. to manipulate osmotic pressure or enable drug delivery) allows this technique to be a powerful single cell analysis tool for a diverse set of applications. We demonstrate the feasibility of this technique through the localized application of low magnetic fields that produce bead displacements in the micrometer scale. The effects of larger induced magnetic fields in the displacement field are also presented, along with validation and viability studies, and a range of practical applications for the study of single cells

Multiple trap optical tweezers for cell force measurements

Adherent cells establish transient adhesion sites that serve as stable anchors of the cell cytoskeleton to the substrate and concomitantly allow for force transduction and cell motility. We established a multiple trap optical tweezers system for non-invasive cell force measurements at individual adhesion sites. A force study was conducted to analyze the impact of bead functionalization, adhesion site area, location, spacing, and orientation as well as the role of vinculin on adhesion formation

Optical manipulation : advances for biophotonics in the 21st century

We thank the UK Engineering and Physical Sciences Research Council for funding (Grant Nos. EP/P030017/1 and EP/R004854/1).Significance: Optical trapping is a technique capable of applying minute forces that has been applied to studies spanning single molecules up to microorganisms. AIM: The goal of this perspective is to highlight some of the main advances in the last decade in this field that are pertinent for a biomedical audience. Approach: First, the direct determination of forces in optical tweezers and the combination of optical and acoustic traps, which allows studies across different length scales, are discussed. Then, a review of the progress made in the direct trapping of both single-molecules, and even single-viruses, and single cells with optical forces is outlined. Lastly, future directions for this methodology in biophotonics are discussed. Results: In the 21st century, optical manipulation has expanded its unique capabilities, enabling not only a more detailed study of single molecules and single cells but also of more complex living systems, giving us further insights into important biological activities. Conclusions: Optical forces have played a large role in the biomedical landscape leading to exceptional new biological breakthroughs. The continuous advances in the world of optical trapping will certainly lead to further exploitation, including exciting in-vivo experiments.Publisher PDFPeer reviewe

Techniques to stimulate and interrogate cell–cell adhesion mechanics

Cell–cell adhesions maintain the mechanical integrity of multicellular tissues and have recently been found to act as mechanotransducers, translating mechanical cues into biochemical signals. Mechanotransduction studies have primarily focused on focal adhesions, sites of cell-substrate attachment. These studies leverage technical advances in devices and systems interfacing with living cells through cell–extracellular matrix adhesions. As reports of aberrant signal transduction originating from mutations in cell–cell adhesion molecules are being increasingly associated with disease states, growing attention is being paid to this intercellular signaling hub. Along with this renewed focus, new requirements arise for the interrogation and stimulation of cell–cell adhesive junctions. This review covers established experimental techniques for stimulation and interrogation of cell–cell adhesion from cell pairs to monolayers

Biomechanical Characterization at the Cell Scale: Present and Prospects

The rapidly growing field of mechanobiology demands for robust and reproducible characterization of cell mechanical properties. Recent achievements in understanding the mechanical regulation of cell fate largely rely on technological platforms capable of probing the mechanical response of living cells and their physico–chemical interaction with the microenvironment. Besides the established family of atomic force microscopy (AFM) based methods, other approaches include optical, magnetic, and acoustic tweezers, as well as sensing substrates that take advantage of biomaterials chemistry and microfabrication techniques. In this review, we introduce the available methods with an emphasis on the most recent advances, and we discuss the challenges associated with their implementation

SINGLE-MOLECULE STUDY OF THE FORCE-INDUCED ACTIN POLYMERIZATION MEDIATED BY FORMIN

Ph.DPH.D. IN MECHANOBIOLOGY (FOS

Manipulating Cardiovascular Cellular Interactions and Mechanics: A Multidimensional and Multimodal Approach

The goal of this dissertation is to better understand cellular mechanics across length scales for the development of computational models of tissue behavior. To this end, we had two major approaches, multidimensional and multimodal. Firstly, to use a model that better mimics in vivo like cellular environment, microtissue (spheroid) cell culture system was used to study cell mechanics. Secondly, a novel technique was designed to study single cell mechanics in multiple dimensions. Cell mechanical properties are directly related to the composition and organization of the cytoskeleton, which is physically coupled to neighboring cells through adherens junctions and to extracellular matrix through focal adhesion complexes. As such, we hypothesize that the variations in cellular interactions affects cell mechanics. To test our hypothesis, cardiomyocytes and vascular smooth muscle microtissues were cultured under several conditions that limited the cell-cell and cell-matrix interactions. Cell interactions facilitated by integrin β1, connexin 43, and N-cadherin was inhibited and their effect on cell stiffness was characterized by atomic force microscopy (AFM). Currently, there does not exist a single technique that can measure mechanics of a single cell in two different dimensions. To address this gap, we designed a novel set up that combines two different single cell mechanics measurement techniques, AFM and carbon fiber. This combination allows for characterization of mechanical properties of single cells in multiple dimensions. The results of these studies provide insights from a basic science perspective. The results provide information regarding cell mechanics in multiple dimensions at both single cell as well microtissue level. The ultimate fulfillment of this work would be its incorporation into a multiscale model, leading to the ability to tie macro- scale behaviors to nano- scale phenomenon. Such models may help to better understand tissue behavior and further our understanding of the etiology of many diseases

REGULATION OF SHAPE DYNAMICS AND ACTIN POLYMERIZATION DURING COLLECTIVE CELL MIGRATION

This thesis aims to understand how cells coordinate their motion during collective migration. As previously shown, the motion of individually migrating cells is governed by wave-like cell shape dynamics. The mechanisms that regulate these dynamic behaviors in response to extracellular environment remain largely unclear. I applied shape dynamics analysis to Dictyostelium cells migrating in pairs and in multicellular streams and found that wave-like membrane protrusions are highly coupled between touching cells. I further characterized cell motion by using principle component analysis (PCA) to decompose complex cell shape changes into a serial shape change modes, from which I found that streaming cells exhibit localized anterior protrusion, termed front narrowing, to facilitate cell-cell coupling. I next explored cytoskeleton-based mechanisms of cell-cell coupling by measuring the dynamics of actin polymerization. Actin polymerization waves observed in individual cells were significantly suppressed in multicellular streams. Streaming cells exclusively produced F-actin at cell-cell contact regions, especially at cell fronts. I demonstrated that such restricted actin polymerization is associated with cell-cell coupling, as reducing actin polymerization with Latrunculin A leads to the assembly of F-actin at the side of streams, the decrease of front narrowing, and the decoupling of protrusion waves. My studies also suggest that collective migration is guided by cell-surface interactions. I examined the aggregation of Dictyostelim cells under distinct conditions and found that both chemical compositions of surfaces and surface-adhesion defects in cells result in altered collective migration patterns. I also investigated the shape dynamics of cells suspended on PEG-coated surfaces, which showed that coupling of protrusion waves disappears on touching suspended cells. These observations indicate that collective migration requires a balance between cell-cell and cell-surface adhesions. I hypothesized such a balance is reached via the regulation of cytoskeleton. Indeed, I found cells actively regulate cytoskeleton to retain optimal cell-surface adhesions on varying surfaces, and cells lacking the link between actin and surfaces (talin A) could not retain the optimal adhesions. On the other hand, suspended cells exhibited enhanced actin filament assembly on the periphery of cell groups instead of in cell-cell contact regions, which facilitates their aggregation in a clumping fashion