Microengineering 3D Collagen Matrices with Tumor-Mimetic Gradients in Fiber Alignment.

Collagen fibers in the 3D tumor microenvironment (TME) exhibit complex alignment landscapes that are critical in directing cell migration through a process called contact guidance. Previous in vitro work studying this phenomenon has focused on quantifying cell responses in uniformly aligned environments. However, the TME also features short-range gradients in fiber alignment that result from cell-induced traction forces. Although the influence of graded biophysical taxis cues is well established, cell responses to physiological alignment gradients remain largely unexplored. In this work, fiber alignment gradients in biopsy samples are characterized and recreated using a new microfluidic biofabrication technique to achieve tunable sub-millimeter to millimeter scale gradients. This study represents the first successful engineering of continuous alignment gradients in soft, natural biomaterials. Migration experiments on graded alignment show that HUVECs exhibit increased directionality, persistence, and speed compared to uniform and unaligned fiber architectures. Similarly, patterned MDA-MB-231 aggregates exhibit biased migration toward increasing fiber alignment, suggesting a role for alignment gradients as a taxis cue. This user-friendly approach, requiring no specialized equipment, is anticipated to offer new insights into the biophysical cues that cells interpret as they traverse the extracellular matrix, with broad applicability in healthy and diseased tissue environments.

Microengineering 3D Collagen Matrices with Tumor-Mimetic Gradients in Fiber Alignment.

In the tumor microenvironment (TME), collagen fibers facilitate tumor cell migration through the extracellular matrix. Previous studies have focused on studying the responses of cells on uniformly aligned or randomly aligned collagen fibers. However, the in vivo environment also features spatial gradients in alignment, which arise from the local reorganization of the matrix architecture due to cell-induced traction forces. Although there has been extensive research on how cells respond to graded biophysical cues, such as stiffness, porosity, and ligand density, the cellular responses to physiological fiber alignment gradients have been largely unexplored. This is due, in part, to a lack of robust experimental techniques to create controlled alignment gradients in natural materials. In this study, we image tumor biopsy samples and characterize the alignment gradients present in the TME. To replicate physiological gradients, we introduce a first-of-its-kind biofabrication technique that utilizes a microfluidic channel with constricting and expanding geometry to engineer 3D collagen hydrogels with tunable fiber alignment gradients that range from sub-millimeter to millimeter length scales. Our modular approach allows easy access to the microengineered gradient gels, and we demonstrate that HUVECs migrate in response to the fiber architecture. We provide preliminary evidence suggesting that MDA-MB-231 cell aggregates, patterned onto a specific location on the alignment gradient, exhibit preferential migration towards increasing alignment. This finding suggests that alignment gradients could serve as an additional taxis cue in the ECM. Importantly, our study represents the first successful engineering of continuous gradients of fiber alignment in soft, natural materials. We anticipate that our user-friendly platform, which needs no specialized equipment, will offer new experimental capabilities to study the impact of fiber-based contact guidance on directed cell migration.

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment.

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (<40 µm in thickness) of COL1 fibers that extend over distances less than 500 µm and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 µm in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

The Modular µSiM Reconfigured: Integration of Microfluidic Capabilities to Study In Vitro Barrier Tissue Models under Flow.

Microfluidic tissue barrier models have emerged to address the lack of physiological fluid flow in conventional "open-well" Transwell-like devices. However, microfluidic techniques have not achieved widespread usage in bioscience laboratories because they are not fully compatible with traditional experimental protocols. To advance barrier tissue research, there is a need for a platform that combines the key advantages of both conventional open-well and microfluidic systems. Here, a plug-and-play flow module is developed to introduce on-demand microfluidic flow capabilities to an open-well device that features a nanoporous membrane and live-cell imaging capabilities. The magnetic latching assembly of this design enables bi-directional reconfiguration and allows users to conduct an experiment in an open-well format with established protocols and then add or remove microfluidic capabilities as desired. This work also provides an experimentally-validated flow model to select flow conditions based on the experimental needs. As a proof-of-concept, flow-induced alignment of endothelial cells and the expression of shear-sensitive gene targets are demonstrated, and the different phases of neutrophil transmigration across a chemically stimulated endothelial monolayer under flow conditions are visualized. With these experimental capabilities, it is anticipated that both engineering and bioscience laboratories will adopt this reconfigurable design due to the compatibility with standard open-well protocols.

A miniaturized 3D printed pressure regulator (µPR) for microfluidic cell culture applications.

