Double-layer micro-fluidic chip for in-situ monitoring of intercellular communication signal molecules

Abstract

The invention discloses a double-layer micro-fluidic chip for in-situ monitoring of intercellular communication signal molecules, the double-layer micro-fluidic chip comprises a culture layer, a porous polycarbonate film, a channel layer and a glass substrate, the porous polycarbonate film is arranged below the culture layer and is bonded and sealed with the culture layer through PDMS, and the channel layer is arranged below the culture layer. And the sealed culture layer and the channel layer are subjected to oxygen plasma bonding with the glass substrate through curing and sealing. Cells are inoculated into the upper cell culture chamber and the lower cell culture chamber of the double-layer micro-fluidic chip respectively, so that co-culture of the cells is realized, a tumor microenvironment on the chip is formed, substance molecules among the cells are fully exchanged, and in-situ monitoring of communication signal molecules among the cells is realized. According to the double-layer micro-fluidic chip disclosed by the invention, micro-fluid and external stimulation conditions can be effectively controlled through the closed micron-sized sample channel, and the interference of an external environment on a cell communication process and a detection process is reduced.

Description

Technical Field

[0001] The invention relates to microfluidic chip technology, and in particular to a double-layer microfluidic chip for in-situ monitoring of intercellular communication signal molecules. Background Art

[0002] In recent years, the tumor microenvironment (TME) has attracted increasing attention due to its important role in tumor immunosuppression, distant metastasis, local drug resistance and targeted therapy response. TME is a highly complex system, mainly composed of tumor cells, infiltrating immune cells (such as macrophages, dendritic cells and lymphocytes), cancer-associated stromal cells (such as cancer-associated fibroblasts CAFs), endothelial cells and adipocytes, as well as extracellular matrix (ECM) and a variety of signaling molecules. The development, metastatic ability and response to treatment of cancer depend not only on the characteristics of cancer cells, but also on the interaction between cancer cells and the complex surrounding microenvironment. Different cells in the tumor microenvironment achieve intercellular communication through direct cell interactions or the release of soluble signaling factors such as paracrine and endocrine. Therefore, the analysis and research of the mechanism of intercellular communication is not only of great significance for revealing the mechanisms of disease occurrence and development, but also plays an important role in the diagnosis, prevention and prognosis of the disease.

[0003] Microfluidic chips are widely used in the study of intercellular communication. Compared with traditional cell co-culture methods, microfluidic chips can effectively control the spatial distribution of cells through cell patterning culture, and the different designs of chip structure and function can control liquid flow, penetration, apply stimulation, or achieve the separation or integration of cell communication signal molecules and metabolites. However, this in vitro culture method is bound to cause the signal molecules in the cell communication process to be lost during separation, digestion, and purification, which greatly restricts the accuracy of cell communication research. Therefore, the ability to achieve in situ monitoring is crucial for the study of intercellular communication mechanisms. Summary of the invention

[0004] In view of the above existing problems, the purpose of the present invention is to provide a double-layer microfluidic chip for in situ monitoring of intercellular communication signal molecules, which can simultaneously realize cell co-culture and in situ research on the intercellular communication mechanism in the tumor microenvironment.

[0005] To achieve the above object, the present invention adopts the following technical solution: A double-layer microfluidic chip for in-situ monitoring of intercellular communication signal molecules is characterized in that the microfluidic chip comprises a culture layer, a porous polycarbonate membrane, a channel layer and a glass substrate, the culture layer is provided with a circular cell culture chamber, a cell inlet and a cell outlet; the porous polycarbonate membrane is placed below the culture layer, corresponds to the circular cell culture chamber of the culture layer, and is sealed by PDMS bonding; the channel layer is provided with a circular cell culture chamber, a cell inlet, a cell outlet and a sample channel, the cell inlet, the cell outlet and the circular cell culture chamber of the channel layer are connected by a sample channel, the circular cell culture chamber of the sealed culture layer corresponds to the circular cell culture chamber of the channel layer, the sealed culture layer and the channel layer share the cell inlet and the cell outlet, the sealed culture layer and the channel layer are sealed by curing, and are bonded to the glass substrate by oxygen plasma.

[0006] Furthermore, the chip material uses porous transparent polydimethylsiloxane material PDMS, wherein the ratio of PDMS monomer to initiator is 10:1; the culture layer is prepared by PDMS casting, and the channel layer is prepared by PDMS spin coating.

[0007] Furthermore, the diameter of the circular cell culture chamber is 4-6 mm.

[0008] Furthermore, the culture layer height is 2-3 mm; the channel layer height is 45-55 μm.

[0009] Furthermore, the length of the channel layer sample channel is 8-10 mm, the width is 200-300 μm, and the height is 20-25 μm.

