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Microbial activity in planktonic systems creates a dynamic and heterogeneous microscale seascape that harbors a diverse community of microorganisms and ecological interactions of global significance. In recent decades great effort has been put into understanding this complex system, particularly focusing on the role of chemical patchiness, while overlooking a physical parameter that governs microbial life and is affected by biological activity: viscosity. Here we reveal spatial heterogeneity of viscosity in planktonic systems by using microrheological techniques that allow measurement of viscosity at length scales relevant to microorganisms. We show the viscous nature and the spatial extent of the phycosphere, the region surrounding phytoplankton. In â¼45% of the phytoplankton cells analyzed we detected increases in viscosity that extended up to 30 µm away from the cell with up to 40 times the viscosity of seawater. We also show how these gradients of viscosity can be amplified around a lysing phytoplankton cell as its viscous contents leak away. Finally, we report conservative estimates of viscosity inside marine aggregates, hotspots of microbial activity, more than an order of magnitude higher than in seawater. Since the diffusivities of dissolved molecules, particles, and microorganisms are inversely related to viscosity, microheterogeneity in viscosity alters the microscale distribution of microorganisms and their resources, with pervasive implications for the functioning of the planktonic ecosystem. Increasing viscosities impacts ecological interactions and processes, such as nutrient uptake, chemotaxis, and particle encounter, that occur at the microscale but influence carbon and nutrient cycles at a global scale.
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Fitoplâncton/crescimento & desenvolvimento , Plâncton/crescimento & desenvolvimento , Reologia/métodos , Quimiotaxia , Ecossistema , Fitoplâncton/metabolismo , Plâncton/metabolismo , Água do Mar/química , ViscosidadeRESUMO
Microrheology with optical tweezers (MOT) is an all-optical technique that allows the user to investigate a materials' viscoelastic properties at microscopic scales, and is particularly useful for those materials that feature complex microstructures, such as biological samples. MOT is increasingly being employed alongside 3D imaging systems and particle tracking methods to generate maps showing not only how properties may vary between different points in a sample but also how at a single point the viscoelastic properties may vary with direction. However, due to the diffraction limited shape of focussed beams, optical traps are inherently anisotropic in 3D. This can result in a significant overestimation of the fluids' viscosity in certain directions. As such, the rheological properties can only be accurately probed along directions parallel or perpendicular to the axis of trap beam propagation. In this work, a new analytical method is demonstrated to overcome this potential artefact. This is achieved by performing principal component analysis on 3D MOT data to characterise the trap, and then identify the frequency range over which trap anisotropy influences the data. This approach is initially applied to simulated data for a Newtonian fluid where the trap anisotropy induced maximum error in viscosity is reduced from ~ 150% to less than 6%. The effectiveness of the method is corroborated by experimental MOT measurements performed with water and gelatine solutions, thus confirming that the microrheology of a fluid can be extracted reliably across a wide frequency range and in any arbitrary direction. This work opens the door to fully spatially and angularly resolved 3D mapping of the rheological properties of soft materials over a broad frequency range.
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On supercooling a liquid, the viscosity rises rapidly until at the glass transition it vitrifies into an amorphous solid accompanied by a steep drop in the heat capacity. Therefore, a pure homogeneous liquid is not expected to display more than one glass transition. Here we show that a family of single-component homogeneous molecular liquids, titanium tetraalkoxides, exhibit two calorimetric glass transitions of comparable magnitude, one of which is the conventional glass transition associated with dynamic arrest of the bulk liquid properties, while the other is associated with the freezing out of intramolecular degrees of freedom. Such intramolecular vitrification is likely to be found in molecules in which low-frequency terahertz intramolecular motion is coupled to the surrounding liquid. These results imply that intramolecular barrier-crossing processes, typically associated with chemical reactivity, do not necessarily follow the Arrhenius law but may freeze out at a finite temperature.
