Preparation method and application of micro-fluidic chip based on hypoxic microenvironment

Abstract

The invention discloses a preparation method and application of a micro-fluidic chip based on a hypoxic microenvironment, and belongs to the field of micro-fluidic chip preparation.The preparation method comprises the steps that a mold is designed and prepared, and the structure of the mold is used for reetching a runner layer comprising the following structures: a central three-dimensional cell culture cavity; the culture medium runner is positioned on one side of the central three-dimensional cell culture cavity; the oxygen control cavity surrounds the central three-dimensional cell culture cavity and the culture medium runner; preparing the runner layer by using the mold; preparing a substrate layer and a cover layer; and bonding the substrate layer, the flow channel layer and the cover layer in sequence to form the micro-fluidic chip. According to the method, the research cost is remarkably reduced, and the experimental period is shortened.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip fabrication, and in particular to a method for fabricating microfluidic chips based on an oxygen-deficient microenvironment and its application. Background Technology

[0002] Solid tumors often grow faster than their vascular system, leading to poor blood circulation within the tumor and creating a unique hypoxic microenvironment. Tumor hypoxia is a key factor contributing to radiotherapy and chemotherapy resistance, promoting tumor invasion and metastasis, and resulting in poor prognosis. Therefore, in-depth research into the impact of the hypoxic microenvironment on tumor cells is crucial. Currently, research on tumor hypoxia mainly relies on animal models and traditional two-dimensional cell culture. Animal models are costly, time-consuming, and difficult to observe and precisely control microenvironment parameters in real time. Traditional two-dimensional cell culture cannot simulate the three-dimensional structure and physiological barriers of tumors, and typically uses chemical reagents (such as cobalt chloride) to simulate hypoxia, which differs significantly from the actual physical oxygen concentration gradient in vivo.

[0003] Microfluidics technology can precisely control fluids at the microliter or even nanoliter level, constructing complex structures including microchannels and chambers on chips, providing an ideal platform for simulating the physiological and pathological microenvironment of the human body in vitro. However, most existing tumor chips focus on simulating blood vessels or drug delivery, and are not accurate enough in simulating the spatially heterogeneous oxygen concentration gradient formed within tumors due to diffusion limitations, and lack convenient integration with in-situ, multiplex detection methods. Summary of the Invention

[0004] One of the objectives of this invention is to provide a method for fabricating microfluidic chips based on hypoxic microenvironments, in order to solve the problem that the simulation of oxygen concentration gradients in spatial heterogeneity is still not accurate enough in the prior art.

[0005] This invention is achieved through the following technical solution: a method for fabricating a microfluidic chip based on a hypoxic microenvironment, comprising the following steps: designing and fabricating a mold, the structure of which is used to replicate a flow channel layer comprising the following structures: a central three-dimensional cell culture chamber; at least one culture medium flow channel located on one side of the central three-dimensional cell culture chamber; and an oxygen control chamber surrounding the central three-dimensional cell culture chamber and the culture medium flow channel; fabricating the flow channel layer using the mold; fabricating a base layer and a capping layer; and sequentially bonding the base layer, the flow channel layer, and the capping layer to form the microfluidic chip.

[0006] Furthermore, the flow channel layer and the capping layer are made of polydimethylsiloxane, and the base layer is made of glass.

[0007] Furthermore, the flow channel layer is fabricated using soft lithography and replication techniques.

[0008] Furthermore, the molds used in soft lithography and mold replication technology are prepared by coating a substrate with SU-8 negative photoresist and then performing exposure and development processes.

[0009] Furthermore, bonding is achieved by treating each bonding surface with plasma to make it hydrophilic before bonding.

[0010] Furthermore, the culture medium flow channel is configured as two channels, located on both sides of the central three-dimensional cell culture chamber; the culture medium flow channel is separated from the oxygen control chamber by a second physical partition, which is a solid thin-walled structure; the culture medium flow channel is separated from the central three-dimensional cell culture chamber by a first physical partition, which is a solid thin-walled structure or a micropillar array structure.

