Micro-fluidic chip for three-dimensional dynamic culture of cells

Abstract

The invention provides a cell three-dimensional dynamic culture micro-fluidic chip which is suitable for cell three-dimensional culture, drug screening, tumor migration research and the like. The cell culture micro-device is composed of three layers including a cell sample injection layer, a cell scaffold layer and a culture solution sample injection layer. The device is characterized in that the cell sampling layer has a concave-convex structure; the culture solution sample injection layer simulates a human vascular structure and is provided with a permeable wall. Wherein the 3D printing cell growth bracket is embedded in a unit chamber of the culture solution sample injection layer and is assembled by a convex structure on the cell sample injection layer in an elastic interference fit manner, so that the bracket is prevented from being scoured and deformed by liquid. The invention has the advantages that: 1, the cells are uniformly injected and distributed in the cell scaffold; 2, a three-dimensional growth space is provided for cells; 3, nutrition is supplied to the cells, and cell metabolism waste liquid is discharged in time; 4, the bracket is stable in structure and can resist liquid scouring deformation; and 5, the micro-fluidic device can become a functional unit of an integrated micro-fluidic device and has wide applicability.

Description

Technical Field

[0001] The present invention relates to the field of micro-device design using bionic means, in particular to the field of cell culture micro-devices. The cell culture micro-devices are used to solve the problems of poor integration between a bracket and a microfluidic channel and easy deformation. Background Art

[0002] In most organisms, the extracellular matrix (ECM) forms a complex three-dimensional (3D) network within which tissue cells reside. A network of capillaries transports nutrients, which penetrate the tissues through convection and diffusion. Within tissues, fluid flow exhibits a slow and steady interstitial flow pattern, crucial for maintaining a stable tissue environment. The ECM not only provides essential physical support for cell growth but also serves as a pathway for nutrient transport and signal transduction. Research has shown that fluid flow can significantly influence cell growth, differentiation, and gene expression. Therefore, when culturing cells in vitro, it is crucial to create a dynamic and stable fluid microenvironment that mimics the in vivo environment. Such a dynamic microenvironment ensures a continuous supply of culture fluid and the timely removal of waste fluids. Furthermore, a stable microenvironment minimizes the adverse effects of external disturbances on cells.

[0003] Traditional cell culture methods in vitro usually use culture flasks or culture plates. These methods are static cultures and cannot replace the culture medium or discharge the waste liquid in time, which affects the normal growth and functional expression of the cells. In contrast, the channel network design of microfluidic devices is very similar to the vascular system in the body, which can achieve dynamic perfusion culture. The application of microfluidic technology makes it possible to simulate and precisely control the cell microenvironment in vitro. However, existing microfluidic culture chips have some limitations. For example, the straight channel design causes a large amount of fluid to directly impact the cells, generating destructive shear forces, which not only causes mechanical damage to the cells, but may also affect the normal physiological functions of the cells. In addition, the high-speed flow in the straight channel may cause impact and deformation to the cell growth space, thereby affecting the cell growth environment.

[0004] To address these issues, the present invention proposes an improved microfluidic chip design. By introducing a concave-convex structure and an interference fit, the shear force of the fluid on the cells is effectively reduced, while maintaining the stability of the cell growth space. The "8"-shaped cell injection channel can inject cells through multiple channels and multiple directions, achieving uniform distribution of cells within the generated scaffold, thereby providing cells with a microenvironment that is closer to physiological conditions. The design of the concave-convex structure can ensure a tight connection between the various layers of the chip through an interference fit, preventing fluid leakage and enhancing the stability and reliability of the chip. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of existing technologies by designing a microfluidic chip that can address issues such as poor integration of scaffolds with microfluidic channels and easy deformation, using a method that extracts microstructures that influence the microenvironment of fluid flow within a living organism. The chip is then fabricated using microfabrication techniques and used for in vitro cell culture. The cell culture microdevice comprises a three-layer structure: from top to bottom, a cell sample layer, a cell growth scaffold layer, and a culture medium sample layer.

