Search

Biomedical research relies on identification and isolation of specific cell types using molecular biomarkers and sorting methods such as fluorescence or magnetic activated cell sorting. Labelling processes potentially alter the cells’ properties and should be avoided, especially when purifying cells for clinical applications. A promising alternative is the label-free identification of cells based on physical properties. Sorting real-time deformability cytometry (soRT-DC) is a microfluidic technique for label-free analysis and sorting of single cells. In soRT-FDC, bright-field images of cells are analyzed by a deep neural net (DNN) to obtain a sorting decision, but sorting was so far only demonstrated for blood cells which show clear morphological differences and are naturally in suspension. Most cells, however, grow in tissues, requiring dissociation before cell sorting which is associated with challenges including changes in morphology, or presence of aggregates. Here, we introduce methods to improve robustness of analysis and sorting of single cells from nervous tissue and provide DNNs which can distinguish visually similar cells. We employ the DNN for image-based sorting to enrich photoreceptor cells from dissociated retina for transplantation into the mouse eye.

Optical trapping is a vital tool in biology, allowing precise optical manipulation of nanoparticles, micro-robots, and cells. Due to the low risk of photodamage and high trap stiffness, fiber-based dual-beam traps are widely used for optical manipulation of large cells. Besides trapping, advanced applications like 3D refractive index tomography need a rotation of cells, which requires precise control of the forces, for example, the acting-point of the forces and the intensities in the region of interest (ROI). A precise rotation of large cells in 3D about arbitrary axes has not been reported yet in dual-beam traps. We introduce a novel dual-beam optical trap in which a multi-core fiber (MCF) is transformed to a phased array, using wavefront shaping and computationally programmable light. The light-field distribution in the trapping region is holographically controlled within 0.1 s, which determines the orientation and the rotation axis of the cell with small retardation. We demonstrate real-time controlled rotation of HL60 cells about all 3D axes with a very high degree of freedom by holographic controlled light through an MCF with a resolution close to the diffraction limit. For the first time, the orientation of the cell can be precisely controlled about all 3D axes in a dual-beam trap. MCFs provide much higher flexibility beyond the bulky optics, enabling lab-on-a-chip applications and can be easily integrated for applications like contactless cell surgery, refractive index tomography, cell-elasticity measurement, which require precise 3D manipulation of cells.

Mechanical proprieties determine many cellular functions, such as cell fate specification, migration, or circulation through vasculature. Identifying factors governing cell mechanical phenotype is therefore a subject of great interest. Here we present a mechanomics approach for establishing links between mechanical phenotype changes and the genes involved in driving them. We employ a machine learning-based discriminative network analysis method termed PC-corr to associate cell mechanical states, measured by real-time deformability cytometry (RT-DC), with large-scale transcriptome datasets ranging from stem cell development to cancer progression, and originating from different murine and human tissues. By intersecting the discriminative networks inferred from two selected datasets, we identify a conserved module of five genes with putative roles in the regulation of cell mechanics. We validate the power of the individual genes to discriminate between soft and stiff cell states in silico, and demonstrate experimentally that the top scoring gene, CAV1, changes the mechanical phenotype of cells when silenced or overexpressed. The data-driven approach presented here has the power of de novo identification of genes involved in cell mechanics regulation and paves the way towards engineering cell mechanical properties on demand to explore their impact on physiological and pathological cell functions.

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples — so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with molecular specificity. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the cell nucleus, we find that it has lower density but higher longitudinal modulus. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample — a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

Many compartments in eukaryotic cells are protein-rich biomolecular condensates demixed from the cyto- or nucleoplasm. Although much has been learned in recent years about the integral roles condensates play in many cellular processes as well as the biophysical properties of reconstituted condensates, an understanding of their most basic feature, their composition, remains elusive. Here we combined quantitative phase microscopy (QPM) and the physics of sessile droplets to develop a precise method to measure the shape and composition of individual model condensates. This technique does not rely on fluorescent dyes or tags, which we show can significantly alter protein phase behavior, and requires 1000-fold less material than traditional label-free technologies. We further show that this QPM method measures the protein concentration in condensates to a 3-fold higher precision than the next best label-free approach, and that commonly employed strategies based on fluorescence intensity dramatically underestimate these concentrations by as much as 50-fold. Interestingly, we find that condensed-phase protein concentrations can span a broad range, with PGL3, TAF15(RBD) and FUS condensates falling between 80 and 500 mg/ml under typical in vitro conditions. This points to a natural diversity in condensate composition specified by protein sequence. We were also able to measure temperature-dependent phase equilibria with QPM, an essential step towards relating phase behavior to the underlying physics and chemistry. Finally, time-resolved QPM reveals that PGL3 condensates undergo a contraction-like process during aging which leads to doubling of the internal protein concentration coupled to condensate shrinkage. We anticipate that this new approach will enable understanding the physical properties of biomolecular condensates and their function.

