CN103952300B - A kind of micro-fluidic chip and cell chemotaxis motion study method - Google Patents

Abstract

The object of the present invention is to provide a microfluidic chip and a method for researching cell chemotaxis, characterized in that: the microfluidic chip is composed of a glass substrate, a first membrane and a second membrane; There are culture units; there are microchannels on the second membrane; there is a layer of porous membrane between the culture units of the first membrane and the microchannels of the second membrane; the number of culture units on the first membrane is on the second membrane The number of microchannels is the same; the microchannels on the second membrane are respectively located above the culture units of the first membrane. The microfluidic chip of the present invention can be used to study the selective chemotactic movement of cells in the microchannel flowing through multiple culture units. Compared with the traditional cell chemotaxis research method, the present invention is similar to a bionic model of a branched microvessel passing through multiple organs, and provides a new method for studying the selective chemotaxis movement of cells in the flow process, which has important Biomedical research value and economic value.

Description

A microfluidic chip and a research method for cell chemotaxis

technical field

The invention relates to the application of microfluidic chip technology to the field of biomedical research. The invention particularly provides a microfluidic chip and a research method for studying cell chemotaxis movement.

Background technique

Chemotaxis refers to the process in which cells move toward stimuli under the action of chemical concentration gradients. Chemotaxis plays an important role in the development of tissues and organs, wound healing and tumor metastasis. For example, injured tissues secrete chemokines to attract leukocytes to the infection site, remove necrotic tissue, and promote wound healing; organs such as the lung, liver, and bone are It secretes a large number of chemokines and becomes the most common target organ of tumor metastasis.

At present, the commonly used in vitro model for studying cell chemotaxis is the Transwell chamber, which is mainly used to study the chemotaxis process of cells in a static state. Its shape is a small cup that can be placed in an orifice plate. Put the Transwell chamber into the culture plate, add culture medium to the chamber and the culture plate at the same time, plant the cells in the chamber, and the culture medium in the culture plate can be It affects the cells in the small chamber and produces chemotactic effects on the cells. However, the Transwell chamber is a large open space, which does not have the characteristics of a closed space of microvessels in vivo; and because the cells in the chamber are in a static culture state, it is impossible to reproduce the chemotaxis process of cells in the microvessel in a flowing state. Therefore, the Transwell chamber cannot be used to study the biological process of cells moving through the microvascular wall for directional chemotaxis under flow conditions.

In order to solve the above problems, the present invention uses microfluidic chip technology to provide a new research platform, which can simulate the flow of cells in microvessels, especially when they flow through different organs, they are attracted by chemokines produced by organs The process of directional chemotaxis occurs. Microfluidic chip technology is a new technology developed on the basis of capillary electrophoresis in the early 1990s. In recent years, it has rapidly penetrated into the field of biomedicine, showing broad application prospects. This technology has been proved to be effective for mammalian cells and It is an ideal platform for microenvironment manipulation, with low consumption, high throughput, and strong bionicity. The microchannels on the microfluidic chip are micron-sized, and the space is relatively closed, which is very similar to the microvascular structure in the body; and the microfluidic chip is an ideal platform for manipulating fluid, which is convenient for simulating the flow of blood in the body. Therefore, the microfluidic chip platform constructed by the present invention is very suitable for studying the directional chemotactic movement of cells in a flowing state through the microvascular endothelial cell barrier.

Contents of the invention

The purpose of the present invention is to provide a microfluidic chip and a research method for cell chemotaxis movement.

The present invention specifically provides a microfluidic chip, which is characterized in that: the microfluidic chip is composed of a glass substrate, a first membrane and a second membrane; there is a culture unit on the first membrane; There is a microchannel; there is a layer of porous membrane between the culture unit of the first membrane and the microchannel of the second membrane; there are n culture units on the first membrane, and n is an integer greater than zero; the second membrane There are n microchannels, and the value of n is the same as that of the culture unit on the first membrane; the microchannels on the second membrane are respectively located above the culture units of the first membrane.

The microfluidic chip of the present invention is characterized in that: the culture units on the first diaphragm are independent of each other, and each culture unit is composed of an independent liquid inlet 1, a culture pool and a liquid outlet 1; One side of each microchannel is collected at the liquid inlet 2 through the connecting channel, and the other side is collected at the liquid outlet 2 through the connecting channel.

The microfluidic chip of the present invention is characterized in that the culture units on the first membrane are arranged parallel to each other, and the microchannels on the second membrane are parallel to each other.

The microfluidic chip of the present invention is characterized in that: the material of the first diaphragm and the second diaphragm is polydimethylsiloxane; the glass substrate, the first diaphragm and the second diaphragm are irreversible sealing; the depth of the culture unit is 1-30 mm; the depth of the microchannel on the second membrane is 10-1000 microns (the bottom of the culture unit on the first membrane can be opened, and the glass substrate and the first membrane After irreversible sealing, the upper surface of the glass substrate can be used as the bottom surface of the culture unit); the porous membrane is a membrane that allows liquid and cytokines to be transported between the culture unit of the first membrane and the microchannel of the second membrane, preferably For polycarbonate film.

