A human-like brain cortex chip, a method for 3D printing a human-like brain cortex and its application

Abstract

This invention discloses a method for rapidly constructing a human-like cerebral cortex organ-on-a-chip using bio-3D printing and its applications. It comprises three parts: a microfluidic chip fabrication method, a human-like cerebral cortex hydrogel preparation method, and the printing of the human-like cerebral cortex. The microfluidic chip comprises five layers: a mixed-flow channel layer, a liquid pool layer, a micropore array layer, a human-like cerebral cortex culture layer, and a culture medium recovery layer. The human-like cerebral cortex hydrogel is composed of gelatin, alginate, and hyaluronic acid. The human-like cerebral cortex is directly printed into the microfluidic chip using a suspension bath extrusion printing method, and then encapsulated to obtain the human-like cerebral cortex organ-on-a-chip. This invention overcomes the shortcomings of traditional cell culture by directly constructing a large-scale human-like cerebral cortex with three interconnected layers in situ within the organ-on-a-chip using bio-3D printing. Perfusion culture mimics cerebrospinal fluid circulation, facilitating substance exchange, maintaining cell viability, and inducing cell differentiation, and can be widely used in drug development for brain diseases.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chips, and more particularly to a human-like brain cortex chip and a method and application for rapidly constructing a human-like brain cortex using bio-3D printing. Background Technology

[0002] The information disclosed in the background section of this invention is intended to enhance understanding of the overall background of the invention, and such disclosure should not necessarily be construed as an admission or in any way implying that the information has become prior art known to those skilled in the art.

[0003] Organ-on-a-chip technology represents a breakthrough in stem cell research in recent years. It involves constructing a microsystem of organ physiology on a slide-sized chip, containing key elements of the organ microenvironment such as living cells, tissue interfaces, biofluids, and mechanical forces. As a novel technology, organ-on-a-chip has attracted increasing attention and holds broad application prospects in life sciences, drug research, personalized medicine, and toxicity prediction.

[0004] Patent CN201811226234.2 discloses a system and method suitable for biological tissue culture and real-time monitoring, including: a bio-3D printer, an organ-on-a-chip, a connecting base, a driving system, and an auxiliary system; the organ-on-a-chip is connected to the driving system via the connecting base; the bio-3D printer is used to construct bio-3D printed tissue; the organ-on-a-chip is used to place culture medium and bio-3D printed tissue, and to culture the bio-3D printed tissue; the connecting base is used to place the organ-on-a-chip and is connected to the driving system; the driving system is used to drive the culture medium to flow within the organ-on-a-chip; the auxiliary system is used to monitor the state of the bio-3D printed tissue; the organ-on-a-chip body further includes: a rigid top layer, a microfluidic layer, and a transparent layer. The system comprises a bottom layer and a sensor chip, wherein the microfluidic layer is disposed between the rigid top layer and the transparent bottom layer, and the sensor chip is in contact with the culture medium of the organ-on-a-chip body; the rigid top layer includes at least one culture chamber, a gas channel, a top surface groove, a bottom surface groove, and a detection area; the bottom surface groove is used to place the microfluidic layer; the microfluidic layer includes at least one culture chamber, a microchannel, a driving groove, a liquid storage groove, a dividing groove, and a fence-like valve; the microchannel connects the at least one culture chamber, the driving groove, the liquid storage groove, and the dividing groove; the fence-like valve separates the dividing groove; wherein the transport unit is matched with at least one culture chamber in the rigid top layer and at least one culture chamber in the microfluidic layer.

[0005] However, the chip structure constructed by the aforementioned patent is complex, making practical application difficult. Furthermore, it cannot support the renewal of the culture medium, easily leading to the accumulation of toxic substances and making long-term culture unsustainable. Most critically, the printing method and organ-on-a-chip described in the patent cannot support the printing of soft tissue, particularly human-like cerebral cortex.