[0006] To achieve the above-mentioned purpose, the present invention adopts the following technical solution: the cell sampling layer adopts an "8"-shaped microchannel structure, which is conducive to the uniform distribution of cells in the cell growth scaffold.

[0007] The cell sampling layer has four through holes, wherein the culture fluid sampling port and the culture fluid sampling port correspond to the liquid storage tank and the waste liquid tank of the culture fluid sampling layer respectively, and are used to realize the inflow and outflow of the culture fluid; the other two through holes are the cell sampling hole and the cell outlet.

[0008] The culture fluid sampling layer simulates the structure of human blood vessels and has a permeable wall.

[0009] The cell-feeding layer is made of a thermosetting polymer, polydimethylsiloxane (PDMS), or a thermoplastic polymer, polymethyl methacrylate (PMMA), polystyrene (PS), or polycarbonate (PC). During fabrication, a negative-resist BN-303 or BN-308 is spin-coated onto a glass slide to enhance the bonding strength of the subsequent SU-8 adhesive. After drying, the SU-8 adhesive is spin-coated. A mold is formed through pre-baking, photolithography patterning, and development. After the mold is fumigated, the pattern is transferred to the corresponding polymer using soft etching or hot pressing.

[0010] The cell sampling layer uses a special puncher for microfluidic chips to punch through holes at positions corresponding to the culture solution inlet and outlet holes and the cell inlet and outlet holes.

[0011] The cell scaffold layer is 3D printed, and the circular glass slide is laser cut or milled to the desired dimensions. 3D printing precisely constructs the three-dimensional structure, providing support for cell attachment and growth. Laser cutting or micro-milling then refines the circular glass slide into the desired shape and size to meet cell culture requirements.

[0012] The culture medium sampling layer is made of thermosetting polymer PDMS or thermoplastic polymers PMMA, PS, or PC. During fabrication, positive photoresist AZ703 or BP212 is spin-coated onto an oxidized glass slide. After drying and photolithographic patterning, the patterned photoresist is used as a mask to dry-etch the silicon dioxide layer to transfer the pattern. Silicon is then etched using the photoresist and oxide layer as masks. After multiple etching passes and parameter adjustments, the photoresist and oxide layers are removed. After fumigating the silicon mold, the pattern is transferred to the corresponding polymer using soft etching or hot pressing.

[0013] The cell sampling layer, cell growth scaffold layer, and culture fluid sampling layer are bonded via oxygen plasma treatment or heat pressing. Oxygen plasma treatment creates hydrophilic hydroxyl groups on the PDMS surface, forming strong oxygen bonds after bonding. Heat pressing bonding uses ultraviolet light to render the thermoplastic material hydrophilic, and then heat and pressure are applied to complete the bonding. During bonding, the cell sampling layer and cell growth scaffold layer, as well as the cell growth scaffold layer and culture fluid sampling layer, must be aligned. The cell growth scaffold is embedded within the cell compartment of the culture fluid sampling layer, assembled with an elastic interference fit using a raised structure on the cell sampling layer. Visual microscopy technology is used to assist in positional adjustment during the alignment process.

[0014] After bonding, the cell culture microdevices were sterilized by passing anhydrous ethanol through the microdevices to maintain hydrophilicity. Performance was tested by measuring fluid velocity using tracer particle velocimetry: The microdevices were filled with deionized water, the cell inlet and outlet wells were sealed, a constant-flow syringe pump and a waste container were connected, and the microsphere trajectory and velocity were recorded under a fluorescence microscope. Flow field changes were analyzed to test disturbance resistance and stability.