[0010] Furthermore, the diameters of the cell inlet and the cell outlet are 0.5-1 mm.

[0011] Furthermore, the pore size of the porous polycarbonate membrane is 1-10 μm; the pore size of the porous polycarbonate membrane is selected so as to allow small molecules involved in intercellular communication to pass freely while cells cannot pass through.

[0012] Furthermore, a trumpet-shaped channel is provided between the sample channel of the channel layer and the circular cell culture chamber to connect the sample and reduce the flow rate of the sample into the culture area, thereby reducing the cell shear stress.

[0013] Through the above technical solution, the double-layer microfluidic chip for in situ monitoring of intercellular communication signal molecules provided by the present invention has the following beneficial effects: The double-layer microfluidic chip provided by the present invention is creatively combined with a scanning electrochemical microscope for the first time for in-situ monitoring of intercellular communication signal molecules. The double-layer microfluidic chip can realize the co-culture of multiple cells to form a tumor microenvironment on the chip; the double-layer microfluidic chip can effectively control microfluidics and external stimulation conditions through a closed micron-level sample channel, effectively reducing the interference of the external environment on the cell communication process and the detection process; the double-layer microfluidic chip culture layer adopts an open structure, which is easy to combine with a scanning electrochemical microscope probe, and has been successfully used for in-situ monitoring of a variety of intercellular communication signal molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is an exploded view of the double-layer microfluidic chip of the present invention; Figure 2 This is a schematic diagram of the structure of a double-layer microfluidic chip of the present invention; Figure 3 This is a flow chart of the method for preparing a double-layer microfluidic chip of the present invention; Figure 4 This is a cross section of a cell culture chamber of a double-layer microfluidic chip of the present invention; Figure 5 This is a diagram of the double-layer microfluidic chip of the present invention being used for in-situ detection of various intercellular communication signal molecules. DETAILED DESCRIPTION

[0015] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0016] See also Figure 1-3 The double-layer microfluidic chip of the present invention comprises a culture layer 1, a porous polycarbonate membrane 2, a channel layer 3 and a glass substrate 4, wherein the culture layer is provided with a cell inlet 6, a circular cell culture chamber 5 and a cell outlet 7; the porous polycarbonate membrane 2 is placed below the culture layer 1, corresponding to the circular cell culture chamber 5 of the culture layer 1, and is sealed by PDMS bonding; the channel layer 3 is provided with a cell inlet, a sample channel 9, a circular cell culture chamber 8 and a cell outlet, and the cell inlet, the cell outlet and the circular cell culture chamber 8 of the channel layer are connected by the channel layer sample channel 9; the circular cell culture chamber 5 of the sealed culture layer and the circular cell culture chamber 8 of the channel layer are in corresponding positions, the sealed culture layer 1 and the channel layer 3 share the cell inlet and the cell outlet, and the sealed culture layer 1 and the channel layer 3 are sealed by curing and oxygen plasma bonded to the glass substrate 4. Example

[0017] See also Figure 3 The preparation method of the double-layer microfluidic chip of this embodiment is as follows: (1) Preparation of culture layer 1: PDMS monomer and initiator were mixed in a mass ratio of 10:1, and cast on a smooth silicon wafer with a casting thickness of about 2-3 mm. A vacuum pump was used to remove bubbles between the PDMS and the silicon wafer, and the mixture was placed in an 80°C constant temperature forced air drying oven for curing for 30 min. The PDMS was cut into the required size with a cutting knife, and a circular cell culture chamber 5 with a diameter of 4-6 mm was punched in the center with a puncher to obtain culture layer 1. (2) Overlay preparation of the channel layer 3 positive mold: spin-coat SU-8 photoresist with a thickness of 20 μm~25 μm on the silicon wafer substrate for preparing the channel layer; pre-bake at 95°C for 4 min; use a film mask for the first exposure, with an exposure time of about 25 s; post-bake at 95°C for 5 min; use the first exposed structure as the substrate and spin-coat SU-8 photoresist with a thickness of 25 μm~30 μm; pre-bake at 95°C for 4 min; use a film mask to align the first layer structure with the second layer structure through the microscope of the photolithography machine, and perform the second exposure, with an exposure time of about 26 s; post-bake at 95°C for 5 min; develop for about 7 min to finally obtain a channel layer chip positive mold with a total height of about 45~55 μm; harden the channel layer chip positive mold at 150°C for 30 min; use trimethylchlorosilane (TMCS) to fumigate the template for 3 min to perform surface silanization treatment; (3) Preparation of channel layer 3: PDMS monomer and initiator were mixed in a mass ratio of 10:1, and PDMS prepolymer with a thickness of about 45-55 μm was spin-coated on the mold; 0.2 MPa N2 was blown to open the circular cell culture chamber 8; and the mixture was cured at 60°C for 2.5-3 min; (4) Culture layer-channel layer assembly: The culture layer 1 is placed in O2 plasma for 1 min; the porous polycarbonate membrane 2 is cut into 4-6 mm circular shapes, aligned with the circular cell culture chamber 5 of the culture layer, and then bonded and sealed; the sealed culture layer 1 corresponds to the circular cell culture chamber 8 of the channel layer and is sealed by curing at 60°C for 15 min; (5) Cut the sealed culture layer-channel layer into the required size and peel it off, and punch holes at the cell inlet 6 and the cell outlet 7 with a puncher; place the punched culture layer-channel layer together with a clean glass substrate in O2 Plasma for 1.5 min for oxygen plasma bonding, and place it in a constant temperature forced air drying oven at 80°C to obtain the double-layer microfluidic chip of this embodiment.

