1,526 research outputs found

    Bioactive Self-Assembled Protein Nanosheets for Stem Cell-Based Biotechnologies

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    Tissue and stem cell culture methods have been dominated by glass and plastic substrates such as Tissue culture plastic. These solid substrates, although widely used, are associated with poor scalability for adherent stem cell expansion in systems such as 3D bioreactors and the design of parallel culture systems. Therefore, investigating strategies to bypass these obstacles in stem cell expansion is essential to enable the wider translation of stem cell technologies. An alternative strategy recently proposed consists in using a liquid surface instead, such as an oil, and associated oil droplets. Indeed, emulsions can be formed using protein nanosheets to stabilise oil/water interfaces to promote the adhesion of stem cells and enable their proliferation. These nanosheets exhibit enhanced interfacial mechanics and allow the introduction of bioactive components via recombinant protein expression to promote bioactivity. Beyond the application of resulting bioemulsions for the expansion of Mesenchymal stem cells, the impact of these bioactive interfaces on the differentiation of iPSCs and the development of cerebral organoids will be presented. The Bovine serum albumin protein was recombinantly modified to attach an N-terminal Avi-Tag, this was expressed and purified from the yeast P. pastoris expression system. The Avi-tag was then biotinylated in vitro by recombinantly expressed BirA. Emulsions of a specific size were formed using the newly biotinylated Bt-BSA protein and functionalized with a cascade of components to mimic cell-cell ligands, this resulted in bioemulsions with a bioactive surface that can interact with surrounding cells. These functionalised droplets were integrated into developing cerebral organoids and their impact on phenotype was studied. The droplets were found not to deform sufficiently to allow mechanical forces to be measured, yet the many of these droplets were retained within the organoids which led to an interesting phenotype within the organoids. The developing rosettes were found to develop enlarged lumens shown by an increase in area, this phenotype did not impact the differentiation into the cerebral lineage depicted by immunohistochemistry of hallmark marker of neuronal differentiation within organoids retaining droplets. The interfacial mechanics of fibrinogen nanosheets treated with varying concentrations of thrombin was studied using interfacial shear rheology. The effect of thrombin significantly altered the interfacial mechanics with the lower concentration of thrombin significantly increasing the toughness multiple folds and decreasing the elasticity of the nanosheets. Additionally, the nanostructure of nanosheets was studied using SEM and TEM and traditional fibrin fibres were found to not form at these interfaces, but local rearrangements and retractions in the thrombin treated nanosheets were observed. Finally, these enhanced mechanical properties promoted the proliferation and expansion of Mesenchymal stem cells on quasi-2D and 3D interfaces

    Patterning of the cell cortex by Rho GTPase Dynamics

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    The Rho GTPases — RHOA, RAC1 and CDC42 — are small GTP binding proteins that regulate basic biological processes such as cell locomotion, cell division and morphogenesis by promoting cytoskeleton-based changes in the cell cortex. This regulation results from active (GTP-bound) Rho GTPases stimulating target proteins that, in turn, promote actin assembly and myosin 2-based contraction to organize the cortex. This basic regulatory scheme, well supported by in vitro studies, led to the natural assumption that Rho GTPases function in vivo in an essentially linear matter, with a given process being initiated by GTPase activation and terminated by GTPase inactivation. However, a growing body of evidence based on live cell imaging, modelling and experimental manipulation indicates that Rho GTPase activation and inactivation are often tightly coupled in space and time via signalling circuits and networks based on positive and negative feedback. In this Review, we present and discuss this evidence, and we address one of the fundamental consequences of coupled activation and inactivation: the ability of the Rho GTPases to self-organize, that is, direct their own transition from states of low order to states of high order. We discuss how Rho GTPase self-organization results in the formation of diverse spatiotemporal cortical patterns such as static clusters, oscillatory pulses, travelling wave trains and ring-like waves. Finally, we discuss the advantages of Rho GTPase self-organization and pattern formation for cell function

    Multiplexed High-Resolution Imaging Approach to Decipher the Cellular Heterogeneity of the Kidney and its Alteration in Kidney Disease and Nephrolithiasis