[0011] Furthermore, when the first physical barrier is a micropillar array structure, the micropillar array is used to restrict the fluid morphology within the central three-dimensional cell culture chamber by utilizing surface tension, while allowing substances within the culture medium channels to diffuse into the central three-dimensional cell culture chamber.

[0012] Another aspect of the present invention provides an application of a microfluidic chip based on a hypoxic microenvironment. The application of the microfluidic chip prepared according to the preparation method described above in establishing a hypoxic microenvironment includes: introducing a biocompatible matrix containing cells into the central three-dimensional cell culture chamber; continuously perfusing cell culture medium into the culture medium channel; and continuously introducing a control gas into the surrounding oxygen control chamber to establish an oxygen concentration gradient within the central three-dimensional cell culture chamber.

[0013] Furthermore, the control gas is an oxygen-low gas, composed of an inert gas and carbon dioxide.

[0014] Furthermore, the inert gas is nitrogen, which accounts for 95% of the volume of the control gas.

[0015] Furthermore, the cells are tumor cells, and the biocompatible matrix is ​​collagen gel.

[0016] Furthermore, the application also includes establishing a dynamic acute hypoxia model, the specific steps of which include: while continuously introducing low-oxygen gas into the oxygen control chamber, perfusing cell culture medium containing a chemical oxygen scavenger into the culture medium channel; utilizing the micropillar array structure between the culture medium channel and the central three-dimensional cell culture chamber, the chemical oxygen scavenger diffuses into the central three-dimensional cell culture chamber, thereby achieving a rapid reduction in oxygen concentration within the central three-dimensional cell culture chamber.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] 1. This invention features a unique three-chamber structure consisting of an oxygen control chamber, a culture medium channel, and a cell culture chamber. By utilizing the diffusion delay effect of gas passing through multiple layers of PDMS physical barriers, combined with the oxygen consumption of the cells within the chamber, a stable and continuous physiological oxygen concentration gradient can be naturally formed in the central culture area, from the edge (near normoxic) to the center (severe hypoxia), thereby more realistically simulating the microenvironment of solid tumors.

[0019] 2. This invention achieves dynamic and precise control of the hypoxic microenvironment by precisely controlling the gas composition and flow rate in the external oxygen control chamber and the liquid flow rate in the culture medium channel, thereby conveniently regulating the distribution range and gradient slope of oxygen concentration in the cell culture chamber, and has good experimental repeatability.

[0020] 3. This invention enables three-dimensional cell culture in a central chamber, embedding cells in extracellular matrix such as collagen, which better simulates the cell growth state in vivo. The cell death phenomenon in the hypoxic core region formed is highly consistent with the necrotic core of solid tumors. Compared with complex animal models, the microfluidic chip fabrication process of this invention is mature, easy to operate, requires less cells and reagents, can achieve high-throughput drug screening, significantly reduces research costs and shortens the experimental cycle. Attached Figure Description

[0021] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0022] Figure 1 This is an overall structural diagram provided for Embodiment 1 of the present invention.

[0023] Figure 2 This is a comparison diagram of hypoxia-induced kinetics provided in Embodiment 2 of the present invention.

[0024] Figure 3 This is a comparison chart of cell survival rates after 1 hour provided in Example 2 of the present invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated herein by reference to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. The terms “comprising,” “including,” “having,” “containing,” etc., as used herein are open-ended terms, meaning that they include but are not limited to. Unless the context clearly indicates otherwise, the expressions “a” and “an” as used herein include plural references. It should be noted that “first,” “second,” etc., are used merely for convenience of description and distinction and should not be construed as indicating or implying relative importance. The term “about” as used herein indicates a range of ±20% of the following numerical value. In some embodiments, the term “about” indicates a range of ±10% of the following numerical value. In some embodiments, the term “about” indicates a range of ±5% of the following numerical value.

[0027] Example 1

[0028] This embodiment provides a microfluidic chip for simulating and detecting the hypoxic microenvironment of tumors. The fabrication steps of this chip include:

[0029] Step 1: Design and manufacturing of microfluidic chips.

