CN105420105B - Biochip and its manufacture method - Google Patents

Abstract

The invention relates to a biochip, comprising a cell culture microstructure layer and a sealed microporous membrane, and the sealed microporous membrane is covered on the cell culture microstructure layer, wherein the cell culture microstructure layer includes a cell culture chamber and the liquid inlet and liquid outlet respectively arranged on both sides of the cell culture chamber, the cell culture chamber is formed by a plurality of microstructures arranged, and a liquid inlet A channel, a liquid outlet channel is provided between the cell culture chamber and the liquid outlet, so that the liquid inlet, the cell culture chamber and the liquid outlet communicate with each other. According to the present invention, a microenvironment closer to the cell growth niche can be provided, and the controllable and high-throughput culture of cells, especially neural stem cells, can be realized in vitro.

Description

Biochip and its manufacturing method

technical field

The invention relates to the field of biomedical engineering, in particular to a biochip and a manufacturing method thereof.

Background technique

Central nervous system diseases, such as Parkinson's syndrome, ALS, Alzheimer's disease, etc., are major problems in the medical field. Medical research shows that the central nervous system can be repaired by its own endogenous stem cells. Through the release of various chemokines after nerve tissue damage, neural stem cells are attracted to gather at the damaged site, and differentiate into different types of cells under the action of the local microenvironment, thereby repairing and replenishing damaged nerve cells. At the same time, neural stem cells can strengthen the connection between synapses and establish new neural circuits. However, because these primitive neural stem cells are rare and in a quiescent state, and lack specific morphology, surface markers and differentiation antigens, they cannot be highly purified and isolated, and are difficult to clone. Therefore, it is the only effective way to culture and transplant neural stem cells in large quantities from the outside world.

Clinical and research results show that neural stem cells have good biological characteristics. They can self-replicate and proliferate in large quantities in vitro. Neural stem cells transplanted in the host have obvious survival, migration and differentiation capabilities, and can significantly promote the recovery of nerve function. At present, the in vitro culture of neural stem cells mostly adopts methods such as cell culture dishes and cell culture well plates. The methods used for transplanting neural stem cells mainly include local injection transplantation, cerebrospinal fluid injection transplantation, and blood circulation injection transplantation. A large number of animal experiments and clinical applications have shown that traditional cell culture methods are difficult to provide the complex and multi-level microenvironment required for the growth and differentiation of neural stem cells, making the research results far from the real situation in vivo, and it is difficult to meet the needs of stem cell research in many aspects. Traditional transplantation methods are inefficient, risky, life-threatening, and costly, hindering the application of neural stem cells. Therefore, seeking a highly simulated and controllable neural stem cell culture microenvironment and a safe and reliable transplantation method have become the difficulties of neural stem cell research.

Microfluidic technology is an emerging interdisciplinary subject involving chemistry, fluid physics, microelectronics, new materials and biomedical engineering. It integrates the functions of biological and chemical laboratories, such as cell culture and detection and other basic operating units, on a controllable tiny chip, analyzes a large number of biomolecules in a short time, and accurately obtains a large amount of information in the sample. The microfluidic chip is a three-dimensional culture system, which can highly simulate the growth environment of cells in vivo, precisely control the cell microenvironment elements such as substance concentration and pH value, and can flexibly combine various operating units to make cell injection, culture, capture, and lysis Processes such as separation and detection can be completed on one chip. Microfluidic chips can provide high-precision and controllable cell research conditions that traditional methods do not have, and it is a leap forward for cell culture in vitro. In recent years, microfluidic technology has brought innovations in in vitro cell culture, and successfully achieved a variety of cell culture in vitro. However, the microenvironment for the growth of neural stem cells is very complex and more demanding than that of ordinary cells. At the same time, the culture and differentiation of neural stem cells are affected by a series of factors, such as the reaction between cells and cells and between cells and matrix, liquid shear force, microchannel flow field distribution, and liquid exchange speed. Manufacturing a highly integrated, high-throughput structure that is more suitable for the cultivation of neural stem cells is the core issue in the development of microfluidics. At the same time, the field of neural stem cell transplantation based on microfluidic chips is also blank.

Contents of the invention

technical problem

The present invention is proposed in view of the above situation, and the purpose is to provide a biochip and its manufacturing method, which can provide a microenvironment closer to the cell growth niche, and realize the in vitro controllable and high-throughput of cells, especially neural stem cells. Quantitative cultivation.

