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Cells mechanical behaviour in physiological environments is mediated by interactions with the extracellular matrix (ECM). In particular, cells can adapt their shape according to the availability of ECM proteins, e.g., fibronectin (FN). Several in vitro experiments usually simulate the ECM by functionalizing the surfaces on which cells grow with FN. However, the mechanisms underlying cell spreading on non-uniformly FN-coated two-dimensional substrates are not clarified yet. In this work, we studied cell spreading on variously functionalized substrates: FN was either uniformly distributed or selectively patterned on flat surfaces, to show that A549, BRL, B16 and NIH 3T3 cell lines are able to sense the overall FN binding sites independently of their spatial arrangement. Instead, only the total amount of available FN influences cells spreading area, which positively correlates to the FN density. Immunocytochemical analysis showed that ß1 integrin subunits are mainly responsible for this behaviour, as further confirmed by spreading experiments with ß1-deficient cells. In the latter case, indeed, cells areas do not show a dependency on the amount of available FN on the substrates. Therefore, we envision for ß1 a predominant role in cells for sensing the number of ECM ligands with respect to other focal adhesion proteins.
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Toward the ambitious goal of manufacturing synthetic cells from the bottom up, various cellular components have already been reconstituted inside lipid vesicles. However, the deterministic positioning of these components inside the compartment has remained elusive. Here, by using two-photon 3D laser printing, 2D and 3D hydrogel architectures are manufactured with high precision and nearly arbitrary shape inside preformed giant unilamellar lipid vesicles (GUVs). The required water-soluble photoresist is brought into the GUVs by diffusion in a single mixing step. Crucially, femtosecond two-photon printing inside the compartment does not destroy the GUVs. Beyond this proof-of-principle demonstration, early functional architectures are realized. In particular, a transmembrane structure acting as a pore is 3D printed, thereby allowing for the transport of biological cargo, including DNA, into the synthetic compartment. These experiments show that two-photon 3D laser microprinting can be an important addition to the existing toolbox of synthetic biology.
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Células Artificiales , Rayos Láser , Impresión Tridimensional , Biología Sintética , Liposomas UnilamelaresRESUMEN
Micro-fabrication and nano-fabrication provide useful approaches to address fundamental biological questions by mimicking the physiological microenvironment in which cells carry out their functions. In particular, 2D patterns and 3D scaffolds obtained via lithography, direct laser writing, and other techniques allow for shaping hydrogels, synthetic polymers and biologically derived materials to create structures for (single) cell culture. Applications of micro-scaffolds mimicking cell niches include stem cell self-renewal, differentiation, and lineage specification. This review moves from technological aspects of scaffold microfabrication for cell biological applications to a broad overview of advances in (stem) cell research: achievements for embryonic, induced pluripotent, mesenchymal, and neural stem cells are treated in detail, while a particular section is dedicated to micro-scaffolds used to study single cells in basic cell biology.
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Células Artificiales , Andamios del Tejido , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Hidrogeles/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogenously coated surfaces, in collagen gels, and on micropatterned substrates. In contrast to homogenously coated surfaces, a structured environment supports a cellular phenotype with invaginated actin arcs even in the absence of NM IIA-induced contractility. A quantitative shape analysis of cells on micropatterns combined with a scale-bridging mathematical model reveals that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A dynamic cell stretch/release experiment in a three-dimensional scaffold confirms these conclusions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.
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Elasticidad , Miosina Tipo II/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Movimiento Celular/fisiología , Homeostasis , Humanos , Modelos Teóricos , Miosina Tipo II/clasificación , Miosina Tipo II/genética , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/genética , Isoformas de ProteínasRESUMEN
Many essential cellular processes are regulated by mechanical properties of their microenvironment. Here, we introduce stimuli-responsive composite scaffolds fabricated by three-dimensional (3D) laser lithography to simultaneously stretch large numbers of single cells in tailored 3D microenvironments. The key material is a stimuli-responsive photoresist containing cross-links formed by noncovalent, directional interactions between ß-cyclodextrin (host) and adamantane (guest). This allows reversible actuation under physiological conditions by application of soluble competitive guests. Cells adhering in these scaffolds build up initial traction forces of ~80 nN. After application of an equibiaxial stretch of up to 25%, cells remodel their actin cytoskeleton, double their traction forces, and equilibrate at a new dynamic set point within 30 min. When the stretch is released, traction forces gradually decrease until the initial set point is retrieved. Pharmacological inhibition or knockout of nonmuscle myosin 2A prevents these adjustments, suggesting that cellular tensional homeostasis strongly depends on functional myosin motors.
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Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) represent the best cell source for cardiac regenerative purposes but retain an immature phenotype after differentiation with significant limitations compared to adult cardiomyocytes. Apart from an incomplete cardiomyocyte-specific structure and microarchitecture, cells show at the level of Ca2+ signaling only slow Ca2+ release and reuptake properties. Here, we investigated the effect of restructuring single iPSC-CMs in specially designed 3D-micro-scaffolds on cell morphology and Ca2+ handling. Using direct laser writing, rectangular-shaped scaffolds were produced and single iPSC-CMs were seeded into these forms. Structural analyses revealed strong sarcolemmal remodeling processes and myofilament reorientation in 3D-shaped cells leading to enhanced clustered expression of L-type Ca2+ channels and ryanodine receptors and consequently, to faster Ca2+ transient kinetics. Spontaneous beating activity was enhanced and Ca2+ handling was more robust compared to non-patterned cells. Overall, our data demonstrate for the first time significant improvement of Ca2+ signaling properties in reshaped iPSC-CMs indicative of functional maturation by structural remodeling.