[0030] In this embodiment, the chip's overall dimensions are defined as 40mm in length and 25mm in width. The chip has a three-layer structure, consisting of a glass substrate, a PDMS channel layer, and a PDMS capping layer from bottom to top.

[0031] The base layer is a 1mm thick glass slide.

[0032] The flow channel layer is made of polydimethylsiloxane (PDMS) material with a thickness of 3mm. This layer contains the core functional structure, and its specific dimensions are as follows:

[0033] Central three-dimensional cell culture chamber: 10 mm long, 2 mm wide, and 150 μm high.

[0034] Culture medium channels on both sides: located on both sides of the central cell culture chamber, separated from the culture chamber by a PDMS wall with a wall thickness of 100μm; each channel is 500μm wide and 150μm high.

[0035] The surrounding oxygen control chamber completely surrounds the above-mentioned culture chamber and culture medium channel. Its inner wall is 500 μm away from the outer wall of the culture medium channel. The chamber itself has a width of 2 mm and a height of 150 μm.

[0036] Cover layer: Made of PDMS material with a thickness of 2mm, it has fluid inlets and outlets that communicate with each chamber / channel, and the aperture of each is 1.0mm.

[0037] Step 2: Chip manufacturing process. This chip is manufactured using standard soft lithography and molding technology.

[0038] The mold preparation includes using software to design a two-dimensional structure diagram of the chip flow channel layer and fabricating a high-resolution photomask.

[0039] A 4-inch silicon wafer was uniformly coated with a 150μm thick layer of SU-82100 negative photoresist using spin coating. The coated silicon wafer was then pre-baked on a hot plate at 95°C. Using a UV lithography machine, the SU-8 photoresist layer was exposed through a photomask at an exposure energy of 300 mJ / cm². After exposure, it was post-baked on a hot plate at 95°C.

[0040] Finally, SU-8 developer was used to develop the mold, removing the photoresist in the unexposed areas. The mold was then cleaned with isopropanol and dried to obtain an SU-8 mold with a raised flow channel structure.

[0041] Step 3: Preparation of PDMS channel layer and capping layer. The PDMS prepolymer and curing agent are thoroughly mixed at a mass ratio of 10:1. The mixture is then placed in a vacuum dryer and evacuated for 30 minutes to remove air bubbles.

[0042] The degassed PDMS mixture was slowly poured onto an SU-8 mold to prepare the flow channel layer. Simultaneously, a portion of the PDMS mixture was poured into a clean petri dish to prepare a smooth cap layer. The mold and petri dish containing the PDMS were then placed in an oven at 80°C for 2 hours to cure.

[0043] After curing, the PDMS layer is peeled off from the mold and petri dish. Using a 1.0mm diameter punch, fluid inlets and outlets are punched on the cap layer at the positions corresponding to each flow channel and chamber. Figure 1 The overall structure diagram of the chip prepared in this embodiment is shown.

[0044] Step 4: Chip bonding and assembly. Place a clean glass slide (substrate layer) and the bonding surfaces of the PDMS layer with the flow channel structure (flow channel layer), as well as the bonding surfaces of the flow channel layer and the PDMS capping layer, together in a plasma cleaner and treat them for 30 seconds in an oxygen environment with a power of 50W to make their surfaces hydrophilic.

[0045] After processing, immediately align and bond the flow channel layer to the substrate layer, then align and bond the cap layer to the flow channel layer, and gently press to ensure tight contact; place the initially bonded chip in an 80°C oven for 30 minutes to enhance the bonding strength.

[0046] Finally, insert appropriately sized polytetrafluoroethylene (PTFE) tubes into the inlet and outlet ports to complete the chip assembly.

[0047] Step 5: Use of the chip and establishment of a tumor hypoxia model

[0048] 1) System preparation and cell culture: Before using the chip, sterilize all channels and chambers by perfusing 75% ethanol through the inlet and outlet for 20 minutes, and then rinse three times with sterile phosphate-buffered saline (PBS).

