Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Summary

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Abstract

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Introduction

Like cell fusion (for an overview see ^1,2) cell migration is another biological phenomenon that is involved in a plethora of physiological processes including embryonic development, wound healing and immune responses (for review see ³). However, the ability to migrate is also a prerequisite for tumor cells to metastasize (for review see ^3,4).

Cell migration is a complex, not yet fully understood process that is directed by the interplay of several signal transduction pathways initiated by various ligand (e.g., cytokines, chemokines, growth factors, hormones, extracellular matrix compo....

Protocol

1. Preparation of Migration Chambers

1. Prepare a paraffin wax/petroleum jelly (1:1) mix and heat until the mixture has melted. Using a paint-brush and draw 2-3 layers of the paraffin wax/petroleum jelly (1:1) mix in the middle of the glass slide in accordance to Figures 1B-1D.
   NOTE: We are using common glass slides (76 x 26 x 1.0-1.5 mm (W/D/H))
2. Apply the melted paraffin wax/petroleum jelly mix rapidly on the glass slide to avoid solidification while drawing. Ensure the paraffin wax/petroleum jelly layer is about 2-2.5 cm in length and 0.3-0.5 cm in width. The thickness of the paraffin wax/petroleum jelly layer sho....

Results

The used 3D-collagen matrix migration assay combined with time-lapse video-microscopy and computer-assisted cell tracking allows for the determination of various cell migration parameters including both population-based parameter (e.g., mean locomotory activity) and single cell-based parameters (e.g., time of active movement, speed, distance migrated). An example of the obtained cell tracking data sets, data processing and data presentation are given in Figure 2. A cell tracking data fi.......

Discussion

The ability to migrate is a hallmark of tumor cells⁴. Without the ability to detach from the primary tumor and to migrate through the surrounding connective tissue tumor cells won’t be able to seed secondary lesions, which are the main cause of death of nearly all cancer patients. Because of this relationship many studies are focusing on cancer cell migration. The aim of these studies is the identification of novel target molecules and target pathways that efficiently block tumor cell migration, thereby .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Leica DM IL inverted microscope	Leica, Wetzlar, Germany
Microscope stage heater	Distelkamp Electronic, Kaiserslautern, Germany
JVC C1431 video camera	JVC, Bad Vilbel, Germany
Axis 241Q video server	Axis communication GmbH, Ismaning, Germany
Mac G5 Computer	Apple Macintosh
iMac	Apple Macintosh
FileMaker Pro	FileMaker GmbH, Unterschleißheim, Germany
Multi-camera video surveillance software(Security Spy)	Bensoftware, London, UK
Runtime Revolution Media 2.9.0	RunRev Ltd., Edinburgh, UK
Paraffin	Applichem GmbH, Darmstadt, Germany	A4264
Petroleum jelly	local drug store
Purecol (liquid collagen)	Nutacon BV, Leimuiden, The Netherlands		contains 2.9 - 3.3 mg/ml bovine collagen (95% collagen type I, 5% collagen type IV)
10x MEM	Sigma Aldrich, Taufkirchen, Germany	M0275
7.5% sodium bicarbonate solution	Sigma Aldrich, Taufkirchen, Germany	S8761
EGF	Sigma Aldrich, Taufkirchen, Germany	E9644
U73122	Merck Millipore, Darmstadt, Germany	662035	dissolve first in CHCl3; reconstitute in DMSO just prior to use