Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

Summary

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

Abstract

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life from embryonic development and wound healing to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell’s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

Introduction

Migration of cells plays a key role in various physiological responses such as embryonic development, haemostasis, and immune response as well as in pathological processes such as vascular diseases, inflammation, and cancer¹. Dissecting the biochemical and biophysical factors underlying cell migration is therefore fundamentally important not only to understand the basic principles of cellular functions, but also to advance various biomedical applications, such as in tissue engineering, anti-metastasis and anti-inflammatory drug development. Since in vivo observation is technically challenging, a lot of efforts has been focused on in vitro ....

Protocol

1. Cell Harvesting

1. Obtain MD-MBA-231 cells from the 37 °C, 5% CO₂ incubator. Detach cells from tissue culture plate using 0.5% Trypsin-EDTA solution. Use 1 ml of Trypsin-EDTA solution for cell cultured in a T25 flask.
2. Pellet cells in a 15 ml conical tube by centrifugation at 200 × g for 4 min, aspirate the supernatant, and resuspend cells in 5 ml of culture media.
3. Count cell density, ρ, using a hemocytometer.
   Note: To prepare the cell-seeded inner gel, the cell suspension will later on be diluted 10× to reach the final cell seeding density. Therefore, 10× concentrated cell suspension is required.
4. Calcu....

Results

The concentric gel assay presented here was performed using highly invasive breast cancer cells, MDA-MB-231, with 2.4 mg/ml inner collagen gel and a cell seeding density of = 2 × 10⁶ cells/ml, as an example. As shown in Figure 2, typically after a few days of culture, the cells breached the inner–outer gel interface and started to invade the outer gel. The cell population spread predominantly radially outwards.

The polymerization conditions of the outer gel can be m.......

Discussion

In this protocol we describe an in vitro assay to study the 3D migrational behavior of cells in matrix environments that topologically resemble ECMs encountered in vivo. There are three main strengths of this assay as compared to other currently available methods. First, this assay allows one to simultaneously examine the cell migration mechanisms at both population level and individual cell level. This opens up possibilities of studying collective cell migration¹³, which has to date been lar.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell culture incubator	Fisher Scientific Pte Ltd	Model: 371, S/No 318854-6055
Confocal microscope	Nikon A1R		Inverted confocal laser scanning microscope equipped with incubator chamber
Dulbecco's Modified Eagle's Medium (DMEM)	Life Technologies	11965-092
Fetal Bovine Serum (FBS)	Life Technologies	10082-147
Fluorescent CellTracker dye CMTMR	Life Technologies	C2927
Glass-bottom dish	IWAKI Cell Biology	3931-035	35 mm diameter dish with 12 mm diameter glass-bottom well
Hemocytometer	iN CYTO	DHC-N01 (Neubauer Improved)
Microprocessor pH meter	Hanna Instruments	pH 211
Nutragen Collagen	Advanced BioMatrix	#5010-D	Acid-solubilized bovine collagen type I (stock pH ~ 2)
Objective lens	Nikon		CFI Super Plan Fluor ELWD ADM 20XC, W.D. 8.2-6.9mm, NA 0.45.
Penicillin-Streptomycin	Life Technologies	15140-122
pH meter	Sartorius	S/No 29153352	Basic pH Meter PB-11
Trypsin-EDTA	Life Technologies	15400-054