[0049] Human breast cancer cells (MCF-7) were cultured under standard cell culture conditions (37°C, 5% CO2 incubator).

[0050] 2) Construction of a three-dimensional tumor model:

[0051] MCF-7 cells in logarithmic growth phase were digested and collected, resuspended in cell culture medium, and the cell density was adjusted to 1×10⁶ cells / year. 7 cells / mL.

[0052] Under ice bath conditions, type I rat tail collagen solution (concentration 5 mg / mL) was rapidly and gently mixed with the above cell suspension at a 1:1 volume ratio until a final collagen concentration of 2.5 mg / mL and a final cell density of 5 × 10⁻⁶ cells / mL were achieved. 6 cells / mL.

[0053] Using a microsyringe, the cell-collagen mixture is rapidly and steadily injected into the central three-dimensional cell culture chamber through the pre-reserved inlet.

[0054] Finally, the chip was incubated in a 37°C incubator for 30 minutes to allow the collagen to gel, thus embedding MCF-7 cells into a three-dimensional structure.

[0055] 3) Establishment and maintenance of hypoxia gradient:

[0056] The chip with the three-dimensional tumor model is connected to a microinjection pump and a gas mixing device.

[0057] Fresh DMEM high-glucose medium (containing 10% fetal bovine serum) was continuously infused into the culture medium channels on both sides at a flow rate of 2 μL / min using an injection pump.

[0058] Simultaneously, premixed humidified gas is introduced into the outermost oxygen control chamber at a flow rate of 5 mL / min.

[0059] Hypoxia group: A mixture of 95% N2 + 5% CO2 gas is introduced to simulate a severe hypoxia environment.

[0060] Control group: A mixture of 95% air and 5% CO2 was introduced to simulate a normal oxygen environment.

[0061] Because oxygen needs to diffuse from the outermost oxygen control chamber, through the PDMS wall and the culture medium channels, to the central cell culture chamber, this physical barrier and diffusion path, combined with the cell's own oxygen consumption, naturally forms a stable physiological oxygen concentration gradient within the culture chamber (from the outer edge near the culture medium channels to the center of the culture chamber). Subsequent testing was performed after the system had been running stably for 24 hours.

[0062] Effect verification and data analysis:

[0063] Quantitative detection of oxygen gradient distribution:

[0064] Method 1 (Fluorescent Probe Method): Add 100 μM of the hypoxia probe Pimonidazole to the perfused culture medium and continue culturing for 2 hours. Afterward, remove the chip, fix the cells with 4% paraformaldehyde, and permeate the membrane with 0.2% Triton X-100.

[0065] Immunofluorescence staining was then performed, and the cells were incubated with anti-Pimonidazole primary antibody and secondary antibody with a fluorescent label (such as AF488).

[0066] Finally, the images were observed using an inverted fluorescence microscope.

[0067] Experimental Results: A significant fluorescence signal gradient was observed. Cells in the central region of the cell culture chamber exhibited bright green fluorescence, indicating a severely hypoxic state (oxygen partial pressure <10 mmHg); while the regions near the two side culture channels (102a, 102b) showed very weak fluorescence signals, indicating a near-normative state. This directly confirms the successful establishment of a stable oxygen concentration gradient.

[0068] Spatial distribution analysis of cell viability:

[0069] After the system has been running stably for 48 hours, a mixed dye containing Calcein-AM (green, for marking live cells) and propidium iodide (PI, red, for marking dead cells) is injected into the culture medium channels.

[0070] After incubation for 30 minutes, observe using a fluorescence microscope.

[0071] Experimental results: In the peripheral region of the culture chamber, the vast majority of cells exhibited green fluorescence, indicating good cell viability; however, in the hypoxic core region, a large number of red fluorescent signals appeared, indicating cell death due to prolonged severe hypoxia and malnutrition. This is highly consistent with the necrotic core phenomenon inside solid tumors.

[0072] Example 2

[0073] This embodiment is an improvement on Embodiment 1, aiming to provide a microfluidic chip and its usage method that can simulate rapid switching between chronic and acute hypoxia and establish a steeper oxygen concentration gradient.

