Biology subjects

Kamm, R. D.

Publications and source records attributed to Kamm, R. D..

16 recordsLinked to original sources

bioRxiv2025

4D force patterning enables spatial control of angiogenesis

Engineering organized microvascular networks remains a critical challenge in tissue engineering and regenerative medicine. While biochemical approaches for patterning angiogenesis via growth factor delivery have shown promise, their inability to pattern sustained growth factors with spatiotemporal control limits effectiveness. Here, we demonstrate that dynamically patterned mechanical forces enable precise spatiotemporal control over angiogenic sprouting. We developed a magnetically actuated human vessel-on-a-chip platform that integrates a perfusable endothelialized microchannel within a collagen matrix and allows non-invasive and tunable mechanical stimulation across three spatial dimensions and time (4D). Using an automated 3-axis actuator, we systematically investigated how strain magnitude, frequency, and direction modulate endothelial cell behavior and vessel morphogenesis. Dynamic mechanical stimulation at physiological strain magnitudes (5-15%) enhanced endothelial alignment and barrier function while promoting angiogenesis in a strain-magnitude-dependent manner: lower dynamic strain (5%) maximized sprout initiation, whereas higher dynamic strain (15%) promoted elongation of sprouts. Sequential reorientation of strain direction reprogrammed sprouting trajectories along X, Y, and Z directions, generating complex sprout geometries such as L-shaped branches. RNA sequencing revealed mechanically induced transcriptional profiles distinct from unstimulated controls, characterized by upregulation of genes associated with angiogenesis, mechanotransduction, and extracellular matrix remodeling. Functional perturbation of Piezo1 reduced strain-induced sprouting without altering barrier stabilization, indicating that dynamic mechanical stimulation engages multiple mechanotransduction pathways to regulate angiogenesis. Collectively, these findings establish a strategy for spatiotemporally controlled angiogenesis through 4D force patterning to program vascular morphogenesis while preserving function. This approach provides a foundation for engineering hierarchically organized vascular networks for tissue regeneration. SignificanceGeneration of spatially organized, perfusable microvascular networks is essential for building functional human tissues. Biochemical approaches to pattern angiogenesis rely on diffusive growth factors, which limit control over spatiotemporal sprouting dynamics. Here, we demonstrate that dynamically patterned mechanical forces direct vascular morphogenesis across three spatial dimensions and time (4D). Using a magnetically actuated human vessel-on-a-chip, we show how strain magnitude and orientation govern angiogenic sprouting and reveal transcriptional programs linking mechanical cues to observed functional changes. For the first time, we show that dynamic reorientation of imposed forces can reprogram angiogenic trajectories in real-time. This platform enables programmable mechanical control of angiogenesis and systematic dissection of mechanotransduction pathways, advancing strategies for tissue vascularization, and modeling mechanically regulated vascular diseases.

bioengineering↗

bioRxiv2025

ETV2 mediated differentiation of human pluripotent stem cells results in functional endothelial cells for engineering advanced vascularized microphysiological models

Patient-specific microphysiological models, exemplified by organs-on-a-chip and organoids, have become a valuable tool for broad applications, revolutionizing biomedical research. However, limitations persist, with functional vasculature being a significant challenge. Generating functional human induced pluripotent stem cell (h-iPSC) derived endothelial cells (h-iECs) represent an urgent need. With the discovery of ETV2s determinant role in specifying EC lineages during differentiation, researchers have adopted techniques involving ETV2 overexpression to produce h-iECs more efficiently and consistently. However, the capacity of these cells to form functional vasculatures has not yet been thoroughly investigated. Here, we generated multiple h-iPSC lines with inducible ETV2 expression, and subsequently differentiated them into h-iECs, which were validated functionally and by key endothelial markers and RNA-seq analysis. These cells are capable of self-organizing into stable microvascular networks (MVNs) in a microfluidic chip reproducibly, forming lumenized and functional vessels that mimic the in vivo capillary bed in both morphology and function - a result not achieved using h-iECs differentiated with conventional two-step methods using the same h-iPSC lines. Furthermore, complex microphysiological models featuring perfusable vasculature were also successfully developed using ETV2 activated h-iECs, demonstrated with vascularized tumor and blood-brain barrier (BBB) models. Additionally, by pooling genetically engineered h-iPSCs with inducible ETV2, we effectively employed an orthogonally induced differentiation approach to enhance vascularization of an organoid model. Our methodology opens avenues in precision medicine, leading to personalized microphysiological models with perfusable vasculature for various applications.