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    Indiana University-Purdue University Indianapolis (IUPUI)Kidney disease and nephrolithiasis both present a major burden on the health care system in the US and worldwide. The cellular and molecular events governing the pathogenesis of these diseases are not fully understood. We propose that defining the cellular heterogeneity and niches in human and mouse kidney tissue specimens from controls and various models of renal disease could provide unique insights into the molecular pathogenesis. For that purpose, a multiplexed fluorescence imaging approach using co-detection by Indexing (CODEX) was used, using a panel of 33 and 38 markers for mouse and human kidney tissues, respectively. A customized computational analytical pipeline was developed and applied to the imaging data using unsupervised and/or semi-supervised machine learning and statistical approaches. The goal was to identify various cell populations present within the tissues, as well as identify unique cellular niches that may be altered with disease and/or injury. In mice, we examined disease models of acute kidney injury (AKI) and in human tissues we analyzed specimens from patients with AKI, IgA nephropathy, chronic kidney disease, systemic lupus erythematosus, and nephrolithiasis. In both mice and humans, the disease and reference samples show similar broad cell populations for the main segments of the nephron, endothelium, as well as similar groups of immune cells, such as resident macrophages and neutrophils. When comparing between health and disease, however, a change in the distribution of few sub-populations occurred. For example, in human kidney tissues, the abundance and distribution of a subpopulation of proximal tubules positive for THY1 (a marker of differentiation and repair), was markedly reduced with disease. Changes observed in mouse tissues included shifts in the immune cell population types and niches with disease. We propose that our analytical workflow and the observed changes in situ will play an important role in deciphering the pathogenesis of kidney disease

    Dissecting polarity formation in the Drosophila oocyte

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    The polarization of cells is a prerequisite for many fundamental biological processes, such as asymmetric cell division, neuronal polarization, and morphogenesis. In Drosophila melanogaster, both main body axes are established during oogenesis through polarization of the oocyte and as a result of crosstalk between the oocyte and surrounding cells

    Biofidelic simulations of embryonic joint growth and morphogenesis

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    During skeletal development, the opposing surfaces in the joint mould into interlocking and reciprocal shapes in a process called morphogenesis. Morphogenesis is critical to the health and function of the joint, and yet, little is known about the process of joint morphogenesis. For example, how do different joints acquire their specific shapes? Which cellular processes underlie joint shaping and how are they regulated? However, it is known that fetal movements are critical to joint development, with alterations or absences of movement being implicated in multiple pre- and post-natal musculoskeletal conditions. This doctorate explored the cell-level dynamics governing joint growth and the implication of movements in regulating them, using novel biofidelic and mechanobiological models of joint growth. Cell-level data from wild type zebrafish larvae were tracked and synthesised in a biofidelic simulation of zebrafish jaw joint growth. Growth characteristics were quantified revealing a strong anisotropy (Chapter 3). Next, zebrafish larvae were immobilised using drug treatment. The material properties of the zebrafish jaw cartilage were measured using nano-indentation in the presence or absence of movement showing a delay in cartilage stiffening in immobilised larvae (Chapter 4). Then, I developed a novel mechanobiological model of zebrafish jaw joint growth, which identified a correlation between growth characteristics and the dynamic patterns of mechanical stimuli experienced by joint elements over jaw motion (Chapter 5). Finally, local growth rates were characterised in the mouse elbow in the presence or absence of skeletal muscles. Spatial heterogeneity in the growth rates correlated with the emergence of specific shape features at the level of the condyles. Immobilisation led to disruption of the local growth rates correlated with failed shape differentiation of the condyles. The relative contribution of key cell activities to growth such as cell volume expansion, cell number increases and extracellular matrix expansion, were shown to vary over time in both wild types and muscleless-limbs and to be altered in the absence of skeletal muscles (Chapter 6). This research offers avenues for improvement in simulations of joint development and potentially other organs. It provides fundamental advance in our understanding of mechanoregulation in the developing joint and increases our understanding of the origins of musculoskeletal abnormalities.Open Acces