[0006] The human cerebral cortex is characterized by low elastic modulus and high dissipation, and nerve cells have particularly stringent requirements for their living environment. Developing a high-performance, human-cortical-like bio-ink remains a challenge for bio-3D printing. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a human-like brain cortex chip, a method for 3D printing a human-like brain cortex, and its applications. This invention mainly designs a human-like brain cortex chip and combines it with a bio-3D printing method to directly print a three-layered human-like brain cortex in situ within the chip.

[0008] Specifically, the present invention provides the following technical features, and the combination of one or more of the following technical features constitutes the technical solution of the present invention.

[0009] In a first aspect, the present invention provides a human cortical organ-on-a-chip, which comprises, from top to bottom, a mixed-flow channel layer, a liquid pool layer, a microporous array layer, a human cortical culture layer, and a culture medium recovery layer; adjacent layers are sealed with sealing rings.

[0010] The mixed flow channel layer includes a culture medium inlet, a mixed flow channel, a first blind hole, and a culture medium recovery port;

[0011] The liquid pool layer includes a culture medium storage tank, a first through hole, and a second through hole; wherein the culture medium inlet is used to input culture medium, the mixing channel mixes different components of culture medium evenly, and the mixed culture medium is input into the culture medium storage tank of the liquid pool layer through the blind hole and the first through hole;

[0012] The micropore array layer is provided with a third through-hole;

[0013] The human-like cerebral cortex culture layer comprises a culture chamber, a fourth through-hole, and a micropore array; the culture chamber is a human-like cerebral cortex culture chamber; the culture medium flows from the culture chamber through the micropore array layer into the culture medium recovery layer, allowing the culture medium to pass through slowly;

[0014] The culture medium recovery layer is provided with a culture medium recovery pool and a second blind hole. The bottom of the culture medium recovery pool has a slope. The culture medium passes through the second blind hole, the fourth through hole, the third through hole, and the second through hole in sequence to complete the cycle through the culture medium recovery port.

[0015] Preferably, the bottom slope of the culture medium recovery tank 11 is 1°.

[0016] Preferably, the human-like brain cortex chip is made of ordinary flat glass.

[0017] Preferably, the five-layer structure is sandwiched with rubber sealing rings of the same shape for sealing between the layers.

[0018] Preferably, a PET porous membrane is placed beneath the microporous array layer 6 to buffer the pressure of the culture medium on the human cerebral cortex.

[0019] Secondly, based on the aforementioned human cortical organ-on-a-chip, this invention also proposes a method for in-situ bio-3D printing of human cortical brain tissue within a human cortical organ-on-a-chip. The method involves connecting the culture medium recovery layer and the human cortical brain tissue culture layer using studs and fixing them onto a printing platform. A gelatin support bath is injected into a culture chamber. Cell-carrying bio-ink is stored in the syringe of a bio-3D printer. The human cortical brain tissue is then directly printed in-situ into the gelatin support bath within the culture chamber using the bio-3D printer. After printing, the chip is in-situ encapsulated, and then perfused with culture medium for culturing.

[0020] Furthermore, before assembly, the chip is sterilized using a human brain cortex chip at 120°C under high temperature and pressure.

[0021] As a further technical solution, after printing, excess gelatin support bath is scraped off with a scraper, and the microporous array layer, liquid pool layer, and mixed flow channel layer are installed in sequence; culture medium is connected to the culture medium inlet; and the chip is placed in an incubator for incubation.

[0022] As a further technical solution, the above-mentioned bio-ink components are alginate, gelatin and hyaluronic acid.

[0023] As a further technical solution, the preparation method of the bio-ink includes the following steps: dissolving alginate, gelatin and hyaluronic acid in buffer solution respectively, and then uniformly dispersing cells into the ink.