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Células Madre Pluripotentes Inducidas , Adulto , Diferenciación Celular , Humanos , Miocitos Cardíacos , FenotipoRESUMEN
The ability to selectively remove sections from 3D-printed structures with high resolution remains a current challenge in 3D laser lithography. A novel photoresist is introduced to enable the additive fabrication of 3D microstructures at one wavelength and subsequent spatially controlled cleavage of the printed resist at another wavelength. The photoresist is composed of a difunctional acrylate cross-linker containing a photolabile o-nitrobenzyl ether moiety. 3D microstructures are written by photoinduced radical polymerization of acrylates using Ivocerin as photoinitiator upon exposure to 900 nm laser light. Subsequent scanning using a laser at 700 nm wavelength allows for the selective removal of the resist by photocleaving the o-nitrobenzyl group. Both steps rely on two-photon absorption. The fabricated and erased features are imaged using scanning electron microscopy (SEM) and laser scanning microscopy (LSM). In addition, a single wire bond is successfully eliminated from an array, proving the possibility of complete or partial removal of structures on demand.
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The photochemistry of anthracene, a new class of photoresist for direct laser writing, is used to enable visible-light-gated control over the mechanical properties of 3D microstructures post-manufacturing. The mechanical and viscoelastic properties (hardness, complex elastic modulus, and loss factor) of the microstructures are measured over the course of irradiation via dynamic mechanical analysis on the nanoscale. Irradiation of the microstructures leads to a strong hardening and stiffening effect due to the generation of additional crosslinks through the photodimerization of the anthracene functionalities. A relationship between the loss of fluorescence-a consequence of the photodimerization-and changes in the mechanical properties is established. The fluorescence thus serves as a proxy read-out for the mechanical properties. These photoresponsive microstructures can potentially be used as "mechanical blank slates": their mechanical properties can be readily adjusted using visible light to serve the demands of different applications and read out using their fluorescence.
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Mimicking the properties of the extracellular matrix is crucial for developing in vitro models of the physiological microenvironment of living cells. Among other techniques, 3D direct laser writing (DLW) has emerged as a promising technology for realizing tailored 3D scaffolds for cell biology studies. Here, results based on DLW addressing basic biological issues, e.g., cell-force measurements and selective 3D cell spreading on functionalized structures are reviewed. Continuous future progress in DLW materials engineering and innovative approaches for scaffold fabrication will enable further applications of DLW in applied biomedical research and tissue engineering.
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Matriz Extracelular/metabolismo , Impresión Tridimensional , Andamios del Tejido/química , Biología Celular , Técnicas de Cultivo de Célula , Células Eucariotas/metabolismo , Humanos , Fenómenos Mecánicos , Polímeros/química , Análisis de la Célula Individual/métodos , Ingeniería de Tejidos/métodosRESUMEN
Stimuli-responsive microstructures are critical to create adaptable systems in soft robotics and biosciences. For such applications, the materials must be compatible with aqueous environments and enable the manufacturing of three-dimensional structures. Poly(N-isopropylacrylamide) (pNIPAM) is a well-established polymer, exhibiting a substantial response to changes in temperature close to its lower critical solution temperature. To create complex actuation patterns, materials that react differently with respect to a stimulus are required. Here, we introduce functional three-dimensional hetero-microstructures based on pNIPAM. By variation of the local exposure dose in three-dimensional laser lithography, we demonstrate that the material parameters can be altered on demand in a single resist formulation. We explore this concept for sophisticated three-dimensional architectures with large-amplitude and complex responses. The experimental results are consistent with numerical calculations, able to predict the actuation response. Furthermore, a spatially controlled response is achieved by inducing a local temperature increase by two-photon absorption of focused light.
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Cellular communication in multi-cellular organisms is mediated to a large extent by a multitude of cell-surface receptors that bind specific ligands. An in-depth understanding of cell signaling networks requires quantitative information on ligand-receptor interactions within living systems. In principle, fluorescence correlation spectroscopy (FCS) based methods can provide such data, but live-cell applications have proven extremely challenging. Here, we have developed an integrated dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) technique that greatly facilitates live-cell and tissue experiments. Absolute ligand and receptor concentrations and their diffusion coefficients within the cell membrane can be quantified without the need to perform additional calibration experiments. We also determine the concentration of ligands diffusing in the medium outside the cell within the same experiment by using a raster image correlation spectroscopy (RICS) based analysis. We have applied this robust technique to study the interactions of two Wnt antagonists, Dickkopf1 and Dickkopf2 (Dkk1/2), to their cognate receptor, low-density-lipoprotein-receptor related protein 6 (LRP6), in the plasma membrane of living HEK293T cells. We obtained significantly lower affinities than previously reported using in vitro studies, underscoring the need to measure such data on living cells or tissues.