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Hydrogels have been successfully employed as analogues of the extracellular matrix to study biological processes such as cells' migration, growth, adhesion and differentiation. These are governed by many factors, including the mechanical properties of hydrogels; yet, a one-to-one correlation between the viscoelastic properties of gels and cell fate is still missing from literature. In this work we provide experimental evidence supporting a possible explanation for the persistence of this knowledge gap. In particular, we have employed common tissues' surrogates such as polyacrylamide and agarose gels to elucidate a potential pitfall occurring when performing rheological characterisations of soft-materials. The issue is related to (i) the normal force applied to the samples prior to performing the rheological measurements, which may easily drive the outcomes of the investigation outside the materials' linear viscoelastic regime, especially when tests are performed with (ii) geometrical tools having unbefitting dimensions (i.e., too small). We corroborate that biomimetic hydrogels can show either compressional stress softening or stiffening, and we provide a simple solution to quench these undesired phenomena, which would likely lead to potentially misleading conclusions if they were not mitigated by a good practice in performing rheological measurements, as elucidated in this work.
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Artefatos , Hidrogéis , Fenômenos Mecânicos , Matriz ExtracelularRESUMO
We report on the application of a Fourier transform-based method, 'i-Rheo', to evaluate the linear viscoelastic moduli of hard-sphere colloidal dispersions, both in the fluid and glass states, from a direct analysis of raw step-stress (creep) experimental data. We corroborate the efficacy of i-Rheo by comparing the outputs of creep tests performed on homogenous complex fluids to conventional dynamic frequency sweeps. A similar approach is adopted for a number of colloidal suspensions over a broad range of volume fractions. For these systems, we test the limits of the method by varying the applied stress across the materials' linear and non-linear viscoelastic regimes, and we show that the best results are achieved for stress values close to the upper limit of the materials' linear viscoelastic regime, where the signal-to-noise ratio is at its highest and the non-linear phenomena have not appeared yet. We record that, the range of accessible frequencies is controlled at the higher end by the relative weight between the inertia of the instrument and the elasticity of the complex material under investigation; whereas, the lowest accessible frequency is dictated by the extent of the materials' linear viscoelastic regime. Nonetheless, despite these constrains, we confirm the effectiveness of i-Rheo for gaining valuable information on the materials' linear viscoelastic properties even from 'creep ringing' data, confirming its potency and general validity as an accurate method for determining the material's rheological behaviour for a variety of complex systems.
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Linear cationic antimicrobial peptides are a diverse class of molecules that interact with a wide range of cell membranes. Many of these peptides disrupt cell integrity by forming membrane-spanning pores that ultimately lead to their death. Despite these peptides high potency and ability to evade acquired bacterial drug resistance, there is a lack of knowledge on their selectivity and activity mechanisms. Such an understanding would provide an informative framework for rational design and could lead to potential antimicrobial therapeutic targets. In this paper, we use a high-throughput microfluidic platform as a quantitative screen to assess peptide activity and selectivity by precisely controlling exposure to vesicles with lipid compositions that mimic both bacterial and mammalian cell membranes. We explore the complexity of the lipid-peptide interactions governing membrane-disruptive behaviors and establish a link between peptide pore formation and both lipid-peptide charge and topological interactions. We propose a topological model for linear antimicrobial peptide activity based on the increase in membrane strain caused by the continuous adsorption of peptides to the target vesicle coupled with the effects of both lipid-peptide charge and topographical interactions. We also show the validity of the proposed model by investigating the activity of two prototypical linear cationic peptides: magainin 2 amide (which is selective for bacterial cells) and melittin (which targets both mammalian and bacterial cells indiscriminately). Finally, we propose the existence of a negative feedback mechanism that governs the pore formation process and controls the membrane's apparent permeability.