[0015] Before use, the cell culture microdevice, silicone tubing, and tubing connectors should be rinsed with medical alcohol and triple-distilled water, then sterilized under high temperature and high pressure for one hour. They should then be coated with rat tail collagen or fibronectin and placed in an incubator overnight to facilitate cell attachment. After coating, PBS is passed through the microdevice to rinse away excess protein. The microdevice is then filled with culture medium and the cell suspension. After observation, the microdevice is placed in a CO2 incubator for static incubation until the cells adhere. After attachment, dynamic culture begins: connect the microdevice to a syringe pump, set the flow rate, and start the pump to continuously deliver fresh culture medium and remove metabolic waste.

[0016] The present invention boasts significant benefits: Its "8"-shaped cell inlet channel, mimicking the structure of human blood vessels, enables multi-channel, multi-directional cell injection. Combined with the permeable wall design of the culture medium inlet layer, this significantly improves the uniformity of cell introduction and distribution within the scaffold. The concave-convex structure of the cell inlet layer effectively addresses issues such as poor integration of the scaffold with the microfluidic channel and easy deformation, ensuring the stability and reliability of the scaffold structure and effectively resisting deformation caused by fluid erosion. The unique cell scaffold layer provides a three-dimensional space for cell attachment and growth, and exhibits excellent robustness, resisting external perturbations and reducing flow field fluctuations caused by damage and blockage of the integrated micropumps, microvalves, microchannels, concentration gradient generators, or channels. This allows the biomimetic cell scaffold to seamlessly integrate into a variety of cell manipulation microdevices, becoming a core functional unit of integrated microfluidic devices and possessing wide applicability. The culture medium inlet layer exchanges substances with the cell scaffold layer through the permeable wall, dynamically and stably providing nutrients to the cells and promptly removing cellular metabolic waste. The fluid flow throughout the system, particularly within the cell scaffold layer, is designed to be interstitial, closely simulating the actual fluid dynamics microenvironment in which cells reside in the body. The present invention achieves mass cell culture while stabilizing the cell culture microenvironment. The microdevice has high biocompatibility, is easy to manufacture, and is low-cost, making it suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The three-dimensional structure diagram of the cell culture microdevice of the present invention is intuitively shown. The diagram intuitively shows the relative positional relationship between the cell sampling layer (1), the cell support layer (2) and the culture fluid sampling layer (3). The cell sampling layer (1) is located at the top layer, the cell support layer (2) is located at the middle layer, and the culture fluid sampling layer (3) is located at the bottom layer. The cell sampling layer (1) has a culture fluid sampling hole (4) connected to the liquid reservoir (14), a culture fluid outlet (11) connected to the waste liquid outlet (20), a cell sampling channel (6), an inner layer microcolumn (7) and a fixing foot (8). The cell support layer is composed of a cell growth support (12) and a circular glass slide (13). The culture fluid sampling layer (3) has a liquid reservoir (14), a waste liquid reservoir microcolumn (15), a culture fluid sampling channel (16), an outer layer microcolumn (17), a chamber (18), a waste liquid outlet channel (19), and a waste liquid outlet (20).

[0018] Figure 2 It is a two-dimensional top view of the cell sampling layer (1) in the present invention, wherein the top view includes a schematic diagram of the inner layer microcolumns (7) and the fixing feet (8).

[0019] Figure 3 This is an enlarged schematic diagram of the inner microcolumns (7) and the fixed feet (8), which shows that the arrangement of the inner microcolumns is uniformly circular, and there are microcolumn gaps between several microcolumns; the fixed feet (8) are a trapezoidal structure.

[0020] Figure 4 It is a schematic diagram of a cell growth support (12) and a circular glass slide (13) of a cell growth layer (2).

[0021] Figure 5 It is a schematic diagram of the structure of the cell culture fluid injection layer, including a schematic diagram of the liquid reservoir microcolumn (15) and the outer layer microcolumn (17).

[0022] Figure 6 This is an enlarged schematic diagram of the outer layer microcolumns (17), which shows that the outer layer microcolumns are arranged in a uniform circular shape, and there are microcolumn gaps between several microcolumns.

