Cell culture microfluidic chip based on self-deposition porous gasket of functional microspheres

Abstract

The application discloses a cell culture microfluidic chip based on a functional microsphere self-deposition porous gasket, belongs to the technical field of microfluidic chip preparation, and is suitable for the core demand of a culture solution dynamic stable supply in a cell culture scene (drug analysis). The method aims to solve the problems of poor mechanical stability, complex preparation process and poor controllability of pore structure of an existing gel-based porous gasket, and further affect the cell culture effect. The application does not need photo mask alignment, has simple process and low cost, the porous gasket prepared through functional microsphere self-deposition has high mechanical strength and strong corrosion resistance, the pore structure can be accurately controlled through microsphere parameters, the continuous and uniform supply of the culture solution in the cell culture process can be stably ensured, the influence of fluid fluctuation on cell growth is reduced, and the application is suitable for the mass production and actual application demand of cell culture microfluidic chips.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip fabrication technology, specifically relating to the fabrication and integration technology of porous gaskets for microfluidic chips used in cell culture. Background Technology

[0002] In the application of fluidized bed chips in cell culture, porous gaskets with redundant channels are one of the core structures ensuring a dynamic and stable supply of culture medium. In existing technologies, porous gaskets are mostly made of gel materials such as polyacrylamide, prepared through a photocrosslinking polymerization process: a gel prepolymer must first be prepared, and then a photomask is used to align the chip area for photocrosslinking, ultimately forming a porous gasket structure. However, these methods have many drawbacks: insufficient material properties: the gel materials have low mechanical strength and poor chemical corrosion resistance, making them prone to breakage and swelling with long-term use, affecting chip stability; high process complexity: the photomask alignment requires strict precision, the operation process is cumbersome, and mass production costs are high; poor structural controllability: the pore structure of the gel is determined by the degree of photocrosslinking, which is difficult to precisely control, leading to insufficient stability of the culture medium flow rate, thus affecting cell culture results.

[0003] This invention designs a cell culture microfluidic chip based on a functional microsphere self-deposition porous gasket. It forms a porous gasket by self-deposition and stacking of microspheres, which solves the problems of material defects, high process complexity and poor structural controllability of existing gel gaskets. It realizes the simple preparation, high stability and precise control of pore structure of porous gaskets, thereby improving the efficiency of cell culture. Summary of the Invention

[0004] The problem the invention aims to solve Porous gasket gel materials used in cell culture microfluidic devices generally suffer from low mechanical strength and poor chemical stability. During long-term use, they are prone to structural damage and swelling, significantly reducing the overall stability of the chip. Furthermore, traditional fabrication processes are complex, requiring stringent photomask alignment precision and involving cumbersome steps, making low-cost, large-scale fabrication difficult. In addition, the gel's pore structure is highly dependent on the degree of photocrosslinking, limiting its controllability and precise regulation, which can easily lead to unstable culture medium flow rates, thus affecting the stability and effectiveness of cell culture.

[0005] Solution for solving the problem To address the aforementioned problems, the inventors have conducted extensive research and designed a cell culture microfluidic chip based on the self-deposition of functional microspheres into porous gaskets. In this chip, without external driving, functional microspheres are self-deposited in situ under the constraint of a micropillar array through capillary action and solvent evaporation coupling, thereby forming a porous gasket structure with adjustable hydraulic resistance within the chip. Specifically, the invention includes the following technical solution: A cell culture microfluidic chip based on the self-deposition of functional microspheres to form porous gaskets, characterized in that the chip is composed of three sealed layers: a first layer is a cell sample inlet channel layer (cover sheet), a second layer is an inlet / outlet well layer (intermediate sheet), and a third layer is a culture layer (substrate sheet). The culture layer integrates a culture medium channel, a functional microsphere suspension channel, and a cell culture pool. A porous medium filling material formed by the natural deposition of functional microspheres is formed within the microsphere suspension channel. Micropillar structures are distributed on the inner side of the microsphere suspension channel. The micropillar structures and the porous medium filling material together constitute a porous gasket, and the two form a hydraulic resistance control zone.

[0006] In any of the technical solutions, the microfluidic chip for cell culture has microspheres with a size of 10-50 μm, a microsphere suspension with a mass concentration of 30-70 wt%, an effective deposition concentration of 5-15% after entering the functional microsphere suspension channel through capillary action, and a micron-sized porous structure formed by natural deposition of microspheres with a pore size of 1-5 μm.