    Molecular Control of Actin Cortex Architecture During Cell Division

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    Animal cell shape is controlled by gradients in contractile tension of the actin cortex. The cortex is a thin actomyosin network supporting the plasma membrane. At the molecular level, contractile tension is generated by myosin motors pulling on actin filaments. Along- side myosin, actin connectivity has been shown to be key to cortical tension regulation. Understanding molecular organisation of the actin cortex is thus key to understanding cortical tension. To understand how cortical composition changes when tension changes, and to identify potential molecular regulators of cortical tension, I firstly compared protein composition of interphase and mitotic cortices. Indeed, interphase and mitotic cells were previously shown to di↔er in cortical tension. I isolated cortical fractions from cells in these stages of cell cycle, by isolating cortex-enriched blebs. Using mass spectrometry, we detected over 922 proteins in blebs isolated from synchronised cells. Among 238 actin-related proteins, we showed a role for septins in the regulation of the mitotic cell shape. Overall, we created a comprehensive dataset of potential regulators of cortex mechanics. In the second part of my PhD, I focused on the role of actin crosslinkers in cortex tension regulation. In particular, I focused on the role of actin crosslinker size for their localisation and in tension regulation. To this aim, we created artificial crosslinkers, for which I was able to modulate size independently of other features. We created artificial crosslinkers between 5 and 35 nm long, which successfully localised to actin structures. I investigated the role of artificial crosslinkers in the control of cortical thickness, tension and cell division. Together, in this thesis, I investigate new levels of regulation of cortical organisation and tension at the molecular level

    Controlling Surface-Induced Platelet Activation by Modulation of Contacting Interfaces

