Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Summary

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Abstract

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis¹. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration^2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Introduction

Many cellular processes have been extensively studied in 2 dimensions, thereby forming a collective knowledge of basic cell signaling mechanisms. These studies, however, neglect important cellular cues received from the structural and mechanical properties of the local cellular microenvironment and extracellular matrix (ECM). To better understand how cells interact within a physiological context, it is important to study them in 3-dimensional (3D) assays. The ECM for these 3D assays can either be cell-derived or reconstituted from purified proteins. Regardless of the source of the ECM, 3D matrix assays have proven to be invaluable for understanding how cells navigat....

Protocol

1. Neutralization, Dilution and Polymerization of Collagen Solutions for 3D Investigation and Cellular Contraction Assays

1. On ice in sterile tissue culture hood, neutralize collagen (1:1) with sterile, ice-cold 100 mM HEPES in 2x PBS, pH 7.4, in a 15 ml conical tube. Mix thoroughly with plastic pipette until solution is homogenous and mixing swirls are no longer visible. Be careful not to introduce air bubbles during the mixing process. Store briefly on ice.
2. Dilute neutralized collagen to the appropriate concentration with cell media (such as RPMI, DMEM, etc.).
   1. To calculate the amount of neutralized collagen required to make u....

Results

While 3D assays can be done within the same stiffness of collagen gel, varying the gel stiffness can be used to determine how the cells will respond to mechanical changes in their cellular microenvironment. A stiff collagen hydrogel is defined as a gel where the embedded cells are unable to locally contract the surrounding collagen. The intrinsic contractility of different cell types is unique, and thus it is best to begin with a simple contractility curve to establish the collagen concen.......

Discussion

3D collagen gels are a valuable addition to our toolbox to understand how cells interpret and respond to their local microenvironment. This manuscript has provided a very basic protocol for embedding cells within a 3D collagen matrix, and to reproducibly generate matrices with random or aligned collagen fibers. Both protocols work as adaptable platforms where different collagen isoforms, crosslinkers, or other matrix proteins could potentially be added at the time of polymerization. It is also easy to modify the platform.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
High Concentration Rat Tail Collagen	Corning	354249
SylGard184 elastomer kit	Corning	NC9285739	Elastomer for PDMS channels
HEPES	Fisher	BP310	For HEPES neutralization buffer
KCl	Fisher	BP366	For HEPES neutralization buffer
KH2PO4	Fisher	BP362	For HEPES neutralization buffer
Na2HPO4	Fisher	S374	For HEPES neutralization buffer
NaCl	Fisher	BP358	For HEPES neutralization buffer
Levy Improved Neubauer Hemacytometer	Fisher	15170-208	cell counting
6-well non-tissue culture plate	Corning	351146
50 mm glass bottom dish	MatTek	P50g-1.5-30-f
Bel-Art Plastic Vacuum Desiccator	Bel-Art	F4200-2021	Degassing chamber for PDMS
transparency film	3M	pp2950	Plastic film for pouring pdms channels
ThermoScientific CimaRec	ThermoScientific	HP141925	Hot plate for curing PDMS microchannels
Vacuum regulator	Precision Medical	PM3100	Vacuum regulator for collagen microchannels
8" x 8" rubber sheet	Amazon - Rubber-Cal	Silicone - 60A	rubber sheet for pouring PDMS microchannel
8" x 8" x 0.125" acrylic sheet	Amazon	Plexiglass sheets	for pouring PDMS microchannels
10 lb weights	Amazon	CAP Barbell	for pouring PDMS microchannels
15 ml Conical tubes	Fisher	352097
50 ml Conical tubes	Fisher	352098
Plastic pipets	Dot Scientific	229202B, 229206B, and 667225B	2 ml, 5 ml, and 25 ml
70% EtOH	Fisher	NC9663244