[0024] As a further technical solution, the specific preparation method of the bio-ink is as follows:

[0025] Step 1: Dissolve sodium alginate in 1×PBS, stir at a set temperature for a set time, and degas using an ultrasonic disperser to obtain an alginate solution;

[0026] Step 2: Dissolve gelatin and hyaluronic acid in 1×PBS, stir at a set temperature for a set time, and degas using an ultrasonic disperser to obtain a gelatin composite solution.

[0027] Step 3: Sterilize the alginate precursor solution by irradiating it with ultraviolet light for a set time, and then filter the sterilized gelatin composite solution using a filter at a set temperature.

[0028] Step 4: Mix the alginate solution and gelatin composite solution at a 1:1 ratio at a set temperature to obtain bio-ink, and disperse the cells in the ink.

[0029] Thirdly, based on the above-mentioned printing method, the present invention also provides a human-like cerebral cortex obtained by the aforementioned 3D printing method, which consists of three interconnected layers, wherein the top layer is a tissue plate with cells arranged longitudinally, the middle layer is a sparse nerve fiber bundle, and the bottom layer is a nerve tissue plate arranged laterally.

[0030] Fourthly, the present invention also provides an application of the aforementioned human-like cerebral cortex in neuropharmaceutical screening.

[0031] Fifthly, the present invention also provides the application of the aforementioned bio-ink in materials for repairing nerve damage or in the preparation of human-like cerebral cortex.

[0032] One or more technical solutions provided in this invention have at least the following technical effects or advantages:

[0033] 1. The human-like cerebral cortex chip proposed in this invention uses a double-layer porous membrane to separate the culture chamber, mimicking the structure of the human cranial cavity. The external flowing culture medium can provide low shear stress to promote the maturation of the human-like cerebral cortex. The chip is small in size, has low processing cost, and is easy to use.

[0034] 2. A complex human-like cerebral cortex can be directly constructed in situ within an organ-on-a-chip using a suspension bath printing method. After printing, it can be directly encapsulated and perfused for culture, with the culture medium flow mimicking cerebrospinal fluid circulation, enabling long-term culture. The entire printing process is conducted within a temperature range suitable for cells. Furthermore, the human-like cerebral cortex bio-ink in this invention has adjustable elastic modulus and porosity, supporting the survival of nerve cells.

[0035] 3. During the cultivation process, the human-like cerebral cortex chip of the present invention can study the effect of drugs on the human-like cerebral cortex by changing the composition of the culture medium. It can be widely used in medical research on the human cerebral cortex and is of great significance for drug screening.

[0036] 4. The human-like cerebral cortex printed by this invention consists of a three-layer interconnected structure, realistically mimicking the multi-layered structure of the real human cerebral cortex. The organ-on-a-chip has the ability to cultivate large-scale human-like cerebral cortex. Attached Figure Description

[0037] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. The embodiments of this application will be described in detail below with reference to the accompanying drawings, wherein:

[0038] Figure 1 This is a schematic diagram of the human cortical brain chip structure of the present invention;

[0039] Figure 2 This is an exploded view of the human cortical brain chip of the present invention;

[0040] Figure 3 This is a schematic diagram of the structure of the human-like cerebral cortex of the present invention;

[0041] Figure 4(a) shows the microscopic image of the printed structure and the cell state on the first day after immunofluorescence staining.

[0042] Figure 4(b) shows the microscopic image of the printed structure and the cell state on the seventh day after immunofluorescence staining;

[0043] Figure 5 CCK8 staining results for the printed structure.

[0044] In the figure: 1 Mixed flow channel layer, 2 Mixed flow channel, 3 Blind hole, 4 Liquid pool layer, 5 First through hole, 6 Micropore array layer, 8 Human cerebral cortex culture layer, 10 Culture medium recovery layer; 11 Culture medium recovery pool, 12 Bolt; 13 Blind hole, 14 Fourth through hole, 15 Micropore array, 16 Third through hole, 17 Second through hole, 19 Culture medium recovery port, 20 Culture medium inlet. Detailed Implementation

[0045] The present invention will be further described below with reference to specific drawings and embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0047] Example 1

[0048] This embodiment discloses a human-like cerebral cortex chip. The chip is made of ordinary flat glass and measures 50mm×30mm×11mm. The chip needs to be sterilized at 120℃ under high temperature and high pressure for 20 minutes before use.