In order to solve the above problems, the biochip of the present invention is characterized by comprising a cell culture microstructure layer and a sealed microporous membrane, wherein the sealed microporous membrane is covered on the cell culture microstructure layer.

In addition, the manufacturing method of the biochip involved in the present invention includes the following steps: a mold making step, using ultrapure water to clean the silicon chip, and when it is not completely dry, use acetone to ultrasonically clean it, and then use ultrapure water to clean the silicon chip on the silicon chip. Clean the residual liquid and dry it with nitrogen gas for later use. After the chip is modified by adding a few drops of HDMS reagent in a volatile dry cylinder, place the silicon chip in the center of the glue homogenizer to keep it balanced. After spreading the negative glue, stand still for a specified time, and Use the heated electric heating plate, turn on the mercury lamp, start the glue rejection machine to shake the glue, stagnate in the vacuum for a specified time, and restore the center of the silicon wafer to the plane, then place the silicon wafer in the incubator, and then turn on the exposure machine for exposure. And developing with a developing solution; the substrate production step is to mix and stir the silicone rubber elastomer and curing agent evenly in a volume ratio of 10:1, and remove the mixed silicone rubber elastomer and curing agent in a vacuum drying oven. Then pour it on the surface of the prepared male mold, take it out after baking for a specified time, peel off the solidified polydimethylsiloxane after cooling, and punch holes in the predetermined positions to form the liquid inlet and the liquid outlet respectively. Then cut into the specified size, and successively soak in lye and acid solution overnight, rinse with deionized water, dry for later use; seal the microporous membrane manufacturing step, clean the polyethylene terephthalate sealed microporous membrane Disinfection, punch holes at the corresponding entrances and exits, and cut them into specified sizes; in the bonding step, place the manufactured cell culture microstructure layer and sealed microporous membrane on the ultra-clean table, irradiate with ultraviolet light, and then seal the micropores The membrane covers the cell culture microstructure layer and relies on the adsorption force to complete the adhesion.

In addition, the biochip of the present invention can be used for high-efficiency cell culture in vitro and cell transplantation in the form of a biochip.

Beneficial effect

The biochip of the present invention has a cell culture microstructure layer, which can realize an in vitro microenvironment closer to the growth niche of cells, especially neural stem cells, and form a concave-convex mesh structure in the cell culture chamber, which can realize the cultivation of cells, especially neural stem cells. Indoor high-density growth and differentiation, thus solving the problem of harsh requirements for in vitro culture of neural stem cells and low growth and differentiation density.

Since the biochip of the present invention has a sealed microporous membrane, it can ensure the normal exchange between the biochip implanted in the body and the nutrient base fluid of the cells in the body, and ensure the survival rate of cells, especially the survival rate of neural stem cells. In addition, it can also provide a channel for the internal neural stem cells to overlap with the neural tissue in the body, and build a platform for damaged nerve repair and information transmission. Therefore, cells, especially neural stem cells, can be transplanted in a way superior to traditional cell transplantation.

Description of drawings

Fig. 1 is a perspective view of a biochip of the present invention.

Fig. 2 is a side view of the biochip of the present invention.

Fig. 3 is a top view of the biochip of the present invention.

Fig. 4 is a schematic diagram schematically showing the cell culture microstructure layer of the biochip of the present invention, wherein the lower part of the image is an enlarged view of the hexagonal microstructure of the cell culture chamber.

Explanation of reference signs:

1: Cell culture microstructure layer 2: Sealed microporous membrane 11: Liquid inlet 12: Liquid inlet channel 13: Cell culture chamber, 14: Liquid outlet channel, 15: Liquid outlet

detailed description

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

As shown in FIGS. 1 to 4 , the biochip involved in the present invention includes a cell culture microstructure layer 1 and a sealed microporous membrane 2 covering the cell culture microstructure layer.

Wherein, the cell culture microstructure layer includes a liquid inlet 11 , a liquid outlet 15 , a liquid inlet channel 12 , a liquid outlet channel 14 and a cell culture chamber 13 formed by arranging a plurality of microstructures. Wherein, the liquid inlet 11 and the liquid outlet 15 are respectively arranged on both sides of the cell culture chamber 13, and a liquid inlet channel 12 is arranged between the liquid inlet 11 and the cell culture chamber 13, and the liquid outlet 11 is connected to the cell culture chamber 13. A liquid outlet channel 12 is provided between the culture chambers 13 , so that the liquid inlet 11 , the cell culture chamber 13 and the liquid outlet 15 are connected.

