Name: microengineering 3d collagen hydrogels with long-range fiber alignment
Uploaded: 2022-09-07T14:45:00
Description: Watch this Scientific Journal Video about Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment Video at JoVE.com

This work was supported in part by the National Institute of Health under award number R21GM143658 and by the National Science Foundation under grant number 2150798. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

1. Fabrication of the two-piece channel and modular platform base
NOTE: The microfluidic channel is constructed using two parts &#8212; the microfluidic channel &#34;cutout&#34;,&#160;which is razor cut from a poly dimethyl siloxane (PDMS) sheet of defined thickness, and the channel cover, which reversibly bonds to the cutout and forms&#160;the channel. The channel is surrounded by a poly(methyl methacrylate) (PMMA) frame that will acts as a media reservoir (<strong class="xfig"...

Protocols to generate COL1 matrices with aligned fibers have been described using magnetic methods, the direct application of mechanical strain, and microfluidic techniques47. Microfluidic approaches are commonly used to create microphysiological systems because of their well-defined flow and transport characteristics, which enable precise control over the biochemical microenvironment. Since aligned COL1 fibers provide key instructive cues during pathophysiological processes such as wound healing,...

Cells reside in a complex 3D fibrous network called the extracellular matrix (ECM), the bulk of which is composed of the structural protein collagen type I (COL1)1,2. The biophysical properties of the ECM provide guidance cues to cells, and in response, cells remodel the ECM microarchitecture3,4,5. These reciprocal cell-matrix interactions can give rise to aligned COL1 fiber domains6 that promote angiogenesis and cell invasion in the tumor environment7,8,9 and influence cell morphology10,11,12, polarization13, and differentiation14. Aligned collagen fibers also promote wound healing15, play a key role in tissue development16, and contribute to long-range cell communication17,18. Therefore, replicating the native COL1 fiber microarchitecture in vitro is an important step toward developing structured models to study cell responses to aligned microenvironments.
Microfluidic cell culture systems have been established as a preferred technology to develop microphysiological systems (MPS)19,20,21,22,23. Leveraging favorable microscale scaling effects, these systems provide precise control over fluid flows, support the controlled introduction of mechanical forces, and define the biochemical microenvironment within a microchannel21,24,25,26,27. MPS platforms have been used to model tissue-specific microenvironments and study multi-organ interactions28. Simultaneously, hydrogels have been widely explored to recapitulate the 3D mechanics and biological influence of the ECM that are observed in vivo29,30. With a growing emphasis on integrating 3D culture with microfluidic platforms, numerous approaches can combine COL1 hydrogels in microfluidic devices31,32,33. However, the methods to align COL1 hydrogels in microfluidic channels have been limited to thin 2D &#34;mats&#34; (&#60;40 &#181;m in thickness) in channels &#60;1 mm wide, offering limited potential to model cell responses in aligned 3D microenvironments31,34,35,36.
To achieve aligned 3D COL1 hydrogels in a microfluidic system, it has been shown that, when a self-assembling COL1 solution is exposed to local extensional flows (velocity change along the streamwise direction), the resulting COL1 hydrogels display a degree of fiber alignment that is directly proportional to the magnitude of the extensional strain rate they experience37,38. The microchannel design in this protocol is unique in two ways; first, the segmented design introduces local extensional strain to the COL1 solution, and second, its &#34;two-piece&#34; construction allows the user to align COL1 fibers and then disassemble the channel to directly access the aligned fibers in an open format. This approach can further be adopted to develop modular microfluidic platforms that develop microphysiological systems with ordered COL1 matrices. The following protocol describes the process of fabricating segmented microchannels and details the use of the channels to align bovine atelo COL1. This protocol also provides instructions for culturing cells on COL1 in an open well format and discusses adding functionality to the platform using a modular, magnetic base layer.

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			<td>New Era Syringe Pumps</td>
			<td>NE-1002X</td>
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			<td>Microman E Single Channel Pipettor, Gilson, Model M1000E</td>
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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" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m 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.

When a self-assembling COL1 solution flows through a channel with decreasing cross-sectional area, the streamwise velocity (vx) of the COL1 solution increases locally by a magnitude, &#8706;vx, along the length of the constriction between the two segments (&#8706;x), resulting in an extensional strain rate (&#949;&#775;) where &#949;&#775; = &#8706;vx/&#8706;x. The extensional strain rate can be calculated from the fluid velocity, which is measured using particle image velocimetry (PIV), ...

Watch this Scientific Journal Video about Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment Video at JoVE.com

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment Video (Video) | JoVE

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

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 ...

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