[0049] like Figure 1 , Figure 2 As shown, the human-like brain cortex chip is assembled with five layers stacked sequentially: a mixed flow channel layer 1, a liquid pool layer 4, a micropore array layer 6, a human-like brain cortex culture layer 8, and a culture medium recovery layer 10. In particular, rubber sealing rings of the same shape are sandwiched between the five layers for sealing between each layer.

[0050] The mixed flow channel layer 1 includes a culture medium inlet 20, a mixed flow channel 2, a blind hole 3, and a culture medium recovery port 19; wherein the culture medium inlet 20 is used to input culture medium, the mixed flow channel 2 mixes different components of culture medium evenly, and the mixed culture medium is input into the culture medium storage tank 18 through the blind hole 3;

[0051] The liquid pool layer 4 is provided with a culture medium storage pool 18, a second through hole 17, and a first through hole 5. Blind holes 3 communicate with the culture medium storage pool 18 through the first through hole 5. The micropore array layer 6 connects the culture medium storage pool 18 with the human-like cerebral cortex culture layer 8, allowing the culture medium to pass through slowly.

[0052] The human-like brain cortex culture layer 8 includes a culture chamber 9, a fourth through hole 14, and a micropore array 15. The culture chamber 9 is a human-like brain cortex culture chamber.

[0053] The culture medium is fed into the culture medium inlet 20 of the mixed flow channel layer 1, flows through the mixed flow channel 2, and flows into the liquid pool layer 4 through the blind hole 3 and the first through hole 5. The culture medium in the liquid pool layer 4 flows into the human brain cortex culture layer 8 through the micropore array layer 6.

[0054] In particular, the upper and lower surfaces of the microporous array layer 6 are coated with porous elastic films to buffer the impact of the culture medium on the human cerebral cortex.

[0055] After passing through the human-like cerebral cortex culture layer 8, the culture medium flows into the culture medium recovery layer 10 through the micropore array 15, and is recovered through the blind hole 13, the fourth through hole 14, the third through hole 16, the second through hole 17, and the culture medium recovery port 19.

[0056] Specifically, the bottom slope of the culture medium recovery tank 11 is 1°.

[0057] Preferably, the human-like brain cortex chip is made of ordinary flat glass.

[0058] Based on the aforementioned chip, this embodiment also provides a method for preparing human-like cerebral cortex using bio-3D printing, as follows: First, as... Figure 3 As shown, the humanoid cerebral cortex in this embodiment consists of three layers, measuring 30mm × 20mm × 3mm. The three layers are: a bottom layer with horizontally arranged nerve fibers, a middle layer with vertically arranged nerve fiber bundles, and a top layer with longitudinally arranged nerve fiber bundles. These bundles are printed into the aforementioned chip using an extrusion printer. The specific printing method is as follows:

[0059] First, the human-like brain cortex organ-on-a-chip is sterilized by high temperature and high pressure, and then dried at 120°C for 20 minutes.

[0060] Set the printer's low-temperature platform temperature to 15°C. Transfer the cell-loaded bio-ink into the printer syringe, set the syringe temperature to 26°C, and incubate the ink for 10 minutes.

[0061] The human-like cerebral cortex culture layer 8 and the culture medium recovery layer 10 were fixed together using studs 12 and placed on the low-temperature platform of the bioprinter. The gelatin support bath prepared in Example 1 was poured into the culture chamber 9 of the human-like cerebral cortex culture layer 8. After the printing path was planned, the human-like cerebral cortex was directly printed onto the organ-on-a-chip.

[0062] Specifically, the printing needles used are 25G, the layer height is set to 0.2mm, the printing speed is 100mm / min, the moving speed is 900mm / min, the return speed is 2000mm / min, the rim width is 1mm, the rim speed is 100mm / min, and the printing pressure is 30kpa.