bioengineering↗

bioRxiv2025

3D printing and bioprinting for miniaturized and scalable hanging-drop organoids culture

Three-dimensional (3D) cell culture systems rely on the manipulation of a biologically derived matrix, typically soluble Basement Membrane Extract (sBME), in which cells or cellular aggregates, such as organoids, are suspended. This matrix provides mechanobiological support, promoting cellular processes. However, the handling of sBME-based matrices containing cellular constructs poses significant challenges due to their rheological properties. We developed an integrated bioprinting system to surpass the conventional pipetting, seeding and culture in multiwell plates. The system combines a fluidic cartridge with innovative 3D-printed biocompatible culture tools designed to host and preserve high-throughput microcultures of Patient-Derived Organoids (PDOs) in sBME. The miniaturized hanging-drop configuration enables extended culture periods and high-throughput imaging screenings. This comprehensive approach overcomes common issues associated with sBME, including sedimentation of cellular aggregates, premature gelation, and structural collapse, which negatively impact culture quality and reproducibility throughout the entire 3D culture workflow, from seeding to culture maintenance, and post-culture analyses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/678315v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19f976eorg.highwire.dtl.DTLVardef@8eeefcorg.highwire.dtl.DTLVardef@1ebe6c7org.highwire.dtl.DTLVardef@7c4403_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Miniaturized 3D hanging-drop matrix-embedded organoid culture in a 384-well plate - Custom cartridge enables homogeneous bioprinting of organoids in sBME-based matrix - 3D-printed tools support compact, scalable multiwell culture systems - System suited for miniaturized culture organoids for high-throughput drug screening - Scalable miniaturized culture system for extended periods of time

bioengineering↗

bioRxiv2025

A Vascularized Liver Microphysiological System Captures Key Features of Hepatic Insulin Resistance and Monocyte Infiltration

Three-dimensional in vitro liver models are a promising means to recapitulate key aspects of human liver disease pathologies, thereby aiding therapeutic development. Spheroidal aggregates of hepatocytes, sometimes including non-parenchymal cells, are an established approach for modeling facets of metabolism and drug responses, yet these models often lack dynamic interactions with vascular and immune cells that also contribute to disease development and progression. To address this, we developed a microphysiological system (MPS) that integrates multicellular human hepatic spheroids with self-organized microvascular networks. We show extensive interaction between primary human spheroids and functional vasculature while maintaining key hepatic functions. We demonstrate the utility of this MPS by modeling an insulin resistance state through chronic exposure to disease-mimetic media conditions. This disease model displays altered hepatocyte metabolism, dysregulated vascular features, and increased inflammation state. Enabled by the functional vasculature, we further extend this disease model to capture changes in immune cell recruitment. When culturing CD14+ monocytes in our liver MPS, a subset of monocytes extravasate, localize to hepatic spheroids, and begin differentiating into CD163+ macrophages. These cells infiltrate with greater frequency in insulin resistant samples, consistent with known clinical findings. All together, this vascularized MPS model captures disease-relevant liver biology including inflammatory features.

bioengineering↗

bioRxiv2025

Myofibroblasts reduce angiogenesis and vasculogenesis in a vascularized microphysiological model of lung fibrosis

Lung fibrosis, characterized by chronic and progressive scarring, has no cure. Hallmarks are the accumulation of myofibroblasts and extracellular matrix, as well as vascular remodeling. The crosstalk between myofibroblasts and vasculature is poorly understood, with conflicting reports on whether angiogenesis and vessel density are increased or decreased in lung fibrosis. We developed a microphysiological system that recapitulates the pathophysiology of lung fibrosis and disentangles myofibroblast-vascular interactions. Lung myofibroblasts maintained their phenotype in 3D without exogenous TGF-{beta} and displayed anti-angiogenic and anti-vasculogenic activities when cultured with endothelial cells in a microfluidic device. These effects, including decreased endothelial sprouting, altered vascular morphology, and increased vascular permeability, were mediated by increased TGF-{beta}1 and reduced VEGF secretion. Pharmacological interventions targeting these cytokines restored vascular morphology and permeability, demonstrating the potential of this model to screen anti-fibrotic drugs. This system provides insights into myofibroblast-vascular crosstalk in lung fibrosis and offers a platform for therapeutic development.