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Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/química , Eletricidade Estática , Lipídeos de Membrana/metabolismoRESUMO
Atomic force microscopy rheological measurements (Rheo-AFM) of the linear viscoelastic properties of single, charged colloids having a star-like architecture with a hard core and an extended, deformable double-stranded DNA (dsDNA) corona dispersed in aqueous saline solutions are reported. This is achieved by analyzing indentation and relaxation experiments performed on individual colloidal particles by means of a novel model-free Fourier transform method that allows a direct evaluation of the frequency-dependent linear viscoelastic moduli of the system under investigation. The method provides results that are consistent with those obtained via a conventional fitting procedure of the force-relaxation curves based on a modified Maxwell model. The outcomes show a pronounced softening of the dsDNA colloids, which is described by an exponential decay of both the Young's and the storage modulus as a function of the salt concentration within the dispersing medium. The strong softening is related to a critical reduction of the size of the dsDNA corona, down to ≈70% of its size in a salt-free solution. This can be correlated to significant topological changes of the dense star-like polyelectrolyte forming the corona, which are induced by variations in the density profile of the counterions. Similarly, a significant reduction of the stiffness is obtained by increasing the length of the dsDNA chains, which we attribute to a reduction of the DNA density in the outer region of the corona.
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Coloides/química , DNA/química , Elasticidade , Microscopia de Força Atômica , Reologia , Módulo de Elasticidade , Sais/química , Fatores de Tempo , ViscosidadeRESUMO
Nishi et al. have presented a new analytical method for transforming the time-dependent materials' compliance into their frequency-dependent complex shear modulus, without the need of a preconceived fitting function nor the use of Kramers-Kronig transformations. They claim that their method significantly improves the accuracy of the outcomes, especially at high frequencies, up to "almost" the Nyquist frequency. Here, I corroborate that their method is actually able to provide a close estimation of the materials' complex shear modulus over the 'entire' range of explored frequencies (i.e. beyond the Nyquist frequency), as long as the compliance values are linearly spaced in the time-domain and its value at time zero is included as the first data point in the input file. Moreover, as a means of comparison, I employ the analytical method introduced by Tassieri et al. [New J. Phys., 2012, 14, 115032] for performing the Fourier transform of any generic time-dependent function that vanishes for negative times, is sampled at a finite rate, need not be equally spaced and extends over a finite time window. This existing method does not need preconceived fitting functions nor the use of Kramers-Kronig transformations; yet it shows a higher degree of accuracy compared to the one proposed by Nishi et al.
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Optical tweezers have been successfully adopted as exceptionally sensitive transducers for microrheology studies of complex fluids. Despite the general trend, in this article I explain why a similar approach should not be adopted for microrheology studies of living cells. This conclusion is acheived on the basis of statistical mechanics principles that indicate the unsuitability of optical tweezers for such purpose.
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Pinças Ópticas , Reologia , Animais , Elasticidade , Géis/química , Humanos , Parasitos/fisiologia , Termodinâmica , ViscosidadeRESUMO
BACKGROUND: The Cryptococcus neoformans polysaccharide capsule is a well-characterized virulence factor with immunomodulatory properties. The organism and/or shed capsule is postulated to raise intracranial pressure (ICP) in cryptococcal meningitis (CM) by mechanical obstruction of cerebrospinal fluid (CSF) outflow. Little is known regarding capsule phenotype in human cryptococcosis. We investigated the relationship of ex vivo CSF capsular phenotype with ICP and CSF immune response, as well as in vitro phenotype. METHODS: In total, 134 human immunodeficiency virus (HIV)-infected Ugandan adults with CM had serial lumbar punctures with measurement of CSF opening pressures, quantitative cultures, ex vivo capsule size and shedding, viscosity, and CSF cytokines; 108 had complete data. Induced capsular size and shedding were measured in vitro for 48 C. neoformans isolates. RESULTS: Cryptococcal strains producing larger ex vivo capsules in the baseline (pretreatment) CSF correlated with higher ICP (P = .02), slower rate of fungal clearance (P = .02), and paucity of CSF inflammation, including decreased CSF white blood cell (WBC) count (P < .001), interleukin (IL)-4 (P = .02), IL-6 (P = .01), IL-7 (P = .04), IL-8 (P = .03), and interferon γ (P = .03). CSF capsule shedding did not correlate with ICP. On multivariable analysis, capsule size remained independently associated with ICP. Ex vivo capsular size and shedding did not correlate with that of the same isolates grown in vitro. CONCLUSIONS: Cryptococcal capsule size ex vivo is an important contributor to virulence in human cryptococcal meningitis.