As the cell culture microstructure layer, materials such as silicon, glass, quartz, polymethacrylate, and polydimethylsiloxane can be used. Among them, polydimethylsiloxane (PDMS) has high light transmittance and gas permeability. Good stability, high chemical stability, high biocompatibility, can be used as implant material, so polydimethylsiloxane (PDMS) is preferred.

In order to ensure that the culture fluid flows smoothly inside the channel and the operation of changing the culture fluid is convenient, the depth of the liquid inlet, liquid outlet, liquid inlet channel, liquid outlet channel and cell culture chamber of the cell culture microstructure layer is preferably the same, for example, the depth can be 80 μm to 150 μm, preferably 100 μm.

The shape of the liquid inlet and the liquid outlet can be the same, preferably both are circular. The diameters of the two can be the same, for example, 0.45 to 0.7 mm, preferably both are 0.45 mm. On the basis of satisfying the size of the existing cell liquid inlet pipe, the smaller the diameter of the liquid inlet and the liquid outlet, the better, so that the size of the prepared biochip meets the requirements for implantation.

The shape of the liquid inlet channel and the liquid outlet channel can be the same, preferably both are rectangular. In this case, the length can be 250 to 400 μm, and the width can be 200 to 300 μm. Among them, the preferable lengths are both 335 μm and the widths are both 240 μm.

The cell culture chamber can be designed in various shapes, such as circular, polygonal, oval, etc. Cell culture chambers having a hexagonal structure are preferred in the present invention.

The cell culture chamber can also be provided with multiple microstructures, preferably hexagonal microstructures in the present invention. The size of the hexagonal microstructure is that the diameter of the inscribed circle is 10 μm to 40 μm, preferably 20 μm; the depth is 10 μm to 20 μm, preferably 10 μm; the distance between the hexagonal microstructures is 20 μm to 40 μm, preferably 20 μm. According to the size of the cells, a concave-convex mesh structure is formed in the culture chamber to simulate the growth environment of the cells, so as to achieve high-density growth and differentiation of cells in the culture chamber.

4125 to 8625 microstructures, preferably 6895 hexagonal microstructures, can be provided as required. In this way, the complex niche in which cells, especially neural stem cells, can grow can be better simulated, and a more suitable structure can be provided for cell growth and differentiation.

It can be known from the above that the microstructure is a three-dimensional microporous structure. The physical factors of this three-dimensional microporous structure have an impact on cell growth and development, and provide a different cell growth microenvironment from conventional culture. The two-sided structure of the micropore provides a good space for cells to attach. With the help of such a spatial structure, the cells have two faces in contact with the surface of the material and can grow more stably on the surface of the material, enabling the controllable and high-throughput culture of cells, especially neural stem cells, in vitro.

As the sealing microporous membrane, it is preferable to use implantable materials with good hydrophilicity, high chemical stability and good biocompatibility, such as polyethersulfone membrane (PES), polyethylene terephthalate membrane ( PET), organic nylon film, hydrophilic polyvinylidene fluoride film (PVDF) and other materials, among which polyethylene terephthalate film (PET ). Moreover, since the channel in the PET film is a cylindrical straight through hole with regular geometric shape and uniform pore size, it can realize the exchange of culture medium, and also provide a channel for the cell synapse inside the biochip to overlap with the tissue in the body.

The micropore density of the sealed microporous membrane may be 10 ⁴ -10 ¹¹ /cm ² , preferably 1.7× ^{10 5} /cm ² . The pore size can be selected according to the cell size, as long as it can ensure that the cells will not leak out. For example, it can be 8 μm to 40 μm, preferably 8 μm.

In this way, after the chip is implanted in the body, the sealed microporous membrane of the present invention can normally exchange with the nutrient base fluid of the cells in the body, ensuring that the cells implanted in vitro, such as neural stem cells, can grow and differentiate normally in the body. Furthermore, the sealed microporous membrane can realize the overlap between the neural dendrites of the implanted neural stem cells and the neural stem cell tissue in the body while ensuring that the neural stem cells inside the chip do not leak out.

The thickness of the biochip comprising the cell culture microstructure layer and the sealing microporous membrane of the present invention can be 0.3 to 0.7mm, preferably 0.5mm, the width can be 1 to 5mm, preferably 1mm, and the length can be 3 to 5mm, preferably is 3mm. Therefore, the size of the biochip of the present invention is smaller than the traditional size, which can make the in vitro experiment operation convenient and the implantation effect is good.

Next, the method of manufacturing the biochip of the present invention will be described in detail. Specifically, the following steps are included.