bioengineering↗

bioRxiv2024

miR126-mediated impaired vascular integrity in Rett syndrome

Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by mutations in melty-CpG binding protein 2 (MeCP2). MeCP2 is a non-cell type-specific DNA binding protein, and its mutation influences not only neural cells but also non-neural cells in the brain, including vasculature associated with endothelial cells. Vascular integrity is crucial for maintaining brain homeostasis, and its alteration may be linked to the pathology of neurodegenerative disease, but a non-neurogenic effect, especially the relationship between vascular alternation and Rett syndrome pathogenesis, has not been shown. Here, we recapitulate a microvascular network using Rett syndrome patient-derived induced pluripotent stem (iPS) cells that carry MeCP2[R306C] mutation to investigate early developmental vascular impact. To expedite endothelial cell differentiation, doxycycline (DOX)-inducible ETV2 expression vectors were inserted into the AAVS1 locus of Rett syndrome patient-derived iPS cells and its isogenic control by CRISPR/Cas9. With these endothelial cells, we established a disease microvascular network (Rett-dMVNs) and observed higher permeability in the Rett-dMVNs compared to isogenic controls, indicating altered barrier function by MeCP2 mutation. Furthermore, we unveiled that hyperpermeability is involved in the upregulation of miR126-3p in Rett syndrome patient-derived endothelial cells by microRNA profiling and RNAseq, and rescue of miR126-3p level can recover their phenotype. We discover miR126-3p-mediated vascular impairment in Rett syndrome patients and suggest the potential application of these findings for translational medicine.

cell biology↗

bioRxiv2024

Ether lipids influence cancer cell fate by modulating iron uptake

Cancer cell fate has been widely ascribed to mutational changes within proteincoding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with high metastatic ability and cancer stem celllike traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, resulting in significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro threedimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids from plasma membranes also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

cancer biology↗

bioRxiv2023

Patient-Specific Vascularized Tumor Model: Blocking TAM Recruitment with Multispecific Antibodies Targeting CCR2 and CSF-1R

Tumor-associated inflammation drives cancer progression and therapy resistance, with the infiltration of monocyte-derived tumor-associated macrophages (TAMs) associated with poor prognosis in diverse cancers. Targeting TAMs holds potential against solid tumors, but effective immunotherapies require testing on immunocompetent human models prior to clinical trials. Here, we develop an in vitro model of microvascular networks that incorporates tumor spheroids or patient tissues. By perfusing the vasculature with human monocytes, we investigate monocyte trafficking into the tumor and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via TAM-produced CCL7 and CCL2, mediated by CSF-1R. Additionally, we assess a novel multispecific antibody targeting CCR2, CSF-1R, and neutralizing TGF-{beta}, referred to as CSF1R/CCR2/TGF-{beta} Ab, on monocytes and macrophages using our 3D models. This antibody repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and effectively blocks monocyte migration. Finally, we show that the CSF1R/CCR2/TGF-{beta} Ab inhibits monocyte recruitment in patient-specific vascularized tumor models. Overall, this vascularized tumor model offers valuable insights into monocyte recruitment and enables functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment (TME).

bioengineering↗

bioRxiv2023

Engineering microvascular networks using a KLF2 reporter to probe flow-dependent endothelial cell function

Shear stress generated by the flow of blood in the vasculature is a potent regulator of endothelial cell phenotype and vascular structure. While vascular responses to flow are complex and context-dependent, endothelial cell signaling in response to shear stress induced by laminar flows is coordinated by the transcription factor KLF2. The expression of KLF2 in endothelial cells is associated with a quiescent, anti-inflammatory phenotype and has been well characterized in two-dimensional systems, but has not been studied in three-dimensional in vitro systems. Here we develop engineered microvascular networks (MVNs) with a KLF2-based endothelial cell sensor within a microfluidic chip, apply continuous flow using an attached microfluidic pump, and study the effects of this flow on vascular structure and function. We found that culture of MVNs exposed to flow for 48 hours that resulted in increased expression of the KLF2-GFP-reporter display larger vessel diameters and decreased vascular branching and resistance. Additionally, vessel diameters after the application of flow were independent of initial MVN morphologies. Finally, we found that MVNs exposed to flow have improved vascular barrier function and decreased platelet adhesion. The MVNs with KLF2-based flow sensors represent a powerful tool for evaluating the structural and functional effects of flow on engineered three-dimensional vascular systems.

bioengineering↗

bioRxiv2022

On-chip engineered human lymphatic microvasculature for physio-/pathological transport phenomena studies