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    BlutplĂ€ttchen, auch Thrombozyten genannt, sind ein wesentlicher Bestandteil des menschlichen Blutgerinnungssystems. Die Hauptaufgabe der Thrombozyten innerhalb des Körpers besteht in der Blutstillung. Außerhalb des Körpers neigen Thrombozyten jedoch dazu, nach kurzem Kontakt mit synthetischen, nicht physiologischen OberflĂ€chen zu aktivieren, was fĂŒr viele Anwendungen unerwĂŒnscht sein kann, einschließlich der Lagerung von Thrombozyten und der Erforschung der Wechselwirkungen zwischen Thrombozyten und Arzneimitteln. Normalerweise werden Thrombozyten-Konzentrate in handelsĂŒblichen Plastikbeuteln aufbewahrt, die eine große Menge an Weichmachern enthalten, um die FlexibilitĂ€t des Beutels zu erhöhen und die Möglichkeit eines Bruchs wĂ€hrend der Handhabung und des Transports zu vermeiden. Bei lĂ€ngerer Exposition können die giftigen Weichmacher in das Thrombozyten-Konzentrat entweichen. Aktivierte Thrombozyten setzen eine Vielzahl von Proteinen frei, die den Prozess der oberflĂ€cheninduzierten Thrombozytenaktivierung (SIPA) weiter unterstĂŒtzen. SIPA ist eines der Hauptprobleme von Medizinprodukten mit Blutkontakt und TransfusionsgerĂ€ten, und ein entscheidender Faktor fĂŒr die verkĂŒrzte Haltbarkeit gelagerter Thrombozyten. Um SIPA zu vermeiden, werden den Thrombozyten-Konzentraten Antikoagulantien zugesetzt, so dass sie bis zu 5 Tage gelagert werden können. Diese Antikoagulantien greifen in die Aktivierungswege der Thrombozyten ein und beeintrĂ€chtigen so ihre FunktionalitĂ€t. Das hĂ€ufigste Problem bei der Lagerung von Thrombozyten ist schließlich die Gefahr einer bakteriellen Kontamination. Um dieses Problem zu lösen, werden verschiedene UV-Behandlungen eingesetzt, um das Risiko einer Kontamination mit Krankheitserregern zu minimieren. Studien zeigen jedoch, dass diese Strahlung mit kurzer WellenlĂ€nge die Bestandteile der Thrombozytenmembran zerstören und zu einer Aktivierung der Thrombozyten fĂŒhren kann. Diese zahlreichen, oft miteinander verknĂŒpften Probleme verdeutlichen den dringenden Bedarf an einer effizienten Lösung zur Optimierung der Lagerungsbedingungen fĂŒr Thrombozyten und zur Maximierung ihrer LagerfĂ€higkeit. Ziel dieser Doktorarbeit ist es, OberflĂ€chen zu entwickeln, die die AdhĂ€sion von Thrombozyten hemmen und somit ihre Aktivierung und Aggregation verhindern - ohne dass der Zusatz von Antikoagulantien erforderlich ist. FĂŒr die VerĂ€nderung der OberflĂ€cheneigenschaften stehen drei verschiedene AnsĂ€tze zur VerfĂŒgung: Biophysikalische, physikochemische oder biochemische Strategien können Verwendet werden, um eine plĂ€ttchenfreundliche OberflĂ€che zu gestalten. In der ersten Phase dieses Projekts wurde eine Kombination aus physikochemischen und biophysikalischen AnsĂ€tzen angewandt, um Hydrogele aus Gelatine und Agarose herzustellen, die anschließend durch Integration von Eisennanopartikeln zu Nanokompositen verarbeitet wurden. Agarose-basierte Hydrogel-Filme erwiesen sich dabei durch die Kombination von OberflĂ€chenbenetzbarkeit und besseren mechanischen Eigenschaften als ideale OberflĂ€chen. Mikroskopaufnahmen zeigten, dass die Anzahl der BlutplĂ€ttchen, die an solchen OberflĂ€chen adhĂ€rieren, deutlich reduziert und die Ausbreitung der BlutplĂ€ttchen verhindert wurde. Hergestellte Agarose-Filme und ihre Nanokomposite konnten darĂŒber hinaus bakterielles Wachstum erfolgreich hemmen: Von allen getesteten Proben wurde der höchste Prozentsatz an toten Bakterien auf den Nanokomposit-Filmen gemessen. Die Topographie des Substrats spielt eine entscheidende Rolle fĂŒr das Verhalten der Zellen und die Kontrolle ihrer Physiologie und Morphologie. FĂŒr die VerĂ€nderung der OberflĂ€chentopografie stehen zahlreiche komplexe Techniken zur VerfĂŒgung. In dieser Arbeit wurden zwei Techniken mit individuellen Vorteilen zur Herstellung von Nanostrukturen eingesetzt. Bei der ersten handelt es sich um ein auf der Rasterkraftmikroskopie (AFM) basierendes Fluidiksystem namens FluidFM, bei dem eine Monomere enthaltende Tinte aus der Öffnung des Cantilever Spitze auf die OberflĂ€che extrudiert wird. Nach dem Druckvorgang wird die Tinte polymerisiert, um 3D-Strukturen zu erhalten. Mit Hilfe von kontinuierlichen und diskontinuierlichen Topografien wurden hexagonale Bienenstock- bzw. halbkugelförmige Gitterstrukturen hergestellt. Dabei zeigte sich, dass die Thrombozyten diese Strukturierung mechanisch wahrnehmen und ihr Zytoskelett umorganisieren, was zu einer geringeren Ausbreitung der BlutplĂ€ttchen fĂŒhrt. DarĂŒber hinaus wurde die Technik zum Drucken einer modifizierten biofunktionalisierten Tinte verwendet, die so modifiziert wurde, dass MolekĂŒle mit unterschiedlichen funktionellen Gruppen in die Basistinte integriert wurden. Diese Modifikation fĂŒhrte nur zu einer geringfĂŒgigen VerĂ€nderung der mechanischen Eigenschaften der gedruckten Strukturen, wĂ€hrend ihre FunktionalitĂ€t erhalten blieb. Die Möglichkeit, Bindungsmotive fĂŒr spezifische Wechselwirkungen zu integrieren, demonstriert die Vielseitigkeit der FluidFM und ebnet den Weg fĂŒr die weitere Erforschung des biochemischen/topographischen Ansatzes im Bereich der Entwicklung plĂ€ttchenfreundlicher OberflĂ€chen. Das Drucken von Mikro- und Nanostrukturen stellt eine schnelle, kostengĂŒnstige und effiziente Methode zur Herstellung verschiedener geometrischer Prototypen dar und kann nicht nur zur Untersuchung verschiedener Strukturformen, sondern auch ihrer GrĂ¶ĂŸe und anderer topografischer Parameter eingesetzt werden. Die zweite verwendete Technik war die thermische Nanoimprint-Lithografie (T-NIL), mit der ein breiteres Spektrum an OberflĂ€chentopologien untersucht werden konnte, einschließlich Punkt, Kette, Pille und Quadrat-förmiger. Diese Nanomuster wurden auf Siliziumscheiben geĂ€tzt und auf einen PDMS-basierten Stempel ĂŒbertragen, der so zum PrĂ€gen von Hydrogelen verwendet werden konnte. Verschiedene Topologien wurden auf die OberflĂ€che von Agarosegelen geprĂ€gt, um ihre zuvor beobachtete, hemmende Wirkung auf die ThrombozytenadhĂ€sion zu verbessern. Das pillenförmige Nanomuster war dabei am besten geeignet, um die ThrombozytenadhĂ€sion zu hemmen, was auf die Höhe der Struktur zurĂŒckgefĂŒhrt werden kann. Zusammenfassend lĂ€sst sich festhalten, dass in diesem Projekt Hydrogelfilme auf Agarosebasis, insbesondere in Form von Nanokompositen mit integrierten antibakteriellen Eisennanopartikeln, entwickelt wurden, die Lagerungsbedingungen fĂŒr Thrombozyten deutlich verbessern, indem sie die SIPA und das Risiko einer bakteriellen Kontamination verringern. UV-Behandlungen von Thrombozyten-Konzentraten werden dadurch ĂŒberflĂŒssig. Durch die EinfĂŒhrung verschiedener OberflĂ€chentopologien kann die AdhĂ€sion von Thrombozyten gehemmt werden: Das FluidFM-basierte vielseitig einsetybare Nanodrucksystem wurde fĂŒr die Erforschung und Entwicklung von Prototypen effektiver Geometrien eingesetzt, wĂ€hrend T-NIL fĂŒr die PrĂ€gung ausgewĂ€hlter Strukturen auf die OberflĂ€che von Agarose-Filmen verwendet werden kann, um eine einheitliche OberflĂ€chentopographie zu schaffen

