Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

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 (<250 µm in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

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.

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 microarchitecture^3,4,5. These reciprocal cell-matrix interactions can give rise to aligned COL1 fiber domains⁶ that promote angiogenesis and cell invasion in the tumor environment⁷

1. Fabrication of the two-piece channel and modular platform base

NOTE: The microfluidic channel is constructed using two parts — the microfluidic channel "cutout", 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 the channel. The channel is surrounded by a poly(methyl methacrylate) (PMMA) frame that will acts as a media reservoir (

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

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,.......

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.