[0063] After printing, use a scraper to remove excess support ink, and then install the microporous array layer 6, liquid pool layer 4, and mixed flow channel layer 1 in sequence. Inoculate the culture medium containing 10% (w / v) fetal bovine serum and 1% (w / v) penicillin / streptomycin into the culture medium inlet 20, and place the chip in an incubator at 37°C and 5% CO2 for incubation.

[0064] Furthermore, the aforementioned cell-carrying bio-ink is a composite solution composed of alginate, gelatin, and hyaluronic acid as monomers, and the corresponding preparation method is as follows:

[0065] Step 1: Dissolve sodium alginate in 1×PBS, stir at 37℃~40℃ for 2 hours, and degas using an ultrasonic disperser to obtain an alginate solution;

[0066] Step 2: Dissolve gelatin and hyaluronic acid in 1×PBS, stir at 37°C for 1 hour, and degas using an ultrasonic disperser to obtain a gelatin composite solution.

[0067] Step 3: Sterilize the alginate precursor solution by irradiating it with 260nm ultraviolet light for 24 hours. Filter the sterilized gelatin composite solution through a 0.22μm filter at 40℃.

[0068] Step 4: At 37℃, the alginate solution and gelatin composite solution are mixed at a 1:1 ratio to obtain the bio-ink, using 1×10 6 ml -1 The density disperses the cells in the ink.

[0069] In this embodiment, the preparation of cell-loaded bio-ink is as follows:

[0070] Preparation of cell-loaded hydrogel precursor solution:

[0071] Weigh 0.5 g of sodium alginate powder and dissolve it in 10 ml of 1×PBS buffer. Stir the solution at 920 rpm for 2 hours using a magnetic stirrer, while simultaneously heating the solution to 40°C (and maintaining this temperature) until the alginate is completely dissolved, yielding a 5% (w / v) alginate solution. Next, degas the sodium alginate solution using an ultrasonic cleaner for 2 minutes, then transfer it to a clear glass bottle and sterilize it by irradiation with 260 nm UV light for 24 hours. After sterilization, store at 4°C.

[0072] Weigh 1.2g of gelatin powder and 4mg of hyaluronic acid, dissolve them in 10ml of 1×PBS, and stir with a magnetic stirrer at 700r / min for 1h. While stirring, heat the solution to 37℃ (and maintain this temperature) until the solvent is completely dissolved to obtain a gelatin complex solution. After stirring, degas using an ultrasonic cleaner for 2min, and then filter and sterilize using a 0.22um filter.

[0073] The prepared alginate solution and gelatin composite solution were heated to 40°C and mixed in a 1:1 ratio. The mixture was then thoroughly mixed using a vortex mixer, and the ink was kept at 40°C until ready for use.

[0074] NE-4C mouse neural stem cells were selected and cultured in a medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. The culture was maintained at 37°C in a humidified incubator with 5% CO2, and the medium was changed every other day. Before preparing the bio-ink, the cells were digested, and the cell density was counted to be 3 × 10⁻⁶. 6 ml -1 According to 1×10 in bio-ink 6 ml -1 To determine the cell density, aspirate 2 ml of cell suspension and centrifuge. Then, aspirate 6 ml of bio-ink precursor solution, disperse the cells evenly, and prepare the cell-carrying bio-ink.