    Microtubule and spindle pole body regulation in the budding yeast mitotic spindle

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    The microtubule organizing center (MTOC) is a specialized structure with a main function in microtubule (MT) nucleation and organization. In higher eukaryotes, the main MTOC is known as the centrosome, and its functional equivalent in yeast is the spindle pole body (SPB). During mitosis, the cells build the mitotic spindle, an MT-based structure that mediates accurate chromosome segregation, which is essential to prevent aneuploidy and cancer. In yeast, the assembly and function of the mitotic spindle depend on the SPB, microtubule-associated proteins (MAPs), and motor proteins. In this thesis, we aim to advance our understanding of the general principles that regulate the SPBs, and spindle MTs in budding yeast. In Paper I, we developed a method to separate the old (from the previous cell cycle) and newly synthesized SPB component Spc110 and identified age-specific phosphorylation residues in Spc110. We combined Recombination Induced Tag Exchanged – a genetic method to label old and new proteins differentially- with affinity purification. Using mass spectrometry analyses, we identified two phosphosites, S11 and S36, in old Spc110. We explore the function of these two phosphosites in non-phosphorylatable Spc110 mutants. Cells expressing Spc110S11A showed a distinct spindle phenotype, where tubulin intensity is higher and distributed asymmetrically. Furthermore, the double mutant Spc110S11AS36A was slightly delayed in cell cycle progression and re-entry in G1. Thus, we propose Spc110 phosphorylation at S11 regulates MT dynamics, whereas together with S36 regulates timely cell cycle progression in budding yeast. In Paper II, we explored the role of the MT plus-end tracking protein (+TIP) Bik1 in the nucleus of budding yeast. Bik1 has a nuclear and a cytoplasmic pool, and we found that nuclear Bik1 localizes to the kinetochores in a cell cycle-dependent manner, peaking at metaphase. To explore the role of Bik1 at kinetochores, we added a Nuclear Export Signal (NES) to generate a Bik1-NES mutant that excludes Bik1 from the nucleus without disrupting its cytoplasmic pool. The Bik1-NES mutant had a slower cell-cycle progression, characterized by prolonged metaphase. Furthermore, we demonstrated that the kinetochores in Bik1-NES cells are frequently unclustered and mispositioned towards the spindle midzone in metaphase. By applying proximity-dependent methods, we identified kinesins Cin8 and Kip1 as Bik1 interactors. Bik1 and Cin8 cooperate to regulate kinetochore-MT dynamics for chromosome congression. Hence, the study uncovers a novel role of kinetochore Bik1 in cell cycle progression and chromosome congression. In Paper III, we examined the turnover of Spc110 using cycloheximide (CHX) chase experiments. The results indicated that Spc110 has a very short half-life. Performing CHXchase experiments with the autophagy-defective mutant atg1D, we showed that autophagy is not essential for Spc110 degradation. On the other hand, Spc110 contains a potential Esp1 recognition site (the yeast homolog of Separase). Mutation of this Esp1 recognition site on Spc110 results in partial Spc110 stabilization. Hence, we speculate that Esp1 might be involved in Spc110 degradation

