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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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

Protocol

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 (

Representative Results

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, ∂vx, along the length of the constriction between the two segments (∂x), resulting in an extensional strain rate (ε̇) where ε̇ = ∂vx/∂x. The extensional strain rate can be calculated from the fluid velocity, which is measured using particle image velocimetry (PIV), .......

Discussion

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,.......

Acknowledgements

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.

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Materials

NameCompanyCatalog NumberComments
(3-Aminopropyl)triethoxysilane, 99% (APTES)Sigma Aldrich440140-100ML
20 Gauge IT Series Angled Dispensing TipJensen GlobalJG-20-1.0-90
3/16" dia. x 1/16" thick Nickel Plated MagnetKJ MagneticsD31
3M (TC) 12X12-6-467MPDigiKey3M9726-ND
ACETONE ACS REAGENT ≥99.5%Signa Aldrich179124-4L
BD-20AC LABORATORY CORONA TREATERElectro-Technic Products12051A
Bovine Serum Albumin (BSA), Fraction V, 98%, Reagent Grade, Alfa AesarVWRAAJ64100-09
Clear cast acrylic sheetMcMaster-Carr8560K181
Corning 100 mL Trypsin 10x, 2.5% Trypsin in HBSS [-] calcium, magnesium, phenol red, Porcine Parvovirus TestedVWR45000-666
Countess II Automated Cell CounterThermo Fisher ScientificAMQAX1000
CT-FIRE softwareLOCI - University of Wisconsin
EGM-2 Endothelial Cell Growth Medium-2 BulletKit, (CC-3156 & CC-4176), Lonza CC-3162, 500 mLLonzaCC-3162
Glutaraldehyde 50% in aqueous solution, Reagent Grade, Packaging=HDPE Bottle, Size=100 mLVWRVWRV0875-100ML
Graphtec CELITE-50GraphtecCE LITE-50
HEPES (1 M)Thermo Fisher Scientific15-630-080
High-Purity Silicone Rubber .010" Thick, 6" X 8" Sheet, 55A DurometerMcMaster-Carr87315K62
Human Umbilical Vein Endothelial cellsThermo Fisher ScientificC0035C
Invitrogen Trypan Blue Stain (0.4%)Thermo Fisher ScientificT10282
IsopropanolFisher ScientificA4154
Laser cutterFull Spectrum20x12 H-series
Microfluidics Syringe pumpNew Era Syringe PumpsNE-1002X
Microman E Single Channel Pipettor, Gilson, Model M1000EGilsonFD10006
Molecular Probes Alexa Fluor 488 PhalloidinThermo Fisher ScientificA12379
Molecular Probes Hoechst 33342, Trihydrochloride, TrihydrateThermo Fisher ScientificH3570
Nutragen Bovine Atelo CollagenAdvanced BioMatrix5010-50ML
Pbs (10x), pH 7.4VWR70011044.00
PBS pH 7.4Thermo Fisher Scientific10010049.00
Phosphate-buffered saline (PBS, 10x), with Triton X-100Alfa AesarJ63521
Replacement carrier sheet for graphtec craft ROBO CC330L-20USCUTTERGRPCARSHTN
Restek Norm-Ject Plastic Syringe 1 mL Luer SlipRestek22766.00
Silicon waferUniversity wafer452
Sodium Hydroxide, ACS, Packaging=Poly Bottle, Size=500 gVWRBDH9292-500G
Sylgard 184VWR102092-312
Thermo Scientific Pierce 20x PBS Tween 20Thermo Fisher Scientific28352.00

References

  1. Frantz, C., Stewart, K. M., Weaver, V. M. The extracellular matrix at a glance. Journal of Cell Science. 123 (24), 4195-4200 (2010).
  2. Bosman, F. T., Stamenkovic, I. Functional structure and composition of the extracellular matrix.

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3D Collagen HydrogelsFiber AlignmentMicrofluidicPDMSAminopropyl TriethoxysilaneGlutaraldehydeSurface ModificationTissue EngineeringTopography

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