The present invention also provides a method for using the microfluidic chip to study cell chemotaxis, which is characterized in that a layer of a third predetermined type of material is respectively inoculated on the surface of the porous membrane adjacent to the second diaphragm, and The first predetermined type of material is added into the culture unit, the second predetermined type of material is added into the microchannel of the second membrane, the second predetermined type of material is driven to flow in the microchannel, and the adhesion of the second predetermined type of material is investigated.

The method for studying cell chemotaxis in the present invention is characterized in that: the first predetermined type of material is a cell, tissue, organ, chemokine or growth factor; the second predetermined type of material is a cell; the third predetermined type of material for vascular endothelial cells.

The method for researching cell chemotaxis movement situation described in the present invention, concrete research process is as follows:

——add the third predetermined type of material into the microchannel of the second membrane through the second liquid inlet, and let it stand for 12 to 24 hours, so that the third predetermined type of material is fully attached to the surface of the porous membrane;

- adding a first predetermined type of material into the culture unit of the first membrane through the liquid inlet-;

- adding the second predetermined type of material into the microchannel of the second diaphragm through the second liquid inlet;

- connecting the syringe pump with the liquid inlet port two to drive the second predetermined type of material to flow in the microchannel;

- Place the microfluidic chip under a microscope to investigate the adhesion of the second predetermined type of material.

The microfluidic chip of the present invention can be used to study the selective chemotactic movement of cells in the microchannel flowing through multiple culture units. Compared with the traditional Transwell chamber, the invention is similar to a bionic model of a branched microvessel passing through multiple organs, and provides a new method for studying the selective chemotaxis movement of cells in the flow process, which has great potential in biomedical research Important value and broad application prospects.

Description of drawings

Fig. 1 is a schematic diagram of the microfluidic chip of the present invention, which includes a glass substrate 1, a first membrane 2, a second membrane 3, and a porous membrane 4;

Fig. 2 Schematic diagram of the structure of the first diaphragm, in which there are four relatively independent culture units (5a, 5b, 5c, 5d) on the first diaphragm, and each culture unit has a liquid inlet 6, a culture pool 7, and a liquid outlet Mouth-8;

The schematic diagram of the structure of the second diaphragm in Fig. 3, wherein there are four parallel microchannels (9a, 9b, 9c, 9d) on the second diaphragm, and the microchannels are collected in the inlet through a group of branched connecting channels-10a on one layer. Liquid port 2 11, the microchannel is collected in liquid outlet 2 12 through connecting channel 2 10b on another layer;

The photo of the assembled microfluidic chip of Fig. 4, the chip is connected to the syringe pump 13 through the liquid inlet 2 11;

Fig. 5 shows the vascular endothelial cells seeded on the surface of the porous membrane 4 adjacent to the second membrane 3;

Figure 6 shows that after FITC-Dextran (molecular weight is about 12KD) is added to the culture pool of the first membrane 2, FITC-Dextran enters the microchannel of the second membrane through the porous membrane and vascular endothelial cells (from left to The right is the case of FITC-Dextran entering the microchannel of the second membrane at 0 minutes, 10 minutes, 30 minutes, and 120 minutes);

Figure 7 shows the adhesion of tumor cell line (ACC-M) to vascular endothelial cells after different concentrations of chemokine CXCL12 were added to the culture pool of the first membrane (concentrations from top to bottom in the figure are 0ng/ml, 25ng/ml, 50ng/ml, 100ng/ml).

Figure 8 shows the adhesion of the tumor cell line (ACC-M) to vascular endothelial cells after different cells extracted from mice were added to different culture pools of the first membrane (from top to bottom in the figure are those extracted from mice muscle cells, lung cells, liver cells, bone marrow cells).

detailed description

Example 1

The microfluidic chip used was designed and manufactured by our laboratory.

As shown in Figures 1-3, the microfluidic chip of the present invention consists of a glass substrate 1, a first membrane 2, a second membrane 3 and a porous membrane 4, wherein the first membrane 2 and the second membrane 3 The material is polydimethylsiloxane, and the porous membrane 4 is a polycarbonate membrane. There are four parallel and independent culture units (5a, 5b, 5c, 5d) on the first diaphragm 2, and each culture unit (5a, 5b, 5c, 5d) has an independent liquid inlet-6 and liquid outlet respectively Port one 8, a culture pool 7 is arranged between the liquid inlet one 6 and the liquid outlet one 8; there are four parallel microchannels (9a, 9b, 9c, 9d) on the second diaphragm 3, one of the microchannels One side is collected in the liquid inlet 2 11 through a group of branched connection channels 10a on one floor, and the other side is collected in the liquid outlet 2 12 through the connection channel 2 10b on another layer, and the microchannels (9a, 9b, 9c, 9d) are located above the culture units (5a, 5b, 5c, 5d) of the first membrane 2, respectively.

