A muscle pillar chip for lateral exposure

Abstract

The application discloses a simple preparation method of a muscle column chip through lateral exposure, and comprises the following steps: drawing a lateral mask of the chip, uniformly applying glue, pre-baking, exposure, post-baking, development, hardening, and inverse molding. The method is based on the principle of soft lithography, and replaces the forward exposure with lateral exposure. The preparation of the muscle column chip is realized through only one exposure, the success rate of the inverse molding is improved, and the operation of uniform application of glue and exposure multiple times is avoided. The muscle column chip prepared by the method is completely same as the chip prepared by the previous forward exposure, but the success rate is obviously improved, and the method can effectively replace the forward exposure method and realize the simple preparation of the muscle column chip.

Description

Technical Field

[0001] This invention belongs to the field of organ-on-a-chip fabrication, specifically relating to a simple method for fabricating muscle column chips via a single lateral exposure. Background Technology

[0002] Muscles are highly efficient actuators, possessing superior power-to-weight ratio, force-to-weight ratio, compliance, plasticity, scalability, and controllability. Besides their association with the activity of cardiac, skeletal, and smooth muscle cells, tissue contraction is crucial for many other processes throughout the body, such as connective tissue morphogenesis, gastrulation, and wound contraction. Tissue contraction drives tissue development and homeostasis not only at the tissue level but also controls functions such as receptor signaling, differentiation, and proliferation at the cellular level. Tissue contractile force is the tissue's ability to contract itself, while tissue contraction is the metric contraction of the tissue itself. Because the planar microenvironment of two-dimensional culture directly influences the cytoskeleton and cannot provide the degrees of freedom required for proper in vitro mechanical function, three-dimensional engineered tissue structures offer superior models of tissue contractility and function, as they more closely mimic natural tissues and their microenvironment.

[0003] A common method for studying the contractile behavior of engineered microtissues is to embed flexible polydimethylsiloxane (PDMS) micropillars into 3D microtissues. Tissue contraction measurement is crucial for assessing the contractility, structure, and function of engineered tissues, as well as their specific cellular responses to physicochemical stimuli. The size limitations of 3D tissues impose stringent requirements on measurement systems in terms of precise experimental setup, highly sensitive imaging methods, and measurement accuracy. Currently, a common method for observing muscle tissue contraction involves simultaneously seeding muscle tissue onto two flexible micropillars spaced a certain distance apart. As the muscle tissue grows and contracts, the two micropillars are pulled closer together by the traction force of tissue contraction. By measuring the degree of tilt and approach between the two micropillars, the degree of tissue contraction between the muscle tissues can be inferred. One end of each flexible micropillar needs to be completely fixed; therefore, in practical experiments, the channel connecting the two flexible micropillars to their bottoms is often considered as a single structure and fabricated using soft lithography. This allows for completion with only one PDMS molding process. Since soft lithography cannot achieve molding of structures with a smaller top and larger bottom, previous methods designed the template with a larger top and smaller bottom structure, requiring at least two exposures. This method still suffers from low casting success rate and requires multiple exposures, and is prone to damaging the two flexible micropillars during the casting process. This invention aims to fill this gap. Summary of the Invention

[0004] The purpose of this invention is to provide a simple method for fabricating muscle column chips using side exposure, including operations such as drawing a side mask for the chip, spin coating, pre-baking, exposure, post-baking, development, hardening, and casting. This method is based on the principle of soft lithography, replacing forward exposure with side exposure, achieving muscle column chip fabrication with a single exposure. This avoids multiple spin coating exposure operations while improving the success rate of casting. The muscle column chips obtained by this method are completely identical to those produced by conventional forward exposure, and casting failure is almost impossible. It can effectively replace the forward exposure method and achieve simple fabrication of muscle column chips.

[0005] This invention relates to a simple method for fabricating a lateral exposure muscle column chip, comprising the following steps: (1) drawing a chip mask: the chip mask is a muscle column core composed of a substrate and two flexible micropillars.

[0006] The chip unit, wherein the top of the flexible micropillar is a protruding square platform or semi-cylinder, and multiple muscle pillar chip units are repeatedly placed on a chip mask.

[0007] (2) Spreading: Place the glass slide in the center of the spreader and slowly add SU8 to the center of the square glass slide using the dropper.

[0008] Photoresist homogenization;

[0009] (3) Pre-baking: Immediately place the square glass slide on the preheated 93-97℃ baking table and pre-bake for 20-30 minutes;

[0010] (4) Applying photoresist: Place the glass slide on a preheated baking table at 93-97℃, and slowly apply SU8 photoresist to the center of the square glass slide using a dropper, then dry for 12-24 hours.

[0011] (5) Exposure: Place the chip mask on a square glass slide coated with adhesive and expose it in a UV exposure machine;

[0012] (6) Post-baking: Immediately after exposure, place the coated square glass slide on a preheated baking table at 93-97℃ and bake for 30-60 minutes.