<Manufacturing procedure of cell culture microstructure layer>

Taking the polydimethylsiloxane (PDMS) material as an example, the manufacturing steps of the cell culture microstructure layer are specifically described as follows.

1) Mold making.

Clean the silicon wafer with ultra-pure water. When it is not completely dry, use acetone to ultrasonically clean it for 1 minute, then use ultra-pure water to clean the residual liquid on the silicon wafer, and finally blow dry it with nitrogen gas for later use.

Place the chip in a volatile dry cylinder, add 1 to 2 drops of HDMS reagent, and modify for 3 minutes.

Place the silicon wafer in the center of the glue homogenizer, and balance it well; spread the negative glue SU-82015 (MICROCHEM), and let it rest for 1-2 minutes after laying; adjust the 200°C electric heating plate to 95°C; turn on the mercury lamp, and start the glue shaker Spin at 500rpm for 18 seconds, then spin at 1500rpm for 30 seconds, and stagnate in vacuum for 1-2 minutes after the end, so that the center of the silicon wafer will return to the plane and the glue layer will be more uniform.

Next, place the silicon wafer in an incubator at 65°C for 10 minutes and at 95°C for 1 hour, then turn on the exposure machine and expose for 310 seconds.

Then, the silicon wafer was left to stand at 65° C. for 5 minutes and 95° C. for 20 minutes, respectively, and then developed with a developer.

2) Production of polydimethylsiloxane-based sheets.

Use Sylgard184 silicone rubber elastomer and Sylgard184 curing agent (Momentive, USA) to mix and stir evenly at a volume ratio of 10:1.

Degas the mixed silicone rubber elastomer and curing agent in a vacuum drying oven, pour it on the surface of the prepared male mold, bake it at 80°C for 30 minutes, take it out, and put the solidified polydimethylsiloxane after cooling Alkane stripping.

Punch holes at the inlet and outlet of the design to form the liquid inlet and outlet respectively (see Figure 4), then cut to a suitable size, and soak overnight in lye and acid successively, and rinse with deionized water Rinse, dry and set aside.

<Manufacturing steps of sealed microporous membrane>

Clean and disinfect the polyethylene terephthalate (PET) sealed microporous membrane, punch holes corresponding to the liquid inlet and liquid outlet, and cut into appropriate sizes for later use.

<Bonding step>

Place the prepared cell culture microstructure layer and sealed microporous membrane on the ultra-clean table, irradiate for 3 hours at a distance of 3 to 4 cm from the ultraviolet lamp, and then cover the sealed microporous membrane on the cell culture microstructure layer. Or rely on the adsorption force to complete the bonding so as to obtain the biochip of the present invention. In addition, after covering, it can also be pressed with heavy objects to promote the adhesion of the two.

The biological chip of the present invention can be used for culturing high-density cells, especially neural stem cells, in vitro.

In addition, by using the biochip of the present invention, cultured cells can be transplanted into the body in the form of a chip.

Furthermore, using the biochip of the present invention, cells with high density can be accurately transplanted to a predetermined position in the body, for example, high-density neural stem cells can be accurately transplanted to a predetermined position in the brain. For example, a hole is made in the middle and outside of the skull of an animal such as SD rats with a hole diameter of 3 to 4 mm, the dura mater is opened, and the biochip is placed in the hole with the side of the sealing membrane facing down. The hole was then closed with bone wax, and the surgical wound was sutured.

Example

Embodiments of the present invention can be understood more clearly through the following examples.

<Manufacturing of biochips>

A circular liquid inlet and liquid outlet with a diameter of 0.45mm, a rectangular liquid inlet channel and a liquid outlet channel with a length of 335 μm and a width of 240 μm, and a hexagonal cell culture chamber and 6895 cells inside The hexagonal microstructure is used to make a printing mask by AutoCAD drawing software. Wherein, the size of the hexagonal microstructure is that the diameter of the inscribed circle is 20 μm, the depth is 10 μm, and the distance between the hexagonal microstructures is 20 μm.

After cleaning and modifying the silicon wafer, spread the glue SU-82015 evenly on the surface with a glue spreader. After the adhesive layer is uniform, UV exposure is performed for 310 seconds, and the mask pattern is transferred to the photoresist. After developing, the unexposed photoresist is removed.