[0023] Figure 7 This is an enlarged schematic diagram of the liquid reservoir ((14), which shows that the arrangement of the liquid reservoir micropillars (15) is uniformly circular, and there are micropillar gaps between several micropillars.

[0024] Figure 8 This is a schematic diagram of 3D printing of the cell scaffold layer.

[0025] Figure 9 This is a flow chart for making a cell injection layer.

[0026] Figure 10 This is the microfluidic chip packaging flow chart. DETAILED DESCRIPTION

[0027] The present invention will be further described with reference to the accompanying drawings and specific embodiments.

[0028] Example

[0029] like Figure 1As shown, a bionic cell culture microdevice for constructing a dynamic and stable microenvironment for in vitro cell culture is composed of a three-layer structure, namely a cell sampling layer 1, a cell growth scaffold layer 2, and a culture fluid sampling layer 3. The cell sampling layer 1 is located at the top layer, the cell growth scaffold layer 2 is located in the middle, and the culture fluid sampling layer 3 is located at the bottom layer. The culture fluid sampling hole 4 on the cell sampling layer 1 is connected to the liquid reservoir of the culture fluid sampling layer and has a diameter of 0.5-1.5mm. In this embodiment, the diameter of the culture fluid sampling hole is 0.5mm, and the inner diameter of the microcolumns formed in the outer layer of the liquid reservoir is 0.75mm; the cell sampling hole and the cell outlet on the cell sampling layer 1 have the same size, with a diameter of 0.5-1.5mm, and in this embodiment, it is 0.75mm; the size and shape of the circular glass slide 13 on the cell growth scaffold layer 2 are consistent with the size and shape of the cell sampling layer 1 and the culture fluid reservoir. The shape of the sample introduction layer 3 is matched, and the diameter of the inscribed circle is 2-6 mm. In this embodiment, the inscribed circle is a circle with a diameter of 4 mm. The cell sample introduction channel 6 and the cell sample outlet channel 9 on the cell sample introduction layer 1 are symmetrically distributed. The depth of the channel is 30-100 microns, and the width of the channel is 500-1000 microns. In this embodiment, the depth and width of the cell inlet and outlet channels are 30 microns and 700 microns, respectively. The diameter of the culture fluid reservoir 14 on the culture fluid introduction layer 3 is 2-4 mm. In this embodiment, it is 3 mm. The inner diameter of the fence micropillar array 20 is 1-2 mm, the outer diameter of the fence micropillar array is 3-5 mm, the circumferential width of the fence micropillar is 30-150 μm, 110 μm in this embodiment, the height of the fence micropillar is the same as the depth of the culture fluid reservoir 14, 50-200 μm, 150 μm in this embodiment; the width of the culture fluid injection channel 16 is 300-1000 μm, 750 μm in this embodiment; the chamber 18 is circular, and the inscribed circle diameter is 5-10 The outer micropillars 17 surrounding the chamber 18 have a radial width of 100 to 300 microns, 200 microns in this embodiment, and a circumferential width of 50 to 100 microns, 80 microns in this embodiment, with a depth equal to that of the cell scaffold. The waste liquid discharge channel 19 and the culture fluid inlet channel 16 are symmetrically distributed about the cell scaffold's inscribed circle. The biomimetic cell scaffold can be integrated into a hierarchically bifurcated microchannel network calculated according to Murray's law to enable large-scale cell culture.

[0030] The present invention relates to a biomimetic cell culture microdevice. The top and bottom layers of its three-layer structure (the cell sampling layer and the culture medium sampling layer) are both made of a thermosetting polymer, polydimethylsiloxane (PDMS), or a thermoplastic polymer (such as polymethyl methacrylate (PMMA), polystyrene (PS), or polycarbonate (PC). The structural molding method is selected based on the material type: PDMS is molded using soft etching, while thermoplastic polymers are molded using hot pressing. Both methods require the use of a mold.