[0007] In any of the technical solutions, the microfluidic chip for cell culture also has micropillar structures distributed inside the microstructure. The micropillar structures and the micron-sized porous structures together form a porous gasket with a diameter of 1-3 mm. The porous gasket is a hydraulic resistance control zone.

[0008] In any of the technical solutions described for cell culture microfluidic chips, the cover plate, intermediate plate, and substrate of the microstructure need to undergo hydrophilication treatment. The hydrophilication treatment methods include chemical modification, UV / ozone, and oxygen plasma.

[0009] The microfluidic chip for cell culture described in any of the technical solutions is fabricated by a dry etching method, a wet etching method, or a photolithography method.

[0010] The microfluidic chip for cell culture described in any of the technical solutions is made of glass, polymer, silicon, or metal.

[0011] The core function of the hydrophilic treatment of the cover, intermediate, and bottom sheets of the microstructure described in this invention lies in dual regulation, thereby ensuring the quality of microsphere self-deposition and the stability of fluid transport, as well as the uniformity and adhesion of microsphere deposition. The original surfaces of PDMS and conventional polymer substrates are hydrophobic; if they directly contact the functional microsphere suspension, uneven liquid spreading, microsphere aggregation, or deposition detachment can easily occur. After hydrophilic treatment, the surface, primarily composed of hydrophilic groups with hydroxyl (-OH) groups, significantly reduces the surface tension of the liquid, allowing the suspension to quickly wet the inner wall of the channel and form a uniform liquid film. This ensures that the functional microspheres self-deposit in an orderly and dense manner according to the topological structure of the micropillar array during solvent evaporation, avoiding the formation of localized accumulation voids or loose layers, and guaranteeing the integrity and uniformity of the porous gasket structure. This ensures zero leakage and low flow resistance connection of the fluid channel. This invention uses PDMS as the main material, and its hydrophilic treatment is a necessary prerequisite for achieving irreversible bonding. By introducing active groups through methods such as oxygen plasma, chemical bonding reactions can occur on the surfaces of each layer upon contact, thereby forming a high-strength permanent seal. This mechanism ensures that the chip is leak-free under liquid supply pressure, while the hydrophilic surface maintains a stable flow pattern of the culture medium, reduces frictional resistance between the fluid and the wall, provides a stable input pressure source for the porous gasket, and avoids localized vaporization or bubble retention in the flow channel due to hydrophobic interfaces.

[0012] Specifically, this invention uses capillary force as the fluid driving force, and its working principle is as follows: the microsphere suspension flows through the microchannel under the drive of capillary action, and undergoes self-deposition under the coupling effect of capillary action and solvent evaporation, tightly binding with the micropillar structure to form a porous gasket structure. By setting a ring-shaped distribution of microsphere deposition areas around the culture tank, the formed porous gasket surrounds the cell culture tank, thereby constructing a stable liquid supply structure.

[0013] By precisely controlling the particle size of functional microspheres and the concentration of the suspension, porous gaskets can form specific pore structures and generate adjustable hydraulic resistance. Furthermore, the hydraulic resistance of the porous gasket is related to the particle size and deposition density of the functional microspheres: when the microsphere particle size decreases or the deposition density increases, the pore size decreases, thus increasing the hydraulic resistance, and vice versa. Through these adjustments, stable control of the culture medium flow rate can be achieved.

[0014] Under the aforementioned structural and parameter regulation, a stable pressure drop is formed when the culture medium flows through the porous gasket, which is conducive to the stable delivery of the fluid. Utilizing the permeability and pressure-stabilizing properties of the porous gasket, a culture environment with peripheral permeation, stable fluid supply, and metabolic product discharge functions can be created.

[0015] By employing oxygen plasma pretreatment combined with gentle thermal curing, the bonding stability between the microsphere layer and the PDMS substrate can be ensured, while also preserving the good biocompatibility of PDMS, thereby guaranteeing the cell culture effect. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the structure of a microfluidic chip example provided by the present invention; Figure 2 This is a schematic diagram of the cover plate structure of a microfluidic chip example provided by the present invention; Figure 3 This is a schematic diagram of the intermediate chip structure of a microfluidic chip example provided by the present invention; Figure 4 This is a schematic diagram of the substrate structure of an example of a microfluidic chip provided by the present invention; Figure 5 yes Figure 4 A magnified view of a portion of the cell culture pool area; Figure 6 The diagram shows a porous gasket structure formed by self-deposition of functional microspheres under the constraint of a micropillar array, which forms an interconnected pore network inside to provide stable hydraulic resistance.