The human vasculature constitutes an integral part of fluid, protein and cellular transport throughout a variety of physiological processes and pathological events. While the blood vascular system has been the topic of numerous studies in connection to its role in physio-/pathological transport phenomena, our secondary vascular system, the lymphatics, has yet to gain similar attention, in part due to a lack of adequate models to study its biological function. Despite their considerable value, animal models limit the ability to perform parametric studies, whereas current in vitro systems are lacking in physiological mimicry. Here, a microfluidic-based approach is developed that allows for precise control over the transport of growth factors and interstitial fluid flow, which we leverage to recapitulate the in vivo growth of lymphatic capillaries. Using this approach, physiological tissue functionality is validated by characterizing the drainage rate of extracellular solutes and proteins. Finally, lymphatic-immune interactions are studied to affirm inflammation-driven responses by the lymphatics, which recruit immune cells via chemotactic signals, similarly to in vivo, pathological events. Results demonstrate the utility of this platform to study lymphatic biology and disease, as well as use as a screening assay to predict lymphatic absorption of therapeutic biologics.

bioengineering↗

bioRxiv2021

Personalized models of breast cancer desmoplasia reveal biomechanical determinants of drug penetration

Breast cancer desmoplasia heterogeneity contributes to high disease mortality due to discrepancies in treatment efficacy between patients. Personalized in vitro breast cancer models can be used for high throughput testing and ranking of therapeutic strategies to normalize the aberrant microenvironment in a patient-specific manner. Here, tumoroids assembled from patient-derived cells cultured in microphysiological systems including perfusable microvasculature reproduce key aspects of stromal and vascular dysfunction. Increased hyaluronic acid and collagen deposition, loss of vascular glycocalyx and reduced perfusion, and elevated interstitial fluid pressure in the models result in impaired drug distribution to tumor cells. We demonstrate the application of these personalized models as tools to rank molecular therapies for the normalization of the tumoroid microenvironment and to discover new therapeutic targets such as IL8 and CD44, which may ultimately improve drug efficacy in breast cancer patients.

bioengineering↗

bioRxiv2021

A predictive microfluidic model of human glioblastoma to assess trafficking of blood-brain barrier penetrant nanoparticles

The blood-brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood-brain barrier vasculature. Here, we report a vascularized human glioblastoma (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood-brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood-brain barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics. Significance StatementThe blood-brain barrier represents a major therapeutic challenge for the treatment of glioblastoma, and there is an unmet need for in vitro models that recapitulate human biology and are predictive of in vivo response. Here we present a new microfluidic model of vascularized glioblastoma featuring a tumor spheroid in direct contact with self-assembled vascular networks comprised of human endothelial cells, astrocytes, and pericytes. This model was designed to accelerate the development of targeted nanotherapeutics, and enabled rigorous assessment of a panel of surface-functionalized nanoparticles designed to exploit a receptor overexpressed in tumor-associated vasculature. Trafficking and efficacy data in the in vitro model compared favorably to parallel in vivo data, highlighting the utility of the vascularized glioblastoma model for therapeutic development.

bioengineering↗

bioRxiv2021

Modeling the three-way feedback between cellular contractility, actin polymerization, and adhesion turnover resolves the contradictory effects of RhoA and Rac1 on endothelial junction dynamics

The formation and recovery of gaps in the vascular endothelium governs a wide range of physiological and pathological phenomena, from angiogenesis to atherosclerosis and tumor cell extravasation. However, the interplay between the mechanical and signaling processes that drive dynamic behavior in vascular endothelial cells is not well understood. In this study, we propose a chemo-mechanical model to investigate the maintenance of endothelial junctions as dependent on the crosstalk between actomyosin contractility, VE-cadherin bond turnover, and actin polymerization, which mediate the forces exerted on the cell-cell interface. Our theoretical model reveals that active cell tension can stabilize cadherin bonds within an adhesion, but excessive RhoA signaling can drive bond dissociation and junction failure. While Rac1-mediated actin polymerization aids gap closure, high levels of Rac1 may also facilitate junction weakening. Combining the modeling framework with novel experiments, we identify how dynamic rupture and heal cycles emerge and, further, describe why gaps tend to localize at multi-cell contacts. Beyond, our analysis also indicates that a critical balance between RhoA and Rac1 expression is required to maintain junction stability and limit endothelial dysfunction. The model predicts how pharmacological modulation of actin polymerization and cell contractility impacts junction stability, with predictions subsequently validated experimentally. Our proposed framework can help guide the development of therapeutics that target the Rho family of GTPases and downstream active mechanical processes.