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Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Cryptococcus neoformans/citologia , Cryptococcus neoformans/imunologia , Cápsulas Fúngicas/imunologia , Meningite Criptocócica/microbiologia , Infecções Oportunistas Relacionadas com a AIDS/líquido cefalorraquidiano , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Adulto , Análise de Variância , Antifúngicos/farmacologia , Líquido Cefalorraquidiano/citologia , Líquido Cefalorraquidiano/microbiologia , Citocinas , Feminino , Cápsulas Fúngicas/química , Cápsulas Fúngicas/microbiologia , Humanos , Pressão Intracraniana/imunologia , Masculino , Meningite Criptocócica/líquido cefalorraquidiano , Meningite Criptocócica/imunologia , Fenótipo , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Uganda , ViscosidadeRESUMO
Microrheology, the study of material flow at micron scales, has advanced significantly since Robert Brown's discovery of Brownian motion in 1827. Mason and Weitz's seminal work in 1995 established the foundation for microrheology techniques, enabling the measurement of viscoelastic properties of complex fluids using light-scattering particles. However, existing techniques face limitations in exploring very slow dynamics, crucial for understanding biological systems. Here, we present a proof of concept for a novel microrheology technique called "Optical Halo", which utilises a ring-shaped Bessel beam created by optical tweezers to overcome existing limitations. Through numerical simulations and theoretical analysis, we demonstrate the efficacy of the Optical Halo in probing viscoelastic properties across a wide frequency range, including low-frequency regimes inaccessible to conventional methods. This innovative approach holds promise for elucidating the mechanical behaviour of complex biological fluids.
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This study introduces a novel mechanobiology assay, named "i-Rheo-optical assay", that integrates rheology with optical microscopy for analysing the viscoelastic properties of multicellular spheroids. These spheroids serve as three-dimensional models resembling tissue structures. The innovative technique enables real-time observation and quantification of morphological responses to applied stress using a cost-effective microscope coverslip for constant compression force application. By bridging a knowledge gap in biophysical research, which has predominantly focused on the elastic properties while only minimally exploring the viscoelastic nature in multicellular systems, the i-Rheo-optical assay emerges as an effective tool. It facilitates the measurement of broadband viscoelastic compressional moduli in spheroids, here derived from cancer (PANC-1) and non-tumoral (NIH/3T3) cell lines during compression tests. This approach plays a crucial role in elucidating the mechanical properties of spheroids and holds potential for identifying biomarkers to discriminate between healthy tissues and their pathological counterparts. Offering comprehensive insights into the biomechanical behaviour of biological systems, i-Rheo-optical assay marks a significant advancement in tissue engineering, cancer research, and therapeutic development.
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The design of hydrogels as mimetics of tissues' matrices typically disregards the viscous nature of native tissues and focuses only on their elastic properties. In the case of stem cell chondrogenesis, this has led to contradictory results, likely due to unreported changes in the matrices' viscous modulus. Here, by employing isoelastic matrices with Young's modulus of ≈12 kPa, variations in viscous properties alone (i.e., loss tangent between 0.1 and 0.25) are demonstrated to be sufficient to drive efficient growth factor-free chondrogenesis of human mesenchymal stem cells, both in 2D and 3D cultures. The increase of the viscous component of RGD-functionalized polyacrylamide or polyethylene glycol maleimide hydrogels promotes a phenotype with reduced adhesion, alters mechanosensitive signaling, and boosts cell-cell contacts. In turn, this upregulates the chondrogenic transcription factor SOX9 and supports neocartilage formation, demonstrating that the mechanotransductive response to the viscous nature of the matrix can be harnessed to direct cell fate.