[0075] Furthermore, the preparation method of the gelatin support bath described above in this embodiment is as follows:

[0076] Weigh out 2.5g of gelatin granules and 0.5g of CaCl2 granules from 50ml of ultrapure water. Stir at 700 rpm for 1 hour using a magnetic stirrer, while heating the solution to 37°C. After stirring, degas using an ultrasonic cleaner for 2 minutes to obtain a 5% (w / v) gelatin solution. Transfer the gelatin solution to a 50ml centrifuge tube and incubate at 4°C for 24 hours until the gelatin cures. Weigh out 3g of CaCl2 granules and dissolve them in 300ml of ultrapure water to prepare a 1% (w / v) CaCl2 solution. Next, cut the cured gelatin colloid into 10mm pieces. 3The gelatin particles were placed in a crusher, and three times their volume of 1% (w / v) CaCl2 solution was added. The mixture was crushed at 10,000 r / min for 60 s to obtain a gelatin particle solution. The gelatin particle solution was centrifuged at 2000 g to remove the supernatant. Three times their volume of 1% (w / v) CaCl2 solution was added again, and the gelatin particles were washed by centrifugation to obtain a gelatin support bath.

[0077] Example 2

[0078] This embodiment is based on Example 1, and a cytotoxicity experiment is conducted. The specific process is as follows:

[0079] The cell-loaded bio-ink was transferred into the printer syringe, the syringe temperature was set to 26℃, and the ink was incubated for 10 minutes before printing. The printing platform temperature was set to 5℃, the needle size was 22G, the layer height was 0.3mm, the printing speed was 100mm / min, the travel speed was 900mm / min, the printing pressure was 16kPa, and a three-layer mesh structure was printed with a size of 12.0mm×12.0mm, a layer height of 0.3mm, and a spacing of 1mm×1mm.

[0080] After printing, the scaffold was washed twice with 1×PBS and then transferred to a culture medium containing 10% (w / v) fetal bovine serum and 1% (w / v) penicillin / streptomycin. The culture was placed in an incubator at 37°C and 5% CO2.

[0081] Cell viability was analyzed using a live / dead staining kit at days 1, 3, 5, and 7 of culture. Observation was performed using an inverted fluorescence microscope (Figure 4), and ImageJ was used to count live and dead cells. The calculated cell viability after printing was over 96%, indicating that the printing process had almost no impact on the cells. With prolonged culture time, the cells maintained a high viability rate, and significant cell proliferation was observed.

[0082] Cell proliferation was assessed using a CCK8 assay kit on days 1, 3, 5, and 7 of culture. Figure 5 As shown, tests on the first and third days after printing did not show significant changes in absorbance, and the cell count remained largely unchanged. This was mainly due to changes in the extracellular environment during the printing process. Tests on the fifth and seventh days showed a substantial increase in absorbance, indicating significant cell proliferation. This demonstrates that the printing process does not cause irreversible damage to the cell state, and that the biomaterials used have excellent cell compatibility, supporting cell proliferation and migration.

[0083] Example 3

[0084] Human-like cerebral cortex organochips were sterilized by high-temperature autoclaving and dried at 120°C for 20 minutes. Human-like cerebral cortex was then printed onto the human-like cortex chips, resulting in three chips: A, B, and C. One input port of human-like cerebral cortex chip A was connected to a micro-peristaltic pump, which pumped in complete culture medium; the other input port was plugged with PDMS. One input port of human-like cerebral cortex chip B was connected to another micro-peristaltic pump, which pumped in complete culture medium supplemented with 10 ng / ml nerve growth factor (NGF); the other input port was plugged with PDMS. One input port of human-like cerebral cortex chip C was connected to another micro-peristaltic pump, which pumped in complete culture medium supplemented with 10 ng / ml NGF; the other input port was vented with gas, the gas pressure waveform being a square wave at a frequency of 60 Hz.

[0085] On day 14 of culture, the human-like cerebral cortex was removed from the organ-on-a-chip. After slicing the human-like cerebral cortex, immunofluorescence staining was performed, and the synaptic length of neural stem cells was measured using ImageJ to investigate the effects of nerve growth factor and stress on synaptic network formation.