[0013] min; finally, remove the slide and place it on a horizontal table to cool naturally;

[0014] (7) Development: Develop the prepared coated glass slide; place it in a glass culture dish, cover the glass slide with developer, place it on a horizontal shaker and shake it slowly until the edge of the glass slide is clear, then stop developing to obtain the chip template;

[0015] (8) Hardening: Place the chip template in a drying oven preheated to 45-60℃, slowly raise the temperature to 170-190℃, harden the film for 2-3 hours; then close the drying oven and allow it to cool naturally.

[0016] (9) Molding: Pour the prepared PDMS mixture onto the surrounded chip template, use a vacuum dryer to degas for 25-30 minutes, dry at 80°C for 30-60 minutes and then take it out; after the PDMS cools naturally, use a blade to peel the cured chip off the template.

[0017] Furthermore, in the above technical solution, in step (4), the thickness of the photoresist on the dried square glass slide is 1-3 mm.

[0018] Furthermore, in the above technical solution, in step (2) or step (4), the SU8 photoresist is a 2000 or 3000 series SU8 photoresist.

[0019] Furthermore, in the above technical solution, in step (4), the amount of SU8 photoresist added is 4-12 mL.

[0020] Furthermore, in the above technical solution, in step (7), the developer is selected from propylene glycol methyl ether acetate or ethyl lactate.

[0021] Furthermore, in the above technical solution, the exposure time in step (5) is 70-210s.

[0022] Beneficial effects of the invention

[0023] This invention is based on the principle of soft lithography. By modifying the mask and chip design, specifically by designing the mask as a side view of a muscle column, muscle column chips can be fabricated with a single exposure. This avoids multiple spin coating exposures and improves the success rate of die casting. The muscle column chips produced by this method are exactly the same as those produced by conventional forward exposure, but with a significantly improved success rate. It can effectively replace the forward exposure method and achieve simple fabrication of muscle column chips. Attached Figure Description

[0024] Figure 1 This is a mask and its magnified schematic diagram of the designed lateral exposure muscle column chip, where the top of the flexible micropillar is a square platform.

[0025] Figure 2 It is Figure 1 A physical image of a muscle column chip template prepared by side exposure using a middle mask.

[0026] Figure 3 It was taken under a microscope. Figure 2 Image of muscle column microarray fabricated using a template.

[0027] Figure 4 This is a mask and its magnified schematic diagram of the designed lateral exposure muscle column chip, in which the top of the flexible micropillar is a semi-cylinder. Detailed Implementation

[0028] First, design and print the required chip mask (a side view of the muscle column chip). Then, apply SU8 photoresist evenly to a glass slide, pre-bake for 30 minutes, re-apply the photoresist, and continue baking for 12-24 hours. Next, place the chip mask on a square glass slide with the photoresist and expose it. Immediately after exposure, post-bake. After completion, place it in a glass petri dish and develop it with propylene glycol methyl ether acetate until the structure is completely clear. Finally, harden the chip template at 180°C for 2 hours to complete the chip template preparation. After template preparation, casting can be performed. Pour the prepared PDMS mixture into the chip template, vacuum dry for 30 minutes, and then bake at 80°C for half an hour before removing it. After the PDMS cools naturally, use a blade to peel the cured chip from the template to obtain the desired muscle column chip. The invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] Example 1

[0030] A simple method for fabricating a lateral exposure muscle column chip, characterized by the following steps: (1) Mask drawing: First, a mask is drawn, consisting of a substrate (60mm×12mm) and two flexible micropillars (0.23mm×0.6mm) forming a muscle column chip unit, wherein the top of the flexible micropillar is a protruding square platform (0.48mm×0.07mm). Multiple units are repeatedly placed on a mask, see Figure 1 (2) Spin coating: Place the glass slide in the center of the spin coating machine, and slowly add SU8 (3035) photoresist to the center of the square glass slide using the dropper, and then spin coating.

[0031] (3) Pre-baking: Immediately place the square glass slide on the preheated 95℃ baking table and pre-bake for 30 minutes;

[0032] (4) Applying photoresist: Place the glass slide on a preheated 95°C drying table, and slowly apply 8 mL of SU8 (3035) photoresist to the center of the square glass slide using a dropper, and then dry for 24 hours.

[0033] (5) Exposure: Place the chip mask on a square glass slide coated with adhesive and expose it in a UV exposure machine for 140 seconds;

[0034] (6) Post-baking: Immediately after exposure, place the square glass slide with adhesive on a preheated 95°C baking table and bake for 30 minutes; finally, remove the glass slide and place it on a horizontal table to cool naturally.

[0035] (7) Development: Develop the prepared coated glass slide; place it in a glass culture dish, submerge the glass slide in propylene glycol methyl ether acetate, place it on a horizontal shaker and shake it slowly until the edge of the glass slide is clear, then stop developing to obtain the chip template;

[0036] (8) Hardening: Place the chip template in a preheated 50°C drying oven, slowly raise the temperature to 180°C, harden the film for 2 hours; then close the drying oven and allow it to cool naturally.