Well-defined fluid flows are the hallmark feature of microfluidic culture systems and enable precise control over biophysical and biochemical cues at the cellular scale. Microfluidic flow control is generally achieved using displacement-based (e.g., syringe or peristaltic pumps) or pressure-controlled techniques that provide numerous perfusion options, including constant, ramped, and pulsed flows. However, it can be challenging to integrate these large form-factor devices and accompanying peripherals into incubators or other confined environments. In addition, microfluidic culture studies are primarily carried out under constant perfusion conditions and more complex flow capabilities are often unused. Thus, there is a need for a simplified flow control platform that provides standard perfusion capabilities and can be easily integrated into incubated environments. To this end, we introduce a tunable, 3D printed micro pressure regulator (µPR) and show that it can provide robust flow control capabilities when combined with a battery-powered miniature air pump to support microfluidic applications. We detail the design and fabrication of the µPR and: (i) demonstrate a tunable outlet pressure range relevant for microfluidic applications (1-10 kPa), (ii) highlight dynamic control capabilities in a microfluidic network, (iii) and maintain human umbilical vein endothelial cells (HUVECs) in a multi-compartment culture device under continuous perfusion conditions. We anticipate that our 3D printed fabrication approach and open-access designs will enable customized µPRs that can support a broad range of microfluidic applications.

Local extensional flows promote long-range fiber alignment in 3D collagen hydrogels.

Randomly oriented type I collagen (COL1) fibers in the extracellular matrix are reorganized by biophysical forces into aligned domains extending several millimeters and with varying degrees of fiber alignment. These aligned fibers can transmit traction forces, guide tumor cell migration, facilitate angiogenesis, and influence tissue morphogenesis. To create aligned COL1 domains in microfluidic cell culture models, shear flows have been used to align thin COL1 matrices (<50µm in height) in a microchannel. However, there has been limited investigation into the role of shear flows in aligning 3D hydrogels (>130µm). Here, we show that pure shear flows do not induce fiber alignment in 3D atelo COL1 hydrogels, but the simple addition of local extensional flow promotes alignment that is maintained across several millimeters, with a degree of alignment directly related to the extensional strain rate. We further advance experimental capabilities by addressing the practical challenge of accessing a 3D hydrogel formed within a microchannel by introducing a magnetically coupled modular platform that can be released to expose the microengineered hydrogel. We demonstrate the platform's capability to pattern cells and fabricate multi-layered COL1 matrices using layer-by-layer fabrication and specialized modules. Our approach provides an easy-to-use fabrication method to achieve advanced hydrogel microengineering capabilities that combine fiber alignment with biofabrication capabilities.

Microengineered 3D Collagen Gels with Independently Tunable Fiber Anisotropy and Directionality.

Cellular processes, including differentiation, proliferation, and migration, have been linked to the alignment (anisotropy) and orientation (directionality) of collagen fibers in the native extracellular matrix (ECM). Given the critical role that biophysical cell-matrix interactions play in regulating biological functions, several microfluidic-based methods have been used to establish 3D collagen gels with defined fiber properties; these gels have helped to establish quantitative relationships between structural ECM cues and observed cell responses. Although existing microfluidic fabrication methods provide excellent definition over collagen fiber anisotropy, they have not demonstrated the independent control over fiber anisotropy and directionality necessary to replicate in vivo collagen architecture. Therefore, to advance collagen microengineering capabilities, we present a user-friendly technology platform that uses controlled fluid flows within a non-uniform microfluidic channel network to create collagen landscapes that can be tuned as a function of extensional strain rate. Herein, we demonstrate capabilities to i) control the degree of fiber anisotropy, ii) create spatial gradients in fiber anisotropy, iii) independently define fiber directionality, and iv) generate multi-material interfaces within a 3D environment. We then address the practical issue of integrating cells into microfluidic systems by using a peel-off template technique to provide direct access to microengineered collagen gels, and demonstrate that cells respond to the defined properties of the landscape. Finally, the platform's modular capability is highlighted by integrating a sub-micrometer thick porous parylene membrane onto the microengineered collagen as a method to define cell-substrate interactions.

Engineering fiber anisotropy within natural collagen hydrogels.

It is well known that biophysical properties of the extracellular matrix (ECM), including stiffness, porosity, composition, and fiber alignment (anisotropy), play a crucial role in controlling cell behavior in vivo. Type I collagen (collagen I) is a ubiquitous structural component in the ECM and has become a popular hydrogel material that can be tuned to replicate the mechanical properties found in vivo. In this review article, we describe popular methods to create 2-D and 3-D collagen I hydrogels with anisotropic fiber architectures. We focus on methods that can be readily translated from engineering and materials science laboratories to the life-science community with the overall goal of helping to increase the physiological relevance of cell culture assays.