[0017] Explanation of reference numerals in the attached figures: 1. Cover sheet; 2. Intermediate sheet; 3. Negative sheet; 4. Cell injection well; 5. Cell inlet / outlet microchannel; 6. Cell sample inlet well; 7. Functional microsphere suspension injection well; 8. Culture medium injection well; 9. Cell inlet / outlet sample passage; 10. Culture medium inlet / outlet sample passage; 11. Functional microsphere suspension inlet / outlet sample passage; 12. Culture medium sample inlet area; 13. Culture medium microchannel; 14. Functional microsphere suspension sample inlet area; 15. Functional microsphere microchannel; 16. Microcolumn array; 17. Cell culture pool; 18. Functional microsphere.

Claims

1. A cell culture microfluidic chip based on functional microsphere self-deposition porous gaskets, characterized in that, The chip consists of three layers: a cover plate (1), an intermediate plate (2), and a substrate (3). The substrate contains a culture medium channel (13), a functional microsphere suspension channel (15), and a cell culture pool (17). The upper, middle, and lower layers are sealed together to form a fluid channel or chamber. A micropillar array (16) is distributed in a ring around the cell culture pool. Functional microspheres (18) self-deposit between the micropillar array under capillary action and solvent evaporation to form a porous structure. The porous structure and the micropillar array (16) together constitute a porous gasket surrounding the cell culture pool.

2. The cell culture microfluidic chip based on a functional microsphere self-deposition porous gasket according to claim 1, characterized in that, The solvent of the functional microsphere suspension is one of anhydrous ethanol, isopropanol, or deionized water containing a dispersant. The mass concentration of the microsphere suspension is 30-70 wt%. During the preparation process, the mixing system needs to be stirred and ultrasonically treated for 10-20 minutes to ensure that the functional microspheres (18) are uniformly dispersed. The effective deposition concentration after entering the functional microsphere suspension channel (15) through capillary action is 5-15%.

3. The cell culture microfluidic chip based on a functional microsphere self-deposition porous gasket according to claim 1, characterized in that, The solvent of the functional microsphere suspension is one of anhydrous ethanol, isopropanol, or deionized water containing a dispersant. The mass concentration of the microsphere suspension is 30-70 wt%. During the preparation process, the mixing system needs to be stirred and ultrasonically treated for 10-20 minutes to ensure that the functional microspheres (18) are uniformly dispersed. The effective deposition concentration after entering the functional microsphere suspension channel (15) through capillary action is 5-15%.

4. A cell culture microfluidic chip based on a functional microsphere self-deposition porous gasket according to claim 1, characterized in that, The diameter of the micropillar array (16) is 20-30 μm, and the center distance between adjacent micropillars is 50-100 μm.

5. A cell culture microfluidic chip based on a functional microsphere self-deposition porous gasket according to claim 1, characterized in that, The cover sheet (1), intermediate sheet (2), and bottom sheet (3) with microstructures are made of one or more of glass, polymer, silicon material, and metal, respectively, wherein the polymer is preferably PDMS.

6. A cell culture microfluidic chip based on a functional microsphere self-deposition porous gasket according to claim 1, characterized in that, The functional microspheres (18) are made of one or more of silica, polystyrene, and hydrogel, and the particle size of the functional microspheres is 10-50 μm.

7. A cell culture microfluidic chip based on a functional microsphere self-deposition porous gasket according to claim 1, characterized in that, The cover sheet (1), intermediate sheet (2), and bottom sheet (3) with microstructures need to be hydrophilized. The hydrophilization treatment method is one of chemical modification, UV / ozone, or oxygen plasma.

8. A cell culture microfluidic chip based on a functional microsphere self-deposition porous gasket according to claim 1, characterized in that, The microstructure fabrication method is one of dry etching, wet etching, or photolithography.

9. A cell culture microfluidic chip based on a functional microsphere self-deposited porous gasket according to claim 1, characterized in that, The porous gasket is a micron-sized porous structure formed by the self-deposition of microspheres after the microsphere suspension flows into the channel, due to capillary force and solvent evaporation. It has a greater hydraulic resistance than the culture medium channel, creating a pressure drop as the culture medium flows in to stabilize the flow rate. Under the pressure difference between the inlet and outlet of the culture medium, a steady flow passes through the saturated porous gasket and enters the culture tank area, providing a dynamic and stable supply of culture medium for adherent cells. Simultaneously, cell metabolic products flow towards the outlet with the flow direction and can be discharged promptly.

10. A cell culture microfluidic chip based on a functional microsphere self-deposition porous gasket according to claim 1, characterized in that, The microfluidic chip described above has the advantages of simple manufacturing process, low cost, and easy mass production.

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