Study of Cell Migration in Microfabricated Channels

Summary

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

Abstract

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

Introduction

Migration is a complex cellular function that is important for many physiological processes in multicellular organisms, including development, immune responses, and tissue regeneration. In addition, certain pathological situations such as tumor invasion and metastasis rely on cell motility¹. For these reasons, cell migration has become a major field of study in the context of both fundamental and translational research. In vivo, most tissues are characterized by a rich extracellular matrix and high cell density. Cell migration therefore, under physiological conditions, occurs in a complex confined environment. Classically, most likely due to histor....

Protocol

Important note: This protocol assumes that the mold containing the shape for the desired microchannels has been already made. Further information on the preparation of the mold has been already published¹⁰. This protocol also assumes that bone marrow DCs culture is known.

1. Chip Fabrication

1. Mix PDMS oil and curing agent at a weight ratio 10:1 in a plastic cup. Mix both compounds thoroughly.
2. Cast the mix over the mold bearing microchannels. The total height must be between 0.5-1 cm.
3. Remove air bubbles in a vacuum jar bell during 1 hr.
4. Harden PDMS in the mold by placing th....

Results

In each experiment the surface of the PDMS is coated with a molecule adapted to the interest of the study. Figure 2 shows channels coated with a fluorescent molecule, PLL-g-PEG, before and after washing (step 2.4). Such an experiment allows the control of the homogeneity of the coating in the channels.

After cell loading, video microscopy can be performed to follow cell migration. Figure 3A shows an example of DC migrating in microchannels at a density appropr.......

Discussion

Here we describe a device composed of microchannels as a method to study the migratory properties of large number of cells in single experiments. This experimental system mimics the confined environmental constraints found in tissues by endogenous migratory cells. However, by forcing migration in a single dimension, it facilitates automatic cell tracking and the extraction of measurables (Figure 5). We also show that our device is compatible with fluorescence microscopy and can therefore be adapted to st.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polydimethylsiloxane (PDMS)	GE Silicones	RTV615	Package of 90% silicone base and 10% curing agent
Core sample cutter	Ted Pella Int.	Harris Uni-Core	Diameter 2.5 mm
Glass-bottom dish	WPI	Fluorodish FD 35-100
Ultrasonic cleaner	Branson Ultrasonics	Branson 200
Plasma cleaner	Harrick Plasma	PDC 32 G	For small samples (35 dishes). A bigger version is also available
Fibronectin from bovine plasma	Sigma Aldrich	F0895
PolyLysine grafted PEG (Pll-g-PEG)	Susos	PLL(20)-g[3.5]-PEG(5)
Hoechst 33342	Sigma Aldrich	B2261
Y27632	TOCRIS	1254