Interstitial fluid pressure gradients and interstitial flow have been shown to drive morphogenic processes that shape tissues and influence progression of diseases including cancer. The advent of porous media microfluidic approaches has enabled investigation of the cellular response to interstitial flow, but questions remain as to the critical biophysical and biochemical signals imparted by interstitial fluid pressure gradients and resulting flow on resident cells and extracellular matrix (ECM). Here, we introduce a low-cost method to maintain physiological interstitial fluid pressures that is built from commonly accessible laboratory equipment, including a laser pointer, camera, Arduino board, and a commercially available linear actuator. We demonstrate that when the system is connected to a microfluidic device containing a 3D porous hydrogel, physiologic pressure is maintained with sub-Pascal resolution and when basic feedback control is directed using an Arduino, constant pressure and pressure gradient can be maintained even as cells remodel and degrade the ECM hydrogel over time. Using this model, we characterized breast cancer cell growth and ECM changes to ECM fibril structure and porosity in response to constant interstitial fluid pressure or constant interstitial flow. We observe increased collagen fibril bundling and the formation of porous structures in the vicinity of cancer cells in response to constant interstitial fluid pressure as compared to constant interstitial flow. Collectively, these results further define interstitial fluid pressure as a driver of key pathogenic responses in cells, and the systems and methods developed here will allow for future mechanistic work investigating mechanotransduction of interstitial fluid pressures and flows.
Somatic activating mutations of PIK3CA are associated with development of vascular malformations (VMs). Here, we describe a microfluidic model of PIK3CA-driven VMs consisting of human umbilical vein endothelial cells expressing PIK3CA activating mutations embedded in three-dimensional hydrogels. We observed enlarged, irregular vessel phenotypes and the formation of cyst-like structures consistent with clinical signatures and not previously observed in cell culture models. Pathologic morphologies occurred concomitant with up-regulation of Rac1/p21-activated kinase (PAK), mitogen-activated protein kinase cascades (MEK/ERK), and mammalian target of rapamycin (mTORC1/2) signaling networks. We observed differential effects between alpelisib, a PIK3CA inhibitor, and rapamycin, an mTORC1 inhibitor, in mitigating matrix degradation and network topology. While both were effective in preventing vessel enlargement, rapamycin failed to reduce MEK/ERK and mTORC2 activity and resulted in hyperbranching, while inhibiting PAK, MEK1/2, and mTORC1/2 mitigates abnormal growth and vascular dilation. Collectively, these findings demonstrate an in vitro platform for VMs and establish a role of dysregulated Rac1/PAK and mTORC1/2 signaling in PIK3CA-driven VMs.
TOR Serine-Threonine Kinases , Vascular Malformations , Humans , TOR Serine-Threonine Kinases/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Sirolimus/pharmacology , Human Umbilical Vein Endothelial Cells/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Class I Phosphatidylinositol 3-Kinases/genetics , Class I Phosphatidylinositol 3-Kinases/metabolism , Vascular Malformations/metabolism , rac1 GTP-Binding Protein/metabolism
Efficient delivery of oxygen and nutrients to tissues requires an intricate balance of blood, lymphatic, and interstitial fluid pressures (IFPs), and gradients in fluid pressure drive the flow of blood, lymph, and interstitial fluid through tissues. While specific fluid mechanical stimuli, such as wall shear stress, have been shown to modulate cellular signaling pathways along with gene and protein expression patterns, an understanding of the key signals imparted by flowing fluid and how these signals are integrated across multiple cells and cell types in native tissues is incomplete due to limitations with current assays. Here, we introduce a multi-layer microfluidic platform (MµLTI-Flow) that enables the culture of engineered blood and lymphatic microvessels and independent control of blood, lymphatic, and IFPs. Using optical microscopy methods to measure fluid velocity for applied input pressures, we demonstrate varying rates of interstitial fluid flow as a function of blood, lymphatic, and interstitial pressure, consistent with computational fluid dynamics (CFD) models. The resulting microfluidic and computational platforms will provide for analysis of key fluid mechanical parameters and cellular mechanisms that contribute to diseases in which fluid imbalances play a role in progression, including lymphedema and solid cancer.
Lymphatic Vessels , Microfluidics , Microfluidics/methods , Stress, Mechanical
Extravasation of circulating cells is an essential process that governs tissue inflammation and the body's response to pathogenic infection. To initiate anti-inflammatory and phagocytic functions within tissues, immune cells must cross the vascular endothelial barrier from the vessel lumen to the subluminal extracellular matrix. In this work, we present a microfluidic approach that enables the recreation of a three-dimensional, perfused endothelial vessel formed by human endothelial cells embedded within a collagen-rich matrix. Monocytes are introduced into the vessel perfusate, and we investigate the role of luminal flow and collagen concentration on extravasation. In vessels conditioned with the flow, increased monocyte adhesion to the vascular wall was observed, though fewer monocytes extravasated to the collagen hydrogel. Our results suggest that the lower rates of extravasation are due to the increased vessel integrity and reduced permeability of the endothelial monolayer. We further demonstrate that vascular permeability is a function of collagen hydrogel mass concentration, with increased collagen concentrations leading to elevated vascular permeability and increased extravasation. Collectively, our results demonstrate that extravasation of monocytes is highly regulated by the structural integrity of the endothelial monolayer. The microfluidic approach developed here allows for the dissection of the relative contributions of these cues to further understand the key governing processes that regulate circulating cell extravasation and inflammation.
