Technical field

The invention relates to a method for determining cell migration and invasion. Prior art

Microfluidic technology provides precise spatial and temporal control, high-throughput analysis, low fabrication costs ve portability. Used material and waste volumes can be as low as picoliters. Using small volumes of unknown or toxic materials provides safe experimental study. Moreover, microfluidic technology can provide means to mimic physiological microenvironments. This feature can help us more realistically study cells in both health and disease states and improve drug testing approaches. It can also help reduce animal testing.

Cell migration and invasion is most commonly analyzed with the method named Boyden Chamber. However, this method is an end-point method. It does not ensure continuous investigation of cell migration and invasion. There are membranes with certain hole sizes in the mechanism used. These hole sizes are needed to be chosen according to different cells. What is more, using a membrane negatively affects providing a physiological environment.

Microfluidic device-based devices and methods are present for chemotactic tropism of cells. However, a microfluidic-based method which is able to imitate the Boyden Chamber method, is more useful than that, is able to better mimic the physiological environment and ensure obtaining more data, is not present.

Technical problems to be solved by the invention

The purpose of the invention is to examine cell migration and invasion in time at intervals or continuously by using a microfluidic device.

Method steps having the specified features are shown in the drawing below.

Drawings are not to scale, unless necessary. Description of drawings

Figure 1: Method steps.

a) Top view of microfluidic device (1).

b) Matrix (3) is loaded into the central channel (2).

c) Agent (5) is loaded to the test channel (4).

d) Cells (7) are loaded to the control channel (6).

e) Cells (7) are ensured to settle down at the interface between the central channel (2) and the control channel (6) by keeping the microfluidic device (1) upright.

f) Cell migration and invasion is determined in the setup maintained in suitable culture conditions.

Description of the references in drawings

1. Microfluidic device.

2. Central channel

3. Matrix

4. Test channel

5. Agent

6. Control channel

7. Cell

8. Column

9. Hole

Description of the invention

The invention is a method for determining cell migration and invasion. This method comprises the stages of adding a matrix (3) to the central channel (2) of the microfluidic device (1), adding an agent (5) to the test channel (4) of the microfluidic device (1), adding cells (7) to the control channel (6) of the microfluidic device (1), by positioning the microfluidic device (1) such that the control channel (6) remains at the top, under suitable culture conditions, ensuring cells (7) to settle down at the interface of the test channel (4) and the central channel (2) and culture of them, determining migration and invasion of cells (7) in a matrix channel.

Matrix (3) loaded to central channel can consist of matrigel, collagen, laminin, agarose, polyacrylamide, biocompatible matrices, puramatrix, alginate, fibrin or a combination thereof. Necessary environment is provided after the loading for polymerization in accordance with the type of the matrix (3), if necessary.

For example, matrigel is loaded to the central channel (2) in a cold state, then the matrigel is ensured to polymerize by maintaining the microfluidic device (1) at 37 C. However, puramatrix polymerizes rapidly in salt solution, therefore its polymerization can be completed at the moment it is loaded to the central channel (2) when added to a salt solution.

Agent (5) can be cells, cell-laden or cell-free matrix, polymer, culture medium, physiological buffer solution, cell conditioned culture medium, one or more biological or chemical molecules or a combination thereof. Cells in the control channel (7) and cells used as agent (5) can be the same or different.

Cells (7) can be cell lines, primary culture cells, biopsy cells, stem cells or a combination thereof. For example, cancerous or normal cell lines can be used, tumor biopsies obtained from patients can be used. After the cells (7) settled down at the interface of the central channel (2) and the control channel (6), the microfluidic device (1) can be continued to be positioned upright or can be maintained horizontally under suitable culture conditions.

In an exemplary practice, matrigel is loaded to the central channel (2). After the matrigel polymerizes, an agent (5) estimated to increase cell migration and invasion, for example a growth agent, is added to the test channel (4). Then cells (7), for example cancer cells, are loaded to the control channel (6). Cancerous cells are ensured to settle down at the interface of the central channel (2) and the control channel (6) by positioning the microfluidic device (1) upright such that the control channel is at the top, under culture conditions. Migration and invasion of cancer cells in the central channel (2) can be examined in time continuously or at intervals. If the growth agent is efficient as anticipated, cells will show more migration and invasion compared to the condition without the growth agent.

Cell migration and invasion can be examined by microscopy or spectroscopy. Cells (7) can be altered genetically as to give fluorescent signal or can be labeled with chemical dyes. Cells can also be examined by standard light microscope techniques in the event that they do not give fluorescent signal.

Cell migration and invasion can be determined by counting cells (7) that have entered the central channel, by measuring the distance covered by the cells (7) in the central channel (2) in one or more time points, by measuring shapes, speed, persistence of the cells (7) in the central channel (2).

A matrix (3) showing an increase or decrease in fluorescent signal when degraded by cells can be loaded to the central channel (2). Thus, the matrix (3) degradation by cells (7) can be examined.

Cells (7) entering the central channel (2) can be extracted from the microfluidic device (1) and used for out-of-device assays such as polymerase chain reaction, ELISA. Cells (7) entering the central channel (2) can also be fixed and sectioned, separately or within the matrix (5) they are in, for out-of-device assays such as electron microscopy, histological staining.

Microfluidic device (1) used for this method can comprise glass, polydimethylsiloxane (PDMS), polystyrene (PS), polymethylmethacrylate (PMMA), cyclic olefin copolymer (COC) or a combination thereof. Molds to be used for the manufacturing of the microfluidic device (1) can be produced with a method comprising UV lithography, three-dimensional printing, metal casting or a combination thereof.

Central channel (2) in the microfluidic device (1) used for the method for determining cell migration and invasion is between the test channel (4) and the control channel (6). Channels (2, 4, 6) are separated from one another by more than one column (8). Thus cells (7) and for example other agents (5) being able to change places with diffusion can switch between channels (2, 4, 6).

Channels (2, 4, 6) can be at height and width between 50 micrometers and 1 centimeter. Channels (2, 4, 6) can be at length between 500 micrometers and 20 centimeters. Cross sections of the columns (8) can be in different shapes like triangle, trapezoid, circle, ellipse. Longest horizontal axis of columns (8) can be at length between 50 micrometers and 1 centimeter.

There is at least one hole (9) in each of the channels (2, 4, 6) such that loading can be realized. Industrial applicability of the invention

The invention can be used in the pharmaceutical industry for determining agents that can affect cell migration and invasion.