biophysics↗

bioRxiv2021

Physiologic flow-conditioning limits vascular dysfunction in engineered human capillaries

Hemodynamics play a central role in the health and disease of the coronary and peripheral vascular systems. Vessel-lining endothelial cells are known mechanosensors, responding to disturbances in flow - with mechanosensitivity hypothesized to change in response to metabolic demands. The health of our smallest microvessels have been lauded as a prognostic marker for cardiovascular health. Yet, despite numerous animal models, studying these small vessels has proved difficult. Microfluidic technologies have allowed a number of 3D vascular models to be developed and used to investigate human vessels. Here, two such systems are employed for examining 1) interstitial flow effects on neo-vessel formation, and 2) the effects of flow-conditioning on vascular remodelling following sustained static culture. Interstitial flow is shown to enhance early vessel formation via significant remodeling of vessels and interconnected tight junctions of the endothelium. In formed vessels, continuous flow maintains a stable vascular diameter and causes significant remodeling, contrasting the continued anti-angiogenic decline of statically cultured vessels. This study is the first to couple complex 3D computational flow distributions and microvessel remodeling from microvessels grown on-chip (exposed to flow or no-flow conditions). Flow-conditioned vessels (WSS < 1Pa for 30 micron vessels) increase endothelial barrier function, result in significant changes in gene expression and reduce reactive oxygen species and anti-angiogenic cytokines. Taken together, these results demonstrate microvessel mechanosensitivity to flow-conditioning, which limits deleterious vessel regression in vitro, and could have implications for future modeling of reperfusion/no-flow conditions.

bioengineering↗

bioRxiv2020

Integrated in silico and 3D in vitro model of macrophage migration in response to physical and chemical factors in the tumor microenvironment

Macrophages are abundant in the tumor microenvironment (TME), serving as accomplices to cancer cells for their invasion. Studies have explored the biochemical mechanisms that drive pro-tumor macrophage functions, however the role of TME interstitial flow (IF) is often disregarded. Therefore, we developed a three-dimensional microfluidic-based model with tumor cells and macrophages to study how IF affects macrophage migration and its potential contribution to cancer invasion. The presence of either tumor cells or IF individually increased macrophage migration directedness and speed. Interestingly, there was no additive effect on macrophage migration directedness and speed under the simultaneous presence of tumor cells and IF. Further, we present an in silico model that couples chemokine-mediated signaling with mechanosensing networks to explain our in vitro observations. The model proposes IL-8, CCL2 and {beta}-integrin as key pathways that commonly regulate various Rho GTPases. In agreement, in vitro macrophage migration remained elevated when exposed to a saturating concentration of recombinant IL-8 or CCL2, or to the co-addition of a sub-optimal concentration of both cytokines. Moreover, antibody blockade against IL-8 and/or CCL2 inhibited migration that could be restored by IF, indicating cytokine-independent mechanisms of migration induction. Importantly, we demonstrate the utility of an integrated in silico and 3D in vitro approach to aid the design of tumor-associated macrophage-based immunotherapeutic strategies.

systems biology↗

bioRxiv2017

Non-Elastic Remodeling of the 3D Extracellular Matrix by Cell-Generated Forces

The mechanical properties of the extracellular matrix (ECM) - a complex, 3D, fibrillar scaffold of cells in physiological environments - modulate cell behavior and can drive tissue morphogenesis, regeneration, and disease progression. For simplicity, it is often convenient to assume these properties to be time-invariant. In living systems, however, cells dynamically remodel the ECM and create time-dependent local environments. Here, we demonstrate that cell generated contractile forces are capable of producing substantial irreversible changes to the density and architecture of physiologically relevant ECMs - collagen I and fibrin - in a matter of minutes. We measure the 3D mechanical deformation profiles of the ECM surrounding cancer and endothelial cells during stages when force generation is active or inactive. We further correlate these measurements to both discrete fiber simulations that incorporate fiber crosslink unbinding kinetics and continuum-scale modeling. Our findings reveal that plasticity, as a mechanical law in these networks, is fundamentally related to the force-driven unbinding of fiber crosslinks. These results illustrate the dynamic nature of the mechanical environment of physiologically mimicking cell-in-gel systems.

bioengineering↗