Background

Directional cell migration plays an important role in many biological processes and diseases such as host defence, tissue generation and metastatic cancers. Among the various environmental guiding mechanisms, a chemical gradient can direct the migration of different cell types by chemotaxis.

Traditional cell migration assays are labor intensive, slow, and have unstable chemical gradients limiting the types of experiments that can be conducted. Compared with conventional cell migration assays, microfluidic devices provide useful experimental tools for quantitative cell migration and chemotaxis analysis in well‑controlled chemical gradients.

In this direction, a team of researchers from the University of Manitoba have developed a new method for rapid cell type chemotaxis testing as part of a microfluidic system. The integrated standalone microfluidic gradient generator and cell‑docking structure improve the control of the cell migration experiment, which enables easier and more accurate chemotaxis analysis. The method was successfully validated by using isolated human blood neutrophils and two human breast cancer cell lines.

Technology Overview

Chemotaxis, the directional cell migration guided by a soluble chemical gradient, plays a fundamental role in many normal biological and pathological processes. Microfluidics-based chemotaxis assays have been developed to study chemotaxis in vitro, but are difficult to conduct and require time-consuming yet often inaccurate cell tracking analysis, significantly limiting their wider adoption and market potential.

As a solution, University researchers have developed a microfluidic device with a radial channel design to allow for simultaneous chemotaxis tests of different cell types and different gradient conditions. This radial microfluidic device is capable of stand-alone stable gradient generation using passive pumping and pressure-balancing strategies based on the immiscibility between oil and buffers. The device was validated by testing the migration of fast-migrating human neutrophils to demonstrate proof of concept.

Design and fabrication of the microfluidic device

The device pattern was designed using AUTOCAD and printed on a transparent film at high resolution. The SU-8 device master was fabricated on a 3-inch silicon wafer by a two-layer photolithography10. The first layer defines the cell docking structure that is used to align the cells to one side of the gradient channel prior to migration. Its thickness should be cell specific, with it being slightly lower than the cell size but not be too low to prevent the cells from crossing.

Figure 1. Illustration of the radial microfluidic device and the silicone oil-based pressure-balancing strategy.

Design of the radial microfluidic device and oil-based pressure balancing strategy

The radially arranged microfluidic device consists of nine identical gradient units (Figure. 2A-B). Each gradient unit has two chemical inlets merging in a gradient channel, one cell loading inlet, and one waste outlet. The cell docking function was enabled by a thin barrier channel to separate the higher cell loading channel and the main gradient channel, thereby initially trapping cells along one side of the gradient channel as previously described (Figure. 2C).

Figure 2. Illustration of the pressure balancing strategy and validation of gradient generation in the radical microfluidic device.

An oil-based pressure balancing strategy by covering the cell culture media in both chemical inlets with oil (Figure. 3A). Researchers chose silicone oil which is immiscible with the reagents and has a slightly lower density than culture medium, although any suitable oil having a suitable density so as to remain on the surface of the desired reagent may be used, as will be readily apparent to one of skill in the art. The oil on the top of the cell culture media effectively balanced the pressure in the two chemical inlets and this ensured the formation of a stable gradient (Figure. 3A).

Figure 3. Cell trajectories

Chemotaxis of neutrophils and cancer cells in the radial microfluidic device

The researchers validated the radial chemotaxis device using isolated human blood neutrophils and two human breast cancer cell lines. Neutrophils were exposed to different chemoattractant gradients in the radial microfluidic device and cell migration was quantified within a 20-minute migration experiment. The current experimental settings resulted in a more than 3-fold stronger chemotaxis response of human neutrophils with the fMLP gradient than the IL-8 gradient.

The researchers employed the MDA-MB-231 and MCF-7 cells to test the chemotaxis response of these human breast cancer cell lines in the radial microfluidic device. When exposed to a gradient of epidermal growth factor (EGF; 100 ng/mL), MDA-MB-231 cells actively migrated out of the docking structure towards the EGF gradient. By contrast, MDA-MB-231 cells exposed to a uniform field of EGF (100 ng/mL) or normal culture medium remained stationary in the docking structure and failed to show a chemotaxis response. Similar results were obtained for MCF-7 cells. These results confirmed that the new radial microfluidic device was suitable for quantifying directional migration of human breast cancer cells when exposed to an EGF gradient.

This device also allowed us to monitor changes in cell morphology and track directionality of cell movement. When positioned within an area of the gradient with low EGF concentration, the breast cancer cells displayed higher migration directionality and extended lamellipodia towards an area of the gradient with higher EGF concentrations. However, as the tumor cells moved further into the EGF gradient channel, they displayed decreased directionality and more fluctuating lamellipodia orientation.

Summary

Researchers at the University of Manitoba have designed a novel radial microfluidic gradient device which can perform up to nine parallel cell migration experiments simultaneously with independently controlled gradient configuration and cell types. Each of the units includes a docking structure to align the cells at the low concentration area prior to chemotactic migration. A silicone oil-based pressure balancing strategy was employed to ensure identical and stable gradient formation in each of the chemotaxis units. This radial microfluidic platform was validated by demonstrating chemotaxis of human neutrophils in IL-8 and fMLP gradients and human breast cancer cells in an EGF gradient. These chemo attractants have previously been shown to promote chemotaxis in neutrophils and breast cancer cells, respectively.

Stage of Development

Technology Readiness Level (TRL): 7

Benefits

The developed microfluidic cell mobility assay and migration method specifically offers the following advantages:

Stable gradients based on passive gravity flow; no pumps required
Faster, easier and more efficient cell loading
The addition of micropillars increased the structural stability of the device by preventing the barrier channels from collapsing during the bonding process
Can perform up to nine parallel cell migration experiments simultaneously with independently controlled gradient configuration and cell type
Starting gate technology quickly aligns cells by the gradient channel to allow rapid determination of chemotaxis relative to the cells’ identical initial positions
All cells exposed to same gradient allowing greater accuracy with a pressure balancing strategy to ensure identical and stable gradient formation

Applications

One of the most attractive features of this technology is the minimal requirement for assay operation, which is very important for its practical use in life science labs and clinical labs. In addition to neutrophils and cancer cells, there is a growing potential to further apply this technology to study the migration of different immune cell types such as T cells, and stem cells.

Opportunity

The University is actively seeking a licensee to license the patent to move this technology to the market.

The University of Manitoba readily welcomes interactions with any investor or entrepreneur that would like to license this proven technology and the University would be highly supportive of the creation of a start‑up based in Manitoba, or outside of the province.

A Method for the Preparation of Microfluidic Chemotactic Devices

Patents

IP Status

Seeking