[0018] See also Figure 4-5, which is a diagram of the double-layer microfluidic chip of the present invention for in-situ detection of various intercellular communication signal molecules. The channel layer cells 11 are injected into the sample channel 9 through the cell inlet 6, and the channel layer cells 11 are inoculated in the channel layer circular cell culture chamber 8. After the channel layer cells 11 adhere to the wall, the culture layer cells 10 are inoculated in the culture layer circular cell culture chamber 5, and the culture layer cells 10 will grow close to the porous polycarbonate membrane 2. In order to maintain the cell state, fresh culture medium is replaced every 12 to 24 hours through the cell inlet 6, the sample channel 9 and the cell outlet 7. After 48-72 hours of co-culture, the stimulation conditions (such as drugs, etc.) are injected through the cell inlet 6, and act on the cells through the sample channel 9. The double-layer microfluidic chip of this embodiment is combined with a scanning electrochemical microscope, and the scanning electrochemical microscope probe is positioned on the culture layer cells 10, and the in-situ detection of various intercellular communication signal molecules such as reactive oxygen, lactic acid, and glutathione is successfully achieved.

Claims

1. A double-layer microfluidic chip for in situ monitoring of intercellular communication signal molecules, characterized in that: The microfluidic chip comprises a culture layer, a porous polycarbonate membrane, a channel layer and a glass substrate, wherein the culture layer is provided with a circular cell culture chamber, a cell inlet and a cell outlet; the porous polycarbonate membrane is placed below the culture layer, corresponds to the circular cell culture chamber of the culture layer, and is bonded and sealed by PDMS; the channel layer is provided with a circular cell culture chamber, a cell inlet, a cell outlet and a sample channel, the cell inlet, the cell outlet and the circular cell culture chamber of the channel layer are connected by a sample channel, the circular cell culture chamber of the sealed culture layer corresponds to the circular cell culture chamber of the channel layer, the sealed culture layer and the channel layer share a cell inlet and a cell outlet, the sealed culture layer and the channel layer are bonded to the glass substrate by oxygen plasma by curing.

2. The double-layer microfluidic chip for in situ monitoring of intercellular communication signal molecules according to claim 1, characterized in that: The chip material uses porous transparent polydimethylsiloxane material PDMS, wherein the ratio of PDMS monomer to initiator is 10:1; the culture layer is prepared by PDMS casting, and the channel layer is prepared by PDMS spin coating.

3. The double-layer microfluidic chip for in situ monitoring of intercellular communication signal molecules according to claim 1, characterized in that: The diameter of the circular cell culture chamber is 4-6 mm.

4. The double-layer microfluidic chip for in situ monitoring of intercellular communication signal molecules according to claim 1, characterized in that: The culture layer height is 2-3 mm; the channel layer height is 45-55 μm.

5. The double-layer microfluidic chip for in situ monitoring of intercellular communication signal molecules according to claim 1, characterized in that: The length of the channel layer sample channel is 8-10 mm, the width is 200-300 μm, and the height is 20-25 μm.

6. The double-layer microfluidic chip for in situ monitoring of intercellular communication signal molecules according to claim 1, characterized in that: The cell inlet and the cell outlet have diameters of 0.5 to 1 mm.

7. The double-layer microfluidic chip for in situ monitoring of intercellular communication signal molecules according to claim 1, characterized in that: The pore size of the porous polycarbonate membrane is 1-10 μm; the pore size of the porous polycarbonate membrane is selected so as to allow small molecules involved in intercellular communication to pass freely while cells cannot pass through.

8. The double-layer microfluidic chip for in situ monitoring of intercellular communication signal molecules according to claim 1, characterized in that: A trumpet-shaped channel is provided between the sample channel of the channel layer and the circular cell culture chamber to connect the sample, thereby reducing the flow rate of the sample flowing into the culture area and reducing the cell shear stress.

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