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Condrogênese , Células-Tronco Mesenquimais , Humanos , Hidrogéis/farmacologia , Hidrogéis/metabolismo , Células-Tronco , Materiais Biocompatíveis/metabolismo , Diferenciação Celular , Células CultivadasRESUMO
Recently, optical tweezing has been used to provide a method for microrheology addressed to measure the rheological properties of small volumes of samples. In this work, we corroborate this emerging field of microrheology by using these optical methods for the characterization of polyelectrolyte solutions with very low viscoelasticity. The influence of polyelectrolyte (i.e., polyacrylamide, PAM) concentration, specifically its aging, of the salt concentration is shown. The close agreement of the technique with classical bulk rheological measurements is demonstrated, illustrating the advantages of the technique.
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Resinas Acrílicas/química , Elasticidade , Eletrólitos/química , Pinças Ópticas , Reologia , Soluções , ViscosidadeRESUMO
Microrheology, the study of fluids on micron length-scales, promises to reveal insights into cellular biology, including mechanical biomarkers of disease and the interplay between biomechanics and cellular function. Here a minimally-invasive passive microrheology technique is applied to individual living cells by chemically binding a bead to the surface of a cell, and observing the mean squared displacement of the bead at timescales ranging from milliseconds to 100s of seconds. Measurements are repeated over the course of hours, and presented alongside analysis to quantify changes in the cells' low-frequency elastic modulus, G0', and the cell's dynamics over the time window â¼10-2 s to 10 s. An analogy to optical trapping allows verification of the invariant viscosity of HeLa S3 cells under control conditions and after cytoskeletal disruption. Stiffening of the cell is observed during cytoskeletal rearrangement in the control case, and cell softening when the actin cytoskeleton is disrupted by Latrunculin B. These data correlate with conventional understanding that integrin binding and recruitment triggers cytoskeletal rearrangement. This is, to our knowledge, the first time that cell stiffening has been measured during focal adhesion maturation, and the longest time over which such stiffening has been quantified by any means. STATEMENT OF SIGNIFICANCE: Here, we present an approach for studying mechanical properties of live cells without applying external forces or inserting tracers. Regulation of cellular biomechanics is crucial to healthy cell function. For the first time in literature, we can non-invasively and passively quantify cell mechanics during interactions with functionalised surface. Our method can monitor the maturation of adhesion sites on the surface of individual live cells without disrupting the cell mechanics by applying forces to the cell. We observe a stiffening response in cells over tens of minutes after a bead chemically binds. This stiffening reduces the deformation rate of the cytoskeleton, although the internal force generation increases. Our method has potential for applications to study mechanics during cell-surface and cell-vesicle interactions.
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Citoesqueleto , Pinças Ópticas , Citoesqueleto/metabolismo , Membrana Celular/metabolismo , Módulo de Elasticidade , Citoesqueleto de ActinaRESUMO
Fluorescence Lifetime Imaging Microscopy in the time domain is typically performed by recording the arrival time of photons either by using electronic time tagging or a gated detector. As such the temporal resolution is limited by the performance of the electronics to 100's of picoseconds. Here, we demonstrate a fluorescence lifetime measurement technique based on photon-bunching statistics with a resolution that is only dependent on the duration of the reference photon or laser pulse, which can readily reach the 1-0.1 picosecond timescale. A range of fluorescent dyes having lifetimes spanning from 1.6 to 7 picoseconds have been here measured with only ~1 s measurement duration. We corroborate the effectiveness of the technique by measuring the Newtonian viscosity of glycerol/water mixtures by means of a molecular rotor having over an order of magnitude variability in lifetime, thus introducing a new method for contact-free nanorheology. Accessing fluorescence lifetime information at such high temporal resolution opens a doorway for a wide range of fluorescent markers to be adopted for studying yet unexplored fast biological processes, as well as fundamental interactions such as lifetime shortening in resonant plasmonic devices.
