Molds, models, methods, and applications of high-throughput biomimetic in vitro tumor drug screening models

Abstract

This invention discloses a mold for preparing a high-throughput biomimetic in vitro tumor drug screening model, the high-throughput biomimetic in vitro tumor drug screening model, a preparation method, and applications. The mold for preparing the high-throughput biomimetic in vitro tumor drug screening model includes: a basket assembly, a first base, a second base, and a sacrificial material assembly. The basket assembly can be used in conjunction with the first base, the second base, and the sacrificial material assembly to prepare the high-throughput biomimetic in vitro tumor drug screening model. The mold of this invention can rapidly and in batches prepare high-throughput biomimetic in vitro tumor drug screening models.

Description

Technical Field

[0001] This invention relates to the field of biomanufacturing technology, and in particular to a mold for preparing a high-throughput biomimetic in vitro tumor drug screening model, the high-throughput biomimetic in vitro tumor drug screening model, its preparation method, and its application. Background Technology

[0002] Esophageal cancer, also known as esophageal tract cancer, is a common intracavitary malignant tumor in China, typically with a poor prognosis. Therefore, accelerating research into the mechanisms of esophageal cancer development and progression, and developing specific anti-esophageal cancer drugs, is of great significance. Anti-tumor drug screening models are commonly used tools for screening anti-tumor drugs.

[0003] Regarding anti-tumor drug screening models, the current technology still has the following problems: (1) There is no high-throughput 3D bionic sample preparation technology; (2) There is no rapid preparation and personalized tumor drug screening model; (3) It is difficult to prepare bionic tumor chips for micro-samples; (4) The bionic 3D printed chips on the market cannot be matched with traditional equipment and require sample transfer, and cannot be directly detected by traditional instruments such as enzyme readers; (5) The bionic 3D printed chips on the market cannot be batch assembled or separated into tumor drug screening components; (6) The 3D bionic lumen preparation process is complex and has a long cycle.

[0004] Therefore, it is of great significance to develop a mold that can be used for high-throughput preparation of 3D biomimetic samples and tumor drug screening models, so that high-throughput biomimetic in vitro tumor drug screening models can be rapidly prepared, mass-produced, and easily detected. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art, which lacks molds for rapid and high-throughput preparation of high-throughput biomimetic in vitro tumor drug screening models and lacks high-throughput biomimetic in vitro tumor drug screening models.

[0006] To address the aforementioned technical problems, this invention provides a mold for preparing a high-throughput biomimetic in vitro tumor drug screening model, comprising:

[0007] A suspended platform assembly includes multiple suspended platforms, each platform including a frame and a top frame. The frame includes a receiving cavity and a connecting rod. The receiving cavity includes a first side and a second side disposed opposite to each other, with through holes disposed opposite to each other on the first side and the second side. One end of the connecting rod is connected to the upper edge of the opening of the receiving cavity, and the other end of the connecting rod is connected to the lower edge of the opening of the top frame. The opening of the top frame communicates with the opening of the receiving cavity.

[0008] The first base includes a plurality of first grooves, each groove including a first sidewall and a second sidewall disposed opposite to each other, the first sidewall and the second sidewall being provided with through holes opposite to each other, the first grooves matching the suspended basket, and when the suspended basket assembly is close to the first base, the suspended frame can penetrate into the first groove, the top frame can cover the first base, and the perforation and the through hole can communicate with each other.

[0009] The second base includes a plurality of second grooves that match the basket, and when the basket assembly is close to the second base, the hanging frame can penetrate into the second grooves and the top frame can cover the second base.

[0010] A sacrificial material assembly, which is adapted to the perforation and through-hole.

[0011] Optionally, the bottom surface of the receiving cavity is any one of a polycarbonate film, a polyester film, or a polytetrafluoroethylene film.

[0012] Optionally, the first side and the second side are also provided with windows opposite to each other, and the number of windows on the first side and the second side is one or more.

[0013] Optionally, the number of the first groove and the second groove is equal to the number of the basket, and the sacrificial material assembly is a pin array.

[0014] Optionally, the basket is arranged axially and has multiple rows evenly distributed, and the row of needles can pass through the perforations on one side of the basket in sequence and through the perforations on the opposite side to reach the opposite side of the basket.

[0015] Optionally, the number of hanging baskets is 96 or 384.

[0016] Optionally, when the number of hanging baskets is 96, the hanging baskets are arranged in 8 rows and 12 columns, and the arrangement of the first groove and the second groove is the same as the arrangement of the hanging baskets.