The preparation process of the chip is as follows: firstly, the first membrane 2 is sealed with the glass substrate 1 through an irreversible sealing technique; secondly, the porous membrane 4 is covered on the culture unit of the first membrane 2; In three steps, the microchannel of the second membrane 3 is aligned with the culture unit on the first membrane 2 for irreversible sealing.

As shown in Fig. 4, the cell culture solution is added into the culture unit through the liquid inlet one 6, and the cell culture solution is added into the microchannel through the liquid inlet two 11. The vascular endothelial cell line HUVEC is seeded on the surface of the porous membrane 4 through the liquid inlet 2 11 . Then place the chip in a CO2 incubator at 37°C for 24 hours to allow HUVECs to fully attach (Figure 5), ready for use.

Example 2

The chip described in Example 1 was used for research: FITC-Dextran (molecular weight is about 12KD) was added into the culture pool 7 through the liquid inlet 6, and as time went on, FITC-Dextran diffused through the porous membrane 4 and vascular endothelial cells into the microchannel of the second diaphragm 3 (Figure 6).

Example 3

The chip described in Example 1 is used for research: through the liquid inlet 6, add the concentration of 0ng/ml, 25ng/ml, 50ng/ml, and 100ng/ml respectively in the four culture pools 7 of the first membrane 2. Chemokine CXCL12, incubated for 2 hours. Add the red fluorescent probe-labeled tumor cell line (ACC-M) into the microchannel through the liquid inlet 2 11 . Drive the red fluorescent probe-labeled tumor cell line (ACC-M) through the syringe pump 13 connected to the liquid inlet 2 11 to make it flow in the microchannel of the second diaphragm 3, stop after 30 minutes, and use phosphate After gently washing with buffer, photographs were taken under a fluorescent microscope to record the adhesion of ACC-M cells to vascular endothelial cells (Figure 7).

Example 4

The chip described in Example 1 was used for research: add muscle cells, lung cells, liver cells, and bone marrow cells extracted from mice into the four culture pools 7 of the first membrane 2 through the liquid inlet 6, and incubate for 2 Hour. Add the red fluorescent probe-labeled tumor cell line (ACC-M) into the microchannel through the liquid inlet 2 11 . Drive the red fluorescent probe-labeled tumor cell line (ACC-M) through the syringe pump 13 to make it flow in the microchannel of the second diaphragm 3, stop after 30 minutes, wash gently with phosphate buffer, and then Photographs were taken under a fluorescent microscope to record the adhesion of ACC-M cells to vascular endothelial cells (Figure 8).

The above-mentioned embodiments are only for illustrating the technical conception and characteristics of the present invention, and its purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and not to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention shall fall within the protection scope of the present invention.

Claims (5)

1. A method of using a microfluidic chip to study cell chemotactic movement, characterized in that: the microfluidic chip is made up of a glass substrate, a first membrane and a second membrane; unit; there is a microchannel on the second membrane; there is a layer of porous membrane between the culture unit of the first membrane and the microchannel of the second membrane; there are n culture units on the first membrane, and n is greater than zero Integer; there are n microchannels on the second membrane, and the n value is the same as the n value of the culture unit on the first membrane; the microchannels on the second membrane are respectively located above the culture units of the first membrane; the glass substrate , The first diaphragm and the second diaphragm are irreversibly sealed, the depth of the culture unit on the first diaphragm is 1-30 mm; the depth of the microchannel on the second diaphragm is 10-1000 microns; the first diaphragm The above culture units are independent of each other, and each culture unit is composed of an independent liquid inlet 1, a culture pool and a liquid outlet 1; one side of each microchannel on the second membrane is collected in the liquid inlet 2 through a connecting channel, and another One side is collected at the liquid outlet 2 through the connecting channel; The specific research process is as follows: ——add the third predetermined type of material into the microchannel of the second membrane through the second liquid inlet, and let it stand for 12 to 24 hours, so that the third predetermined type of material is fully attached to the surface of the porous membrane; - adding a first predetermined type of material into the culture unit of the first membrane through the liquid inlet-; - adding the second predetermined type of material into the microchannel of the second diaphragm through the second liquid inlet; - connecting the syringe pump with the liquid inlet port two to drive the second predetermined type of material to flow in the microchannel; - placing the microfluidic chip under a microscope to examine the adhesion of the second predetermined type of material; The first predetermined type of material is cells, tissues, organs, chemokines or growth factors; the second predetermined type of material is cells; the third predetermined type of material is vascular endothelial cells. 2. The method according to claim 1, characterized in that: the culture units on the first membrane are arranged parallel to each other, and the microchannels on the second membrane are parallel to each other. 3. The method according to claim 1, wherein the material of the first diaphragm and the second diaphragm is polydimethylsiloxane. 4. The method of claim 1, wherein the porous membrane is a membrane that allows the transfer of liquids and cytokines between the culture units of the first membrane and the microchannels of the second membrane. 5. The method according to claim 4, wherein the porous membrane is a polycarbonate membrane.