Mix Sylgard184 silicone rubber elastomer and Sylgard184 curing agent (Momentive, USA) at a ratio of 10:1 by volume and stir evenly. Then degas the mixed silicone rubber elastomer and curing agent in a vacuum drying oven, pour it on the surface of the prepared male mold, bake it at 80°C for 30 minutes, take it out, and peel off the cured PDMS after cooling, so that the The cell culture chamber, the liquid inlet, the liquid outlet, the liquid inlet channel and the liquid outlet channel are transferred to the PDMS substrate.

A puncher is used to punch holes at selected liquid inlets and liquid outlets on the manufactured PDMS substrate to form the liquid inlets and liquid outlets. Then it is cut into a size of 3mm in length and 1mm in width, soaked in lye and acid solution overnight, rinsed with deionized water, and dried to form a cell culture microstructure layer for later use.

Clean and disinfect the polyethylene terephthalate (PET) sealed microporous membrane, punch holes corresponding to the liquid inlet and liquid outlet, and then cut it into a size of 3mm in length and 1mm in width for later use.

Place the prepared polydimethylsiloxane (PDMS) cell culture microstructure layer and polyethylene terephthalate (PET) sealed microporous membrane on the ultra-clean table, and use ultraviolet lamps to irradiate at close range 3 hours, then layered in turn and pressed with a heavy object to make the two bond. So far, the fabrication of the biochip is completed.

<In vitro culture of neural stem cells on a biochip>

The biochip manufactured above was washed successively with distilled water, 75% alcohol, and deionized water to ensure that there was no material residue inside the channel. After autoclaving at 121° C. for 20 minutes, put the chip into a 60° C. incubator to dry. Then the completely dried biochip was washed with PBS buffer solution through the liquid inlet of the chip with a micro-sampler for 3 times, so as to ensure that the liquid residue in the microchannel was completely replaced by PBS. The chip was then coated with multi-Matrigel, placed in a 37°C incubator for 2 hours, washed three times with PBS, coated with laminin (LN), and placed in a 4°C refrigerator overnight.

The neural stem cells used in the present invention are obtained from the subventricular zone of 0-3 day old SD rats. The neural stem cells were digested with 0.05% trypsin (Trypsin-EDTA) to prepare a cell suspension, and then centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Then use the SD rat neural stem cell medium to resuspend the cells, and adjust the neural stem cell density to 1×10 ⁵ cells/ml for later use.

The components of the SD rat neural stem cell culture medium (purchased from Thermofisher Company) are as follows:

Using a sterilized medical syringe, a capillary tube with an inner diameter of 0.45 mm and a connecting steel needle, slowly inject the neural stem cell suspension into the chip through the liquid inlet of the biochip, and the amount of liquid injected is about 1 μl for cell loading. Observe under a microscope to confirm that the cells are evenly distributed in the cell culture chamber. Then, place the chip in a 37°C, 5% CO ₂ incubator for culture, observe the cell growth after 7 hours, and record the image of the cell growth. Then, observe again 24 hours after the completion of cell loading, and record the image of cell growth status again.

After 24 hours of cell culture, the implantation experiment was performed under the condition of ensuring good growth.

<Implantation experiment of well-grown neural stem cell biochip>

Take 10 healthy adult SD rats, and use 3.6% chloral hydrate to perform abdominal injection anesthesia according to the ratio of body weight 1ml/100g. Then, the anesthetized SD rats were subjected to craniotomy, and a hole was made in the middle and outer sides of the skull with a hole size of 3.5mm. The surgical wound is closed and sutured. Rats were cultured normally and the physiological conditions of the rats were observed.

48 hours after implanting the biochip, 10 rats were sacrificed, the biochip was taken out for microscopic observation, and the microscopic observation results were recorded. The results of statistical analysis of the observation results showed that the survival rate of neural stem cells in the biochip was 30%. Due to the normal cell growth cycle, cells have a 50% apoptosis rate, coupled with inflammatory reactions and other in vivo unmeasured factors, which have a non-negligible impact on cell growth. Therefore, compared with the 20% survival rate of traditional direct injection of neural stem cells, the survival rate of stem cells transplanted with the biochip of the present invention is significantly improved. In addition, the biggest problem with traditional culture lies in cell implantation and location determination, and it is difficult to observe cell growth by staining ventricle slices. Compared with traditional methods, the biochip of the present invention can be implanted with cells at certain positions.