[0074] 1. Improvements in chip structure design:

[0075] Compared with Example 1, this example makes key improvements to the structure of the flow channel layer (intermediate layer), specifically including:

[0076] The partition between the central three-dimensional cell culture chamber and the culture medium flow channel:

[0077] In Example 1, a 100μm thick PDMS solid wall was used. In this example, the solid wall is replaced with a micropillar array structure. The specific dimensions are: micropillar diameter 100μm, micropillar spacing 50μm.

[0078] The micropillar array utilizes the surface tension of the liquid to confine the cell-containing collagen gel within the central culture chamber, preventing it from leaking into the culture medium channels on both sides. At the same time, the gaps between the micropillars allow the fluids in the culture medium channels (including nutrients or chemical oxygen scavengers) to come into direct contact with the gel and undergo rapid convection-diffusion exchange, eliminating the diffusion barrier of the solid PDMS wall to macromolecules and chemical ions.

[0079] The partition between the culture medium flow channel and the oxygen control chamber:

[0080] The design in Example 1 is maintained, and a 100μm thick PDMS solid thin wall is still used. This is to ensure that the oxygen concentration in the outer ring gas channel is regulated only by diffusion, and to prevent liquid leakage into the gas channel.

[0081] 2. Chip fabrication:

[0082] The chip fabrication process (photolithography, soft lithography, bonding) is basically the same as in Example 1, the only difference being that the design pattern of the photolithography mask includes the above-mentioned micropillar array structure.

[0083] 3. Establishment and application of dynamic hypoxia model:

[0084] Step A: Routine culture and establishment of a chronic hypoxia gradient:

[0085] 1. As described in Example 1, MCF-7 cells and a collagen mixture were seeded in the central chamber.

[0086] 2. Introduce 95% N2 + 5% CO2 gas into the oxygen control chamber of the outer ring.

[0087] 3. Pour conventional cell culture medium into the culture medium channels on both sides.

[0088] 4. Results: Due to the physical diffusion of the outer gas and cellular oxygen consumption, the system establishes a smooth physiological gradient from the edge (near normoxic) to the center (moderate hypoxia) after 2-4 hours, simulating the natural growth state of tumors.

[0089] Step B, Triggering of Acute Hypoxic Shock:

[0090] 1. When it is necessary to simulate tumor vascular embolism or acute hypoxia events, the perfusion medium in both sides of the culture medium channel is switched to a culture medium containing a chemical oxygen scavenger (in this example, a serum-free culture medium with 10 mM sodium sulfite Na2SO3 added, or a culture medium containing glucose oxidase / catalase is used).

[0091] 2. Results: Due to the use of a micropillar array structure, the chemical oxygen scavenger did not need to penetrate the solid wall of PDMS, but diffused directly into the gel edge through the gaps between the micropillars. The oxygen concentration in the central culture chamber rapidly decreased to below 0.1% (severe hypoxia) within 10-15 minutes, which is more than 10 times faster than relying solely on external gas diffusion (which usually takes several hours to reach equilibrium).

[0092] Nitrogen gas is introduced into the outer ring to maintain a low background oxygen level, while a powerful oxygen scavenger is introduced into the internal culture medium channels, causing a sudden and sharp drop in oxygen partial pressure in the edge region of the culture chamber (the region that originally had a higher oxygen content). This edge-center dual hypoxia trapping mode successfully simulates the acute diffuse hypoxia phenomenon that occurs when solid tumors are treated with anti-angiogenic drugs. Figure 2 The diagram shows a comparison of hypoxia-induced kinetics in this embodiment, illustrating the differences in biological outcomes between chronic and acute hypoxia. The curve for chronic hypoxia shows a gradual decline, representing slow cell apoptosis, a characteristic of chronic hypoxia. In contrast, the curve for acute hypoxia exhibits a sharp drop, representing acute cell necrosis, caused by the acute impact of chemical oxygen scavengers and the micropillar array. This demonstrates that this chip can simulate specific pathological models such as hypoxia-reoxygenation injury or acute embolism.

