A Customizable Chamber for Measuring Cell Migration

Summary

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

Abstract

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors^1,2. However, methods for making microfluidics based assays can be difficult to learn.

Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.

The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

Introduction

In order for vital processes such as wound healing, immune responses, and embryonic development to occur, cell migration must take place. Cell migration involves the interaction between cells and neighboring cells, the extracellular matrix, and soluble chemical cues (attractants or repellants). As an example, in the process of wound healing, fibroblasts play an integral role in fibrogenesis and wound contraction, where the cells are recruited to the site of injury to synthesize collagen in order to form the extracellular matrix³. Numerous mechanisms behind the migration of fibroblasts to a wound site have been studied, and they include differen....

Protocol

1. Producing a Microchannel Chamber to Create a Concentration Gradient

1. Cutting an acrylic mold piece
   1. Obtain a piece of acrylic of the desired width. Typically use 1/16-inch-thick acrylic sheets. Thicker sheets are difficult to cut well and very thin sheets do not have the requisite mechanical strength, causing them to break or warp during the process.
   2. Create a CAD file with the desired shape of the acrylic piece to produce a cavity within the polydimethylsiloxane (PDMS). Adjust the channel width and length to achieve the desired gradient relevant to individual experimental protocols.
      1. Here, use an acryli....

Results

Figure 2 shows the movement of the cell front across the channel in response to a gradient of fetal bovine serum placed at the opposite end of the channel from where the cells are plated. The cell front is shown at 48 h (Figure 2A), 72 h (Figure 2B), 96 h (Figure 2C), and 120 h (Figure 2D) post plating. The movement of the cell front was tracked with these time-lapse images, and the migration distance and.......

Discussion

Our microchannel chamber may be used for a multitude of purposes, including determining the cell migration rate in response to growth factors and chemoattractants and measuring the diffusion rate of a drug from a polymer matrix. It is possible to utilize our microchannel chamber to grow cells and place a chemoattractant at one end of the chamber. The cells grow in response to the chemoattractant, and the cell migration can be quantified by taking time-lapse images that may be analyzed in order to determine the cell migra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sylgard 184 Silicone Elastomer Kit	Sigma-Aldrich	761036	poly(dimethylsiloxane) 2-part kit including silicone elastomer base and silicone elastomer curing agent
Acrylic Sheets	US Plastic	44200
Disposable Petri Dishes	Falcon	25373-041
Fluorescein isothiocyanate–dextran	Sigma-Aldrich	FD20s-100MG
Agarose, Type I, Low EEO	Sigma-Aldrich	A6013-100G
Dulbecco's Modified Eagle's Medium	Fisher Scientific	11965092	Cell media components
Fetal Bovine Serium	Fisher Scientific	16000036	Cell media components
Penicillin-streptomycin	Fisher Scientific	15140148	Cell media components
Phosphate Buffered Saline (PBS)	Fisher Scientific	BP24384
EVOS XL Cell Imaging System	Thermo Fisher Scientific	AME3300	Instrument used for taking time-lapse images
Versa Laser	Universal Laser Systems, Inc.	Model number VLS2.30	Laser cutter used for cutting plastic