From the above description, it can be known that using the biochip provided by the present invention can provide a cell culture microenvironment simulating cells, especially neural stem cells, so as to realize high-density culture of cells, especially neural stem cells in vitro. In addition, because it has a sealed microporous membrane that can normally exchange cell nutrient solution, it can realize the normal exchange of implanted cells and in vivo cell nutrient base solution, ensuring that the cells implanted in vitro can grow and differentiate normally in vivo. Furthermore, the sealed microporous membrane provides a channel for the neural dendrites of the implanted neural stem cells to overlap with the neural stem cell tissue in the body while ensuring that the cells inside the chip do not leak out. Therefore, using the biochip of the present invention can realize efficient and reliable transplantation of cells, especially neural stem cells, at a lower cost. The present invention is superior to the traditional method, solves the problems at the present stage, and proposes a new method in nerve restoration and information transmission, which has wide application value.

It should be noted that the above-disclosed embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the technical solutions of the present invention, the present invention can be modified. Various modifications or equivalent replacements are made to the technical solution of the invention, so these modification examples are also included in the scope of the claims of the present invention.

Claims (7)

1. A biochip, characterized in that, It includes a cell culture microstructure layer and a sealed microporous membrane, the sealed microporous membrane covers and adheres to the cell culture microstructure layer, and the micropore density of the sealed microporous membrane is 10 ⁴ -10 ¹¹ / cm ² , the pore size is 8μm to 40μm, where, The cell culture microstructure layer includes a cell culture chamber and liquid inlets and liquid outlets respectively arranged on both sides of the cell culture chamber. The cell culture chamber is formed by arranging a plurality of microstructures, and the microstructures It is a hexagonal microstructure, a liquid inlet channel is provided between the liquid inlet and the cell culture chamber, and a liquid outlet channel is provided between the cell culture chamber and the liquid outlet, so that the liquid inlet, cells The cultivation chamber communicates with the liquid outlet. 2. The biochip according to claim 1, characterized in that, The material of the cell culture microstructure layer is selected from one of silicon, glass, quartz, polymethacrylate and polydimethylsiloxane. 3. The biochip according to claim 1, characterized in that, The depth of the liquid inlet, the liquid outlet, the liquid inlet channel, the liquid outlet channel and the cell culture chamber is 80 μm to 150 μm. 4. The biochip according to claim 1, characterized in that, The size of the hexagonal microstructure is that the diameter of the inscribed circle is 10 μm to 40 μm, the depth is 10 μm to 20 μm, and the distance between the hexagonal microstructures is 20 μm to 40 μm. 5. The biochip according to claim 1, characterized in that, The sealed microporous membrane is selected from one of polyethersulfone membrane (PES), polyethylene terephthalate membrane (PET), organic nylon membrane, and hydrophilic polyvinylidene fluoride membrane (PVDF). . 6. The biochip according to any one of claims 1 to 5, characterized in that, The biochip has a thickness of 0.3 to 0.7 mm, a width of 1 to 5 mm, and a length of 3 to 5 mm. 7. A method for manufacturing a biochip according to any one of claims 1-5, characterized in that, comprising the steps of: In the mold making step, use ultrapure water to clean the silicon wafer. When it is not completely dry, use acetone to ultrasonically clean it, then use ultrapure water to clean the residual liquid on the silicon wafer and blow it dry with nitrogen for later use. Place the chip in a volatilization dry tank After adding a few drops of HDMS reagent to the medium for modification, place the silicon wafer in the center of the glue homogenizer to maintain balance. After spreading the negative glue, let it rest for a specified time, then use the heated electric heating plate, turn on the mercury lamp, and start the glue shaker to shake the glue. , stagnate in vacuum for a specified time, so that the center of the silicon wafer returns to the plane, then place the silicon wafer in the incubator, then turn on the exposure machine for exposure, and develop with a developer; The substrate production step is to mix and stir the silicone rubber elastomer and curing agent evenly at a volume ratio of 10:1, degas the mixed silicone rubber elastomer and curing agent in a vacuum oven, and then pour it into the prepared The surface of the positive mold is taken out after baking for a specified time. After cooling, the solidified polydimethylsiloxane is peeled off, and holes are punched at predetermined positions to form liquid inlets and liquid outlets, and then cut into specified sizes. Soak in lye and acid solution overnight, rinse with deionized water, and dry for later use; The manufacturing step of the sealed microporous membrane is cleaning and disinfecting the polyethylene terephthalate sealed microporous membrane, punching holes corresponding to the liquid inlet and the liquid outlet, and cutting into specified sizes; In the bonding step, place the prepared cell culture microstructure layer and sealed microporous membrane on the ultra-clean table, irradiate with ultraviolet light, and then cover the sealed microporous membrane on the cell culture microstructure layer, relying on the adsorption force to complete bonding.