[0031] The molds for the cell and culture medium injection layers are made of SU-8 glue. The production process is as follows: First, a layer of negative resist (BN-303 or BN-308) with a thickness of 0.5 to 2 microns is spin-coated on a flat glass slide to enhance the bonding strength between the SU-8 glue and the substrate. After the negative resist has dried, another layer of SU-8 glue is spin-coated to a thickness equal to the depth of the cell injection channel, 20 to 100 microns (30 microns in this example). Figure 9 After pre-baking, the SU-8 glue was patterned by photolithography and the SU-8 glue mold was obtained after development. To facilitate subsequent demoulding, the dried mold was placed in a drying tower and fumigated with trimethylchlorosilane for 1 hour. The cell growth scaffold layer was produced using 3D printing technology ( Figure 8 ), whose circular glass slides are processed to the target size by laser cutting or micro-milling. This processing method can accurately construct a three-dimensional structure, providing good support for cell attachment and growth. The three-layer structure is made of polydimethylsiloxane (PDMS) and soft etching technology is used. Using a special puncher for microfluidic chips, through holes are punched at the corresponding positions of the culture medium inlet and outlet sample holes and the cell inlet and outlet sample holes. Finally, the three-layer structure is bonded by oxygen plasma treatment or hot pressing ( Figure 10 ). Oxygen plasma treatment is applicable to all-PDMS structures. After treatment, hydrophilic hydroxyl groups (-OH) are generated on the bonding surface, and strong oxygen bonds (-O-) and water (H2O) are formed after bonding to achieve permanent bonding. Hot press bonding is applicable to all-thermoplastic material (PMMA, PS, PC) structures. The hydrophobic groups must be removed by UV treatment first, and then the material is heated and pressurized to reach the glass transition temperature before bonding is completed. This embodiment uses oxygen plasma treatment for bonding, and the parameters are 20W and 50 seconds. During the bonding process, the cell sampling layer and the cell growth scaffold layer, as well as the cell growth scaffold layer and the culture medium sampling layer need to be precisely aligned, and visual microscopy technology is used to assist in adjusting the position to ensure precise alignment between the chip layers.

[0032] The scope of protection claimed in the patent of the present invention is not limited to the above embodiments.

Claims

1. The three-dimensional dynamic cell culture microdevice comprises a three-layer structure, which comprises, from top to bottom, a cell sampling layer (1), a cell support layer (2) and a culture medium sampling layer (3).

2. The cell sampling layer (1) as claimed in claim 1, characterized in that The lower surface of the device has a concave-convex structure, wherein the concave structure forms a cell sampling microchannel (6), and the convex structure protrudes from the lower surface of the device to form an inner microcolumn (7) and a fixing foot (8).

3. The cell sampling layer (1) as claimed in claim 1, characterized in that Cell sampling is performed by symmetrically arranging multiple cell sampling microchannels (6).

4. The cell scaffold layer (2) as claimed in claim 1 comprises a cell growth scaffold (12) and a circular glass slide (13).

5. The culture fluid sampling layer (3) as claimed in claim 1 is composed of a culture fluid sampling channel (16) and an outer layer of microcolumns (17).

6. The cell scaffold layer (2) as claimed in claim 1 is embedded in the unit chamber (18) on the culture fluid sampling layer (3) and is fixed by the inner microcolumns (7) and fixing feet (8) on the cell sampling layer (1) to form an elastic interference fit.

7. The cell sampling layer (1) as claimed in claim 1, wherein the depths of the channels and unit chambers are the same, ranging from 40 μm to 60 μm; the heights of the inner microcolumns and the fixing feet are the same, ranging from 60 μm to 100 μm.

8. The cell scaffold layer (2) as claimed in claim 1, wherein the number of layers in the vertical direction of the scaffold is 8 to 12; and the base is a circular glass slide with a diameter of 6 mm to 10 mm and a thickness of 100 μm to 200 μm.

9. The culture fluid sampling layer (3) as claimed in claim 1, wherein the depths of the channels and unit chambers are the same.

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