Claims

1. A human-like cerebral cortex organ-on-a-chip, characterized in that, The chip consists of, from top to bottom, a mixed flow channel layer, a liquid pool layer, a microporous array layer, a human-like cerebral cortex culture layer, and a culture medium recovery layer, with adjacent layers sealed by a sealing ring; The mixed flow channel layer includes a culture medium inlet, a mixed flow channel, a first blind hole, and a culture medium recovery port; The liquid pool layer includes a culture medium storage tank, a first through hole, and a second through hole; wherein the culture medium inlet is used to input culture medium, the mixing channel mixes different components of culture medium evenly, and the mixed culture medium is input into the culture medium storage tank of the liquid pool layer through the blind hole and the first through hole; The microporous array layer is provided with a third through hole, and a PET porous membrane is covered below the microporous array layer; The human-like cerebral cortex culture layer comprises a culture chamber, a fourth through-hole, and a micropore array; the culture chamber is a human-like cerebral cortex culture chamber; the culture medium flows from the culture chamber through the micropore array layer into the culture medium recovery layer, allowing the culture medium to pass through slowly; The culture medium recovery layer is provided with a culture medium recovery pool and a second blind hole. The bottom of the culture medium recovery pool has a slope. The culture medium passes through the second blind hole, the fourth through hole, the third through hole, and the second through hole in sequence to complete the recycling process through the culture medium recovery port.

2. The method for in-situ bioprinting of human-like cerebral cortex in organ-on-a-chip as described in claim 1, characterized in that, After connecting the culture medium recovery layer and the human-like brain cortex culture layer with a stud, the layers are fixed on the printing platform. A gelatin support bath is injected into the culture chamber. Cell-loaded bio-ink is stored in the syringe of the bio-3D printer. The human-like brain cortex is directly printed in situ in the gelatin support bath of the culture chamber using the bio-3D printer. After printing, the chip is encapsulated in situ, and then perfused with culture medium for culturing.

3. The 3D printing method for human-like cerebral cortex as described in claim 2, characterized in that, After printing, remove excess gelatin support bath, and install the microporous array layer, liquid pool layer, and mixed flow channel layer in sequence; connect the culture medium to the culture medium inlet; and place the chip in an incubator for incubation.

4. The 3D printing method for human-like cerebral cortex as described in claim 3, characterized in that, The human-like cerebral cortex bio-ink is composed of alginate, gelatin, and hyaluronic acid.

5. The 3D printing method for human-like cerebral cortex as described in claim 4, characterized in that, The preparation method of the human-like cerebral cortex bio-ink includes the following steps: alginate, gelatin and hyaluronic acid are dissolved in buffer solution respectively, and then cells are evenly dispersed in the ink.

6. The 3D printing method for human-like cerebral cortex as described in claim 5, characterized in that, The specific preparation method of the human-like cerebral cortex bio-ink is as follows: Step 1: Dissolve sodium alginate in 1×PBS, stir at a set temperature for a set time, and degas using an ultrasonic disperser to obtain an alginate solution; Step 2: Dissolve gelatin and hyaluronic acid in 1×PBS, stir at a set temperature for a set time, and degas using an ultrasonic disperser to obtain a gelatin composite solution. Step 3: Sterilize the alginate precursor solution by irradiating it with ultraviolet light for a set time, and then filter the sterilized gelatin composite solution using a filter at a set temperature. Step 4: Mix the alginate solution and gelatin composite solution at a 1:1 ratio at a set temperature to obtain bio-ink, and disperse the cells in the bio-ink.

7. A human-like cerebral cortex obtained by the 3D printing method for human-like cerebral cortex according to any one of claims 2-6, characterized in that, It consists of three interconnected layers: the top layer is a tissue plate with cells arranged longitudinally, the middle layer is a sparse bundle of nerve fibers, and the bottom layer is a nerve tissue plate arranged transversely.

8. The application of the human-like cerebral cortex as described in claim 7 in neuropharmaceutical screening.

9. The application of the bio-ink in the 3D printing method for human-like cerebral cortex as described in claim 5 in the preparation of materials for repairing nerve damage or in the preparation of human-like cerebral cortex.

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