[0093] Comparative experiments were conducted using the chip (solid wall) from Example 1 and the chip (micropillar array + chemical deoxygenation) from this example, with a hypoxia probe used for detection. Figure 3The graph shows a comparison of cell survival rates after 1 hour for two embodiments. The graph clearly shows the difference in response speed between the two embodiments. Embodiment 1 represents conventional physical diffusion, and the curve rises slowly; while Embodiment 2 represents the improved solution of the present invention, and the curve rises sharply, reaching a plateau at 20 minutes.

[0094] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for fabricating a microfluidic chip based on a hypoxic microenvironment, characterized in that, The preparation method includes: Design and fabricate a mold, the structure of which is used to replicate a flow channel layer comprising the following structure: A central three-dimensional cell culture chamber; At least one culture medium flow channel located on one side of the central three-dimensional cell culture chamber; and An oxygen control chamber surrounding the central three-dimensional cell culture chamber and the culture medium flow channel; The flow channel layer is prepared using the mold described above; Prepare a base layer and a capping layer; and The substrate layer, the channel layer, and the capping layer are bonded sequentially to form the microfluidic chip.

2. The method for fabricating a microfluidic chip based on a hypoxic microenvironment according to claim 1, characterized in that, The flow channel layer and the capping layer are made of polydimethylsiloxane, and the base layer is made of glass.

3. The method for fabricating a microfluidic chip based on a hypoxic microenvironment according to claim 1 or 2, characterized in that, The flow channel layer was fabricated using soft lithography and replication techniques.

4. The method for fabricating a microfluidic chip based on a hypoxic microenvironment according to claim 3, characterized in that, The mold used in the soft lithography and molding technology is prepared by coating a substrate with SU-8 negative photoresist and then performing exposure and development processes.

5. The method for fabricating a microfluidic chip based on a hypoxic microenvironment according to claim 1, characterized in that, The bonding process involves treating each bonding surface with plasma to make it hydrophilic before bonding.

6. The method for fabricating a microfluidic chip based on a hypoxic microenvironment according to claim 1, characterized in that, The culture medium flow channel is configured as two channels, located on both sides of the central three-dimensional cell culture chamber; The culture medium flow channel and the oxygen control chamber are separated by a second physical partition, which is a solid thin-walled structure. The culture medium channel and the central three-dimensional cell culture chamber are separated by a first physical partition; the first physical partition is a solid thin-walled structure or a micropillar array structure.

7. An application of a microfluidic chip based on a hypoxic microenvironment, characterized in that, Application of the microfluidic chip prepared according to any one of claims 1 to 6 in establishing a hypoxic microenvironment for cells. The applications of the microfluidic chip in establishing a hypoxic microenvironment for cells include: A biocompatible matrix containing cells is introduced into the central three-dimensional cell culture chamber; Continuous perfusion of cell culture medium into the culture medium channels; and A control gas is continuously introduced into the surrounding oxygen control chamber to establish an oxygen concentration gradient within the central three-dimensional cell culture chamber.

8. The application of the microfluidic chip based on a hypoxic microenvironment according to claim 7, characterized in that, The control gas is an oxygen-low gas, composed of an inert gas and carbon dioxide.

9. The application of the microfluidic chip based on a hypoxic microenvironment according to claim 8, characterized in that, The inert gas is nitrogen, and its volume fraction in the control gas is 95%.

10. The application of the microfluidic chip based on a hypoxic microenvironment according to claim 7, characterized in that, The cells are tumor cells, and the biocompatible matrix is ​​collagen gel; The application also includes establishing a dynamic acute hypoxia model, the specific steps of which include: While continuously introducing low-oxygen gas into the oxygen control chamber, cell culture medium containing a chemical oxygen scavenger is infused into the culture medium flow channel; By utilizing the micropillar array structure between the culture medium channel and the central three-dimensional cell culture chamber, the chemical oxygen scavenger diffuses into the central three-dimensional cell culture chamber, thereby achieving a rapid reduction in oxygen concentration within the central three-dimensional cell culture chamber.

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