[0037] (9) Molding: Pour the prepared PDMS mixture onto the prepared chip template, degas using a vacuum dryer for 30 minutes, dry at 80°C for 30 minutes, and then remove. After the PDMS cools naturally, use a blade to peel the cured chip off the template. The specific structure of the obtained muscle column chip is shown in [the image / description]. Figure 3 .

[0038] Example 2

[0039] A simple method for fabricating a lateral exposure muscle column chip, characterized by the following steps: (1) Mask drawing: First, a mask is drawn, consisting of a substrate (60mm×12mm) and two flexible micropillars (0.23mm×0.6mm, with a micropillar spacing of 1.18mm) forming a muscle column chip unit, wherein the top of the flexible micropillar is a protruding semi-cylinder (radius of 0.25mm). Multiple units are repeatedly placed on a mask, see Figure 4 ;

[0040] (2) Spin coating: Place the glass slide in the center of the spin coater, and slowly add SU8 (3035) photoresist to the center of the square glass slide using the dropper before spin coating.

[0041] (3) Pre-baking: Immediately place the square glass slide on the preheated 95℃ baking table and pre-bake for 30 minutes;

[0042] (4) Applying photoresist: Place the glass slide on a preheated 95°C drying table, and slowly apply 8 mL of SU8 (3035) photoresist to the center of the square glass slide using a dropper, and then dry for 24 hours.

[0043] (5) Exposure: Place the chip mask on a square glass slide coated with adhesive and expose it in a UV exposure machine for 140 seconds;

[0044] (6) Post-baking: Immediately after exposure, place the square glass slide with adhesive on a preheated 95°C baking table and bake for 30 minutes; finally, remove the glass slide and place it on a horizontal table to cool naturally.

[0045] (7) Development: Develop the prepared coated glass slide; place it in a glass culture dish, submerge the glass slide in propylene glycol methyl ether acetate, place it on a horizontal shaker and shake it slowly until the edge of the glass slide is clear, then stop developing to obtain the chip template;

[0046] (8) Hardening: Place the chip template in a preheated 50°C drying oven, slowly raise the temperature to 180°C, harden the film for 2 hours; then close the drying oven and allow it to cool naturally.

[0047] (9) Molding: Pour the prepared PDMS mixture onto the surrounding chip template, degas it using a vacuum dryer for 30 minutes, dry it at 80°C for 30 minutes, and then remove it; after the PDMS cools naturally, use a blade to peel the solidified chip off the template to obtain the muscle column chip.

Claims

1. A simple method for fabricating a muscle column chip using side exposure, characterized in that, Includes the following steps: (1) Drawing of chip mask: The chip mask is a muscle column chip unit composed of a substrate and two flexible micropillars. The top of the flexible micropillar is a protruding square platform or semi-cylinder. Multiple muscle column chip units are repeatedly placed on a chip mask. (2) Spin coating: Place the glass slide in the center of the spin coater, and slowly add SU8 photoresist to the center of the square glass slide using the dropper before spin coating. (3) Pre-baking: Immediately place the square glass slide on the preheated 93-97℃ baking table and pre-bake for 20-30 minutes; (4) Applying photoresist: Place the glass slide on a preheated baking table at 93-97℃, and slowly apply SU8 photoresist to the center of the square glass slide using a dropper, then dry for 12-24 hours. (5) Exposure: Place the chip mask on a square glass slide coated with adhesive and expose it in a UV exposure machine; (6) Post-baking: Immediately after exposure, place the square glass slide with adhesive on a preheated drying table at 93-97℃ and bake for 30-60 minutes; finally, remove the glass slide and place it on a horizontal table to cool naturally. (7) Development: Develop the prepared coated glass slide; place it in a glass culture dish, cover the glass slide with developer, place it on a horizontal shaker and shake it slowly until the edge of the glass slide is clear, then stop developing to obtain the chip template; (8) Hardening: Place the chip template in a drying oven preheated to 45-60℃, slowly raise the temperature to 170-190℃, harden the film for 2-3 hours; then close the drying oven and allow it to cool naturally. (9) Molding: Pour the prepared PDMS mixture onto the surrounded chip template, use a vacuum dryer to degas for 25-30 minutes, dry at 80°C for 30-60 minutes and then take it out; after the PDMS cools naturally, peel the cured chip off the template.

2. A simple method for fabricating a lateral-exposed muscle column chip according to claim 1, characterized in that: In step (4), the thickness of the photoresist on the square glass slide after drying is 1-3 mm.

3. A simple method for fabricating a lateral-exposed muscle column chip according to claim 1, characterized in that: In step (2) or step (4), the SU8 photoresist is a 2000 or 3000 series SU8 photoresist.

4. A simple method for fabricating a lateral-exposed muscle column chip according to claim 1, characterized in that: In step (4), the amount of SU8 photoresist added is 4-12 mL.

5. A simple method for fabricating a lateral-exposed muscle column chip according to claim 1, characterized in that: In step (7), the developer is selected from propylene glycol methyl ether acetate or ethyl lactate.

6. A simple method for fabricating a lateral-exposed muscle column chip according to claim 1, characterized in that: In step (5), the exposure time is 70-210s.

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