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Biomechanical cues from the extracellular matrix (ECM) are essential for directing many cellular processes, from normal development and repair, to disease progression. To better understand cell-matrix interactions, we have developed a new instrument named 'OptoRheo' that combines light sheet fluorescence microscopy with particle tracking microrheology. OptoRheo lets us image cells in 3D as they proliferate over several days while simultaneously sensing the mechanical properties of the surrounding extracellular and pericellular matrix at a sub-cellular length scale. OptoRheo can be used in two operational modalities (with and without an optical trap) to extend the dynamic range of microrheology measurements. We corroborated this by characterising the ECM surrounding live breast cancer cells in two distinct culture systems, cell clusters in 3D hydrogels and spheroids in suspension culture. This cutting-edge instrument will transform the exploration of drug transport through complex cell culture matrices and optimise the design of the next-generation of disease models.
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Matriz Extracelular , Hidrogéis , Microscopia de Fluorescência , Comunicação CelularRESUMO
In this article we present a new open-access code named "i-RheoFT" that implements the analytical method first introduced in [PRE, 80, 012501 (2009)] and then enhanced in [New J Phys 14, 115032 (2012)], which allows to evaluate the Fourier transform of any generic time-dependent function that vanishes for negative times, sampled at a finite set of data points that extend over a finite range, and need not be equally spaced. I-RheoFT has been employed here to investigate three important experimental factors: (i) the 'density of initial experimental points' describing the sampled function, (ii) the interpolation function used to perform the "virtual oversampling" procedure introduced in [New J Phys 14, 115032 (2012)], and (iii) the detrimental effect of noises on the expected outcomes. We demonstrate that, at relatively high signal-to-noise ratios and density of initial experimental points, all three built-in MATLAB interpolation functions employed in this work (i.e., Spline, Makima and PCHIP) perform well in recovering the information embedded within the original sampled function; with the Spline function performing best. Whereas, by reducing either the number of initial data points or the signal-to-noise ratio, there exists a threshold below which all three functions perform poorly; with the worst performance given by the Spline function in both the cases and the least worst by the PCHIP function at low density of initial data points and by the Makima function at relatively low signal-to-noise ratios. We envisage that i-RheoFT will be of particular interest and use to all those studies where sampled or time-averaged functions, often defined by a discrete set of data points within a finite time-window, are exploited to gain new insights on the systems' dynamics.
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We introduce a novel 3D microrheology system that combines for the first time Optical Tweezers with Integrated Multiplane Microscopy (OpTIMuM). The system allows the 3D tracking of an optically trapped bead, with ~ 20 nm accuracy along the optical axis. This is achieved without the need for a high precision z-stage, separate calibration sample, nor a priori knowledge of either the bead size or the optical properties of the suspending medium. Instead, we have developed a simple yet effective in situ spatial calibration method using image sharpness and exploiting the fact we image at multiple planes simultaneously. These features make OpTIMuM an ideal system for microrheology measurements, and we corroborate the effectiveness of this novel microrheology tool by measuring the viscosity of water in three dimensions, simultaneously.
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Recent advances in 3D bioprinting allow for generating intricate structures with dimensions relevant for human tissue, but suitable bioinks for producing translationally relevant tissue with complex geometries remain unidentified. Here, a tissue-specific hybrid bioink is described, composed of a natural polymer, alginate, reinforced with extracellular matrix derived from decellularized tissue (rECM). rECM has rheological and gelation properties beneficial for 3D bioprinting while retaining biologically inductive properties supporting tissue maturation ex vivo and in vivo. These bioinks are shear thinning, resist cell sedimentation, improve viability of multiple cell types, and enhance mechanical stability in hydrogels derived from them. 3D printed constructs generated from rECM bioinks suppress the foreign body response, are pro-angiogenic and support recipient-derived de novo blood vessel formation across the entire graft thickness in a murine model of transplant immunosuppression. Their proof-of-principle for generating human tissue is demonstrated by 3D bioprinting human airways composed of regionally specified primary human airway epithelial progenitor and smooth muscle cells. Airway lumens remained patent with viable cells for one month in vitro with evidence of differentiation into mature epithelial cell types found in native human airways. rECM bioinks are a promising new approach for generating functional human tissue using 3D bioprinting.