Ascites refers to the abnormal accumulation of fluid in the peritoneum resulting from an underlying pathology, such as metastatic cancer. Among all cancers, advanced-stage epithelial ovarian cancer is most frequently associated with the production of malignant ascites and is the leading cause of death from gynecologic malignancies. Despite decades of evidence showing that the accumulation of peritoneal fluid portends the poorest outcomes for cancer patients, the role of malignant ascites in promoting metastasis and therapy resistance remains poorly understood. This review summarizes the current understanding of malignant ascites, with a focus on ovarian cancer. The first section provides an overview of heterogeneity in ovarian cancer and the pathophysiology of malignant ascites. Next, analytical methods used to characterize the cellular and acellular components of malignant ascites, as well the role of these components in modulating cell biology, are discussed. The review then provides a perspective on the pressures and forces that tumors are subjected to in the presence of malignant ascites and the impact of physical stress on therapy resistance. Treatment options for malignant ascites, including surgical, pharmacological and photochemical interventions are then discussed to highlight challenges and opportunities at the interface of drug discovery, device development and physical sciences in oncology.
Vascularization of large, diffusion-hindered biomaterial implants requires an understanding of how extracellular matrix (ECM) properties regulate angiogenesis. Sundry biomaterials assessed across many disparate angiogenesis assays have highlighted ECM determinants that influence this complex multicellular process. However, the abundance of material platforms, each with unique parameters to model endothelial cell (EC) sprouting presents additional challenges of interpretation and comparison between studies. In this work we directly compared the angiogenic potential of commonly utilized natural (collagen and fibrin) and synthetic dextran vinyl sulfone (DexVS) hydrogels in a multiplexed angiogenesis-on-a-chip platform. Modulating matrix density of collagen and fibrin hydrogels confirmed prior findings that increases in matrix density correspond to increased EC invasion as connected, multicellular sprouts, but with decreased invasion speeds. Angiogenesis in synthetic DexVS hydrogels, however, resulted in fewer multicellular sprouts. Characterizing hydrogel Young's modulus and permeability (a measure of matrix porosity), we identified matrix permeability to significantly correlate with EC invasion depth and sprout diameter. Although microporous collagen and fibrin hydrogels produced lumenized sprouts in vitro, they rapidly resorbed post-implantation into the murine epididymal fat pad. In contrast, DexVS hydrogels proved comparatively stable. To enhance angiogenesis within DexVS hydrogels, we incorporated sacrificial microgels to generate cell-scale pores throughout the hydrogel. Microporous DexVS hydrogels resulted in lumenized sprouts in vitro and enhanced cell invasion in vivo. Towards the design of vascularized biomaterials for long-term regenerative therapies, this work suggests that synthetic biomaterials offer improved size and shape control following implantation and that tuning matrix porosity may better support host angiogenesis. STATEMENT OF SIGNIFICANCE: Understanding how extracellular matrix properties govern angiogenesis will inform biomaterial design for engineering vascularized implantable grafts. Here, we utilized a multiplexed angiogenesis-on-a-chip platform to compare the angiogenic potential of natural (collagen and fibrin) and synthetic dextran vinyl sulfone (DexVS) hydrogels. Characterization of matrix properties and sprout morphometrics across these materials points to matrix porosity as a critical regulator of sprout invasion speed and diameter, supported by the observation that nanoporous DexVS hydrogels yielded endothelial cell sprouts that were not perfusable. To enhance angiogenesis into synthetic hydrogels, we incorporated sacrificial microgels to generate microporosity. We find that microporosity increased sprout diameter in vitro and cell invasion in vivo. This work establishes a composite materials approach to enhance the vascularization of synthetic hydrogels.
Biocompatible Materials , Neovascularization, Physiologic , Animals , Biocompatible Materials/pharmacology , Endothelial Cells , Extracellular Matrix , Hydrogels/pharmacology , Mice , Porosity
Mechanical forces regulate a diverse set of biological processes at cellular, tissue, and organismal length scales. Investigating the cellular and molecular mechanisms that underlie the conversion of mechanical forces to biological responses is challenged by limitations of traditional animal models and in vitro cell culture, including poor control over applied force and highly artificial cell culture environments. Recent advances in fabrication methods and material processing have enabled the development of microfluidic platforms that provide precise control over the mechanical microenvironment of cultured cells. These devices and systems have proven to be powerful for uncovering and defining mechanisms of mechanotransduction. In this review, we first give an overview of the main mechanotransduction pathways that function at sites of cell adhesion, many of which have been investigated with microfluidics. We then discuss how distinct microfluidic fabrication methods can be harnessed to gain biological insight, with description of both monolithic and replica molding approaches. Finally, we present examples of how microfluidics can be used to apply both solid forces (substrate mechanics, strain, and compression) and fluid forces (luminal, interstitial) to cells. Throughout the review, we emphasize the advantages and disadvantages of different fabrication methods and applications of force in order to provide perspective to investigators looking to apply forces to cells in their own research.
