Biology subjects

Joshi, I. M.

Publications and source records attributed to Joshi, I. M..

6 recordsLinked to original sources

bioRxiv2026

Fiber alignment alone is insufficient to predict cellular contact guidance responses in engineered 3D collagen hydrogels

In breast cancer, collagen fiber alignment and the second harmonic generation forward-to-backward ratio (F/B), an optical measurement sensitive to fibril-scale organization within collagen fibers, have each been associated with metastatic outcome. Although both are measured at the tumor-stromal interface, in vitro studies have largely examined them separately, because experimental systems have not allowed the two to be varied independently. To address this technical gap, we used a microfluidic biofabrication approach to generate paired aligned and unaligned regions within 3D collagen hydrogels via extensional strain and varied measured F/B across gels by changing the pre-gel pH. The resulting library covered alignment values reported in breast tumors and F/B values associated with poorer metastatic outcome in invasive ductal carcinoma at matched matrix stiffness, pore diameter, and fiber fraction. In MDA-MB-231 cells, alignment biased migration along the fiber axis without changing migration speed, as expected, but the magnitude of the contact guidance response varied with measured F/B. In contrast, MCF-7 cells showed little overall contact guidance, yet migrated ~30% faster in high F/B than low F/B gels in both aligned and unaligned regions. Together, these results show that cells migrated differently in matrices with similar fiber alignment. Thus, including measured F/B may provide additional information to help anticipate how cells migrate in collagen environments that appear similar based on alignment alone.

bioengineering↗

bioRxiv2023

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.

bioengineering↗

bioRxiv2022

A Miniaturized 3D-Printed Pressure Regulator (μPR) for Microfluidic Cell Culture Applications

Controlled fluid flows are the hallmark feature of microfluidic culture systems and provide precise definition over the biophysical and biochemical microenvironment. Flow control is commonly achieved using displacement-based (e.g., syringe or peristaltic pumps) or pressure-based techniques. These methods offer complex flow capabilities but can be challenging to integrate into incubators or other confined environments due to their large form factors and accompanying peripheral equipment. Since many microfluidic cell culture studies use a single controlled flow rate to maintain or stimulate cells, a portable flow control platform that fits easily into an incubator will benefit the microfluidic community. Here, we demonstrate that a tunable, 3D printed micro pressure regulator (PR), combined with a battery-powered miniature air pump, can operate as a stand-alone pneumatic flow control platform for microfluidic applications. We detail the design and fabrication of the PR and demonstrate: i) a tunable outlet pressure range relevant for microfluidic applications (1-10 kPa), ii) highlight dynamic control in a microfluidic network, and iii) maintain human umbilical vein endothelial cells (HUVECs) in a multi-compartment membrane-based culture device under continuous flow conditions. We anticipate that our 3D-printed fabrication approach and open access designs will allow other laboratories to rapidly customize PRs to support a broad range of applications.

bioengineering↗

bioRxiv2022

The Modular μSiM Reconfigured: Integration of Microfluidic Capabilities to Study in vitro Barrier Tissue Models under Flow

Microfluidic approaches to study tissue barriers have emerged to address the lack of 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, tried-and-true experimental protocols. To advance barrier tissue research, there is a need for a platform that combines the advantages of both conventional open-well and microfluidic systems. Here, we develop a plug-and-play flow module to add on-demand microfluidic capabilities to a modular microfluidic system featuring a silicon membrane "m-SiM" as an open-well device with live-cell imaging capabilities. The magnetic latching assembly of our design enables bi-directional reconfiguration between open-well and fluidic modes. This design feature allows users to conduct an experiment in an open-well format with established protocols and then add or remove microfluidic capabilities as desired. Our work also provides an experimentally-validated flow model to help select desired flow conditions based on the experimental needs. As a proof-of-concept, we demonstrate flow-induced alignment of endothelial cells and visualize different phases of neutrophil transmigration across an endothelial monolayer under flow. We anticipate that our reconfigurable design will be adopted by both engineering and bioscience laboratories due to the compatibility with standard open-well protocols and the simple flow addition capabilities.

bioengineering↗

bioRxiv2022

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

Randomly oriented type I collagen (COL1) fibers in the extracellular matrix (ECM) 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 (<50m in height) in a microchannel. However, there has been limited investigation into the role of shear flows in aligning 3D hydrogels (>130m). 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 platforms 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.

bioengineering↗

bioRxiv2020

Microengineered three-dimensional collagen landscapes with independently tunable fiber anisotropy and directionality

Fibrillar collagens are structural proteins in the extracellular matrix (ECM), and cellular processes, including differentiation, proliferation, and migration, have been linked to the orientation (directionality) and alignment (anisotropy) of collagen fibers. Given the importance of cell-substrate interactions in driving biological functions, several microfluidic approaches have demonstrated three-dimensional (3D) collagen gels with defined fiber properties that enable quantitative correlations between structural cues and observed cell responses. Although existing methods provide excellent definition over collagen fiber anisotropy, independent control over both anisotropy and directionality (that we collectively refer to as the collagen landscape) has not been demonstrated. Therefore, to advance collagen microengineering capabilities, we present a user-friendly approach that uses controlled fluid flows within a non-uniform microfluidic channel network to create well-defined collagen landscapes. We demonstrate capabilities including i) control over fiber anisotropy, ii) spatial gradients in fiber anisotropy, iii) defined fiber directionality, and iv) multi-material interfaces. We then show that cells respond to the microengineered topographic cues by aligning along the anisotropy domains and following fiber directionality. Finally, this platforms modular capability is demonstrated by integrating an ultrathin porous parylene (UPP) membrane on the microengineered collagen as a mask to control cell-substrate interactions.

bioengineering↗