    E-Cadherin Force Transmission and Stiffness Sensing

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    E-cadherin is the chief mediator of cell-cell adhesion between epithelial cells and is a known mechanosensor. Force transmission and stiffness sensing are two crucial aspects of E-cadherin mechanobiology. E-cadherin has an extracellular adhesive region, a transmembrane region and an intracellular region that binds to adhesion-associated proteins. Here, we assessed how different factors affect the level of force transmission (i) from inside the cell such as adhesion-associated proteins, (ii) on the cell membrane, such as growth factor receptors and (iii) outside the cell, such as different binding partners in adhesion. To study the level of force transmission inside the cell, we studied the role of vinculin and α-catenin in transmitting endogenous forces at cell-cell contacts. We found that vinculin, not α-catenin, is pivotal for transmitting high endogenous forces at cell-cell contacts through E-cadherin. To study how the level of force transmission is affected by factors on the cell membrane, we investigated the effect of EGFR on the intercellular forces transmitted at cell-cell contacts. We found that EGFR activity significantly affects the level of intercellular forces. In order to understand how the level of force transmission depends on binding partners from outside the cell, we studied homophilic and heterophilic interactions of cadherins. We found that the intercellular tension for the heterophilic E-cad/N-cad interaction is higher than the homophilic E-cad/E-cad interaction. Additionally, we also devised a modified traction force microscopy method using a novel, simple strategy for coincident immunofluorescence and traction force microscopy. Moreover, E-cadherin adhesions reside in a microenvironment that is comprised of adjacent epithelial cells. We found that E-cadherin adhesions change their organization depending on the magnitude of the epithelial cell-like elasticity of their microenvironment. Such E-cadherin adhesions were of two types: linear shaped adhesions and irregularly shaped adhesions. We found that linear adhesions were dependent on formin-dependent linear actin bundles and irregular adhesions were dependent on high local actin density. Thus, we found that actin is a crucial determinant of how E-cadherin adhesions are organized in response to cell-like soft microenvironments. All these findings have important implications for tissue development (morphogenesis), dysregulation (such as during cancer progression) as well as tissue engineering

    Migration of cytotoxic T lymphocytes in 3D bioprinted PEG-based hydrogels

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    Cytotoxic T lymphocytes (CTLs) are immune cells that can identify and destroy cancer cells. They patrol our body in search for targets without exploiting proteolysis. However, their migration mechanisms are still not entirely understood. One aspect that influences migration is the extracellular matrix (ECM), the environment where cells migrate. However, the ECM in vivo has complex properties and thus, it is difficult to determine how each property affects cell migration. One way to deconvolute the combined effects of ECM properties in cell migration is through reducing the ECM in vivo and mimicking the essential ECM properties in vitro. In this thesis, the aim is to understand how ECM stiffness influences the migration of CTLs in three dimension (3D). CTL migration was investigated in well-defined ECM-mimetic hydrogels based on poly(ethylene glycol) (PEG). Using chemical strategies, the engineered PEG hydrogels achieved a synchronous control over stiffness and presentation of bioactive cues. The PEG-based ECM mimics had stiffness values that mimic a variety of native soft tissues such as brain, kidney, lung, liver, pancreas, and thyroid. Cell migration assays were performed in high-throughput manner using 3D bioprinter, time-lapsed fluorescence microscopy, and computational tools. With these ECM-mimetic hydrogels, it was learned that CTLs migrated faster and killed more target cells in 0.7 kPa than in 1.5 kPa ECM-mimetic hydrogel. Furthermore, high stiffness and the presence of an enzyme-degradable motif in the ECM mimics can trigger proteolytic migration of CTLs. These insights can potentially advance cancer therapy
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