The impulsive stimulated Brillouin microscopy promises fast, non-contact measurements of the elastic properties of biological samples. The used pump-probe approach employs an ultra-short pulse laser and a cw laser to generate Brillouin signals. Modeling of the microscopy technique has already been carried out partially, but not for biomedical applications. The nonlinear relationship between pulse energy and Brillouin signal amplitude is proven with both simulations and experiments. Tayloring of the excitation parameters on the biologically relevant polyacrylamide hydrogels outline sub-ms temporal resolutions at a relative precision of

Cancer cells can switch between signaling pathways to regulate growth under different conditions. In the tumor microenvironment, this likely helps them evade therapies that target specific pathways. We must identify all possible states and utilize them in drug screening programs. One such state is characterized by expression of the transcription factor Hairy and Enhancer of Split 3 (HES3) and sensitivity to HES3 knockdown, and it can be modeled in vitro. Here, we cultured 3 primary human brain cancer cell lines under 3 different culture conditions that maintain low, medium, and high HES3 expression and characterized gene regulation and mechanical phenotype in these states. We assessed gene expression regulation following HES3 knockdown in the HES3-high conditions. We then employed a commonly used human brain tumor cell line to screen Food and Drug Administration (FDA)-approved compounds that specifically target the HES3-high state. We report that cells from multiple patients behave similarly when placed under distinct culture conditions. We identified 37 FDA-approved compounds that specifically kill cancer cells in the high-HES3–expression conditions. Our work reveals a novel signaling state in cancer, biomarkers, a strategy to identify treatments against it, and a set of putative drugs for potential repurposing.—Poser, S. W., Otto, O., Arps-Forker, C., Ge, Y., Herbig, M., Andree, C., Gruetzmann, K., Adasme, M. F., Stodolak, S., Nikolakopoulou, P., Park, D. M., Mcintyre, A., Lesche, M., Dahl, A., Lennig, P., Bornstein, S. R., Schroeck, E., Klink, B., Leker, R. R., Bickle, M., Chrousos, G. P., Schroeder, M., Cannistraci, C. V., Guck, J., Androutsellis-Theotokis, A. Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery. FASEB J. 33, 9235–9249 (2019). www.fasebj.org. © FASEB

The mechanical fingerprint of cells is inherently linked to the structure of the cytoskeleton and can serve as a label-free marker for cell homeostasis or pathologic states. How cytoskeletal composition affects the physical response of cells to external loads has been intensively studied with a spectrum of techniques, yet quantitative and statistically powerful investigations in the form of titration assays are hampered by the low throughput of most available methods. In this study, we employ real-time deformability cytometry (RT-DC), a novel microfluidic tool to examine the effects of biochemically modified F-actin and microtubule stability and nuclear chromatin structure on cell deformation in a human leukemia cell line (HL60). The high throughput of our method facilitates extensive titration assays that allow for significance assessment of the observed effects and extraction of half-maximal concentrations for most of the applied reagents. We quantitatively show that integrity of the F-actin cortex and microtubule network dominate cell deformation on millisecond timescales probed with RT-DC. Drug-induced alterations in the nuclear chromatin structure were not found to consistently affect cell deformation. The sensitivity of the high-throughput cell mechanical measurements to the cytoskeletal modifications we present in this study opens up new possibilities for label-free dose-response assays of cytoskeletal modifications.

Differentiation of oligodendrocyte progenitor cells (OPC) to oligodendrocytes and subsequent axon myelination are critical steps in vertebrate central nervous system (CNS) development and regeneration. Growing evidence supports the significance of mechanical factors in oligodendrocyte biology. Here, we explore the effect of mechanical strains within physiological range on OPC proliferation and differentiation, and strain-associated changes in chromatin structure, epigenetics, and gene expression. Sustained tensile strain of 10-15% inhibited OPC proliferation and promoted differentiation into oligodendrocytes. This response to strain required specific interactions of OPCs with extracellular matrix ligands. Applied strain induced changes in nuclear shape, chromatin organization, and resulted in enhanced histone deacetylation, consistent with increased oligodendrocyte differentiation. This response was concurrent with increased mRNA levels of the epigenetic modifier histone deacetylase Hdac11. Inhibition of HDAC proteins eliminated the strain-mediated increase of OPC differentiation, demonstrating a role of HDACs in mechanotransduction of strain to chromatin. RNA sequencing revealed global changes in gene expression associated with strain. Specifically, expression of multiple genes associated with oligodendrocyte differentiation and axon-oligodendrocyte interactions was increased, including cell surface ligands (Ncam, ephrins), cyto- and nucleo-skeleton genes (Fyn, actinins, myosin, nesprin, Sun1), transcription factors (Sox10, Zfp191, Nkx2.2), and myelin genes (Cnp, Plp, Mag). These findings show how mechanical strain can be transmitted to the nucleus to promote oligodendrocyte differentiation, and identify the global landscape of signaling pathways involved in mechanotransduction. These data provide a source of potential new therapeutic avenues to enhance OPC differentiation in vivo.

The deformability of a cell is the direct result of a complex interplay between the different constituent elements at the subcellular level, coupling a wide range of mechanical responses at different length-scales. Changes to the structure of these components can also alter cell phenotype, thus the critical importance of cell mechano-response for diagnostic applications. The response to mechanical stress depends strongly on the forces experienced by the cell. Here we use cell deformability in both shear-dominant and inertia-dominant microfluidic flow regimes to probe different aspects of the cell structure. In the inertial regime we follow cellular response from (visco-)elastic through plastic deformation to cell structural failure and show a significant drop in cell viability for shear stresses above > 11.8 kN/m2. Comparatively, a shear-dominant regime requires lower applied stresses to achieve higher cell strains. From this regime, deformation traces as a function of time contain a rich source of information including; maximum strain, elastic modulus and cell relaxation times and thus provide a number of markers for distinguishing cell types and potentially disease progression. These results emphasise the benefit of multiple parameter determination for improving detection and will ultimately lead to improved accuracy for diagnosis. We present results for leukemia cells (HL60) as a model circulatory cell as well as for a colorectal cancer cell line SW480 derived from primary adenocarcinoma (Dukes stage B). SW480 were also treated with the actin disrupting drug Latrunculin A (LatA), to test the sensitivity of flow regimes to the cytoskeleton. We show that the shear regime is more sensitive to cytoskeletal changes, and that large strains in the inertial-regime cannot resolve changes to the actin cytoskeleton.