[0017] Optionally, when the number of hanging baskets is 384, the hanging baskets are arranged in 16 rows and 24 columns, and the arrangement of the first groove and the second groove is the same as the arrangement of the hanging baskets.

[0018] In addition, the present invention also provides a method for preparing a high-throughput biomimetic in vitro tumor drug screening model, wherein the preparation method utilizes the mold described above for preparing the high-throughput biomimetic in vitro tumor drug screening model to prepare the high-throughput biomimetic in vitro tumor drug screening model.

[0019] Optionally, the preparation method includes:

[0020] Assemble the basket assembly with the first base, insert the sacrificial material assembly into the perforation and through hole in sequence, inject the photosensitive material and photoinitiator into the receiving cavity of the basket to solidify and shape it, add culture medium to the basket to immerse the photosensitive material, place it in a 37°C cell culture incubator to fill the photosensitive material with culture medium, remove the sacrificial material assembly to form a microchannel;

[0021] At 4°C to 35°C, a thermosensitive hydrogel cell suspension containing tumor cells is injected into the microchannel using a syringe. The microchannel is then placed in a cell culture incubator for incubation. When the thermosensitive hydrogel changes from a liquid state to a gel state, the basket assembly is removed from the first base and placed into the second base. The basket assembly is then incubated in a cell culture incubator for 8-12 hours to confirm that the tumor cells have adhered to the photosensitive material. The microchannel is then placed at 4°C to allow the thermosensitive hydrogel to change from a gel state to a liquid state and flow out. The basket assembly is then removed to obtain the first biochip.

[0022] Take a new second base, add a photosensitive material containing cells and a photoinitiator into the second groove, and let it solidify to form a second biochip;

[0023] Fresh culture medium is added to the second groove of the second biochip and cultured in a cell culture incubator. The first biochip is then placed into the second groove of the second biochip to obtain the high-throughput biomimetic in vitro tumor drug screening model.

[0024] Optionally, the preparation method includes:

[0025] Assemble the basket assembly with the first base, insert the sacrificial material assembly into the perforation and through hole in sequence, inject methacrylamide gelatin and photoinitiator into the basket cavity, irradiate the methacrylamide gelatin with blue-violet light at a wavelength of 405nm to solidify it, add culture medium to the basket to immerse the methacrylamide gelatin, place it in a cell culture incubator at 37°C for 2 hours to fill the culture medium with methacrylamide gelatin, remove the sacrificial material assembly to form a microchannel;

[0026] At 4°C to 35°C, a thermosensitive hydrogel cell suspension containing tumor cells is injected into the microchannel using a syringe. Then, it is placed in a 37°C cell culture incubator and cultured for 5 minutes. When the thermosensitive hydrogel changes from a liquid state to a gel state, the basket assembly is removed from the first base and placed in the second base. It is then cultured in a cell culture incubator for 8-12 hours to confirm that the tumor cells have adhered to the photosensitive material. After that, it is placed at 4°C for 5 minutes to allow the thermosensitive hydrogel to change from a gel state to a liquid state and flow out. The basket assembly is then removed to obtain the first biochip.

[0027] Take another new second base, add methacrylamide gelatin containing cells and a photoinitiator into the second groove, irradiate the methacrylamide gelatin with 405nm blue-violet light to solidify it and obtain the second biochip;

[0028] Fresh culture medium is added to the second groove of the second biochip and cultured in a cell culture incubator at 37°C for 24 hours. The first biochip is then placed into the second groove of the second biochip to obtain the high-throughput biomimetic in vitro tumor drug screening model.

[0029] Furthermore, the present invention also provides a high-throughput biomimetic in vitro tumor drug screening model, which is prepared using the preparation method described above.

[0030] Furthermore, the present invention also provides an application of the above-described high-throughput biomimetic in vitro tumor drug screening model in high-throughput screening of anti-intraluminal tumor drugs.

[0031] Furthermore, the present invention also provides an application of the above-mentioned high-throughput, highly biomimetic in vitro tumor drug screening model in the screening of nano-anti-tumor drugs.

[0032] The mold, high-throughput biomimetic in vitro tumor drug screening model, preparation method, and application provided in this invention have the following advantages compared with the prior art:

[0033] The mold for preparing a high-throughput biomimetic in vitro tumor drug screening model provided in this embodiment of the invention includes a basket assembly, a first base, a second base, and a sacrificial material assembly. The basket assembly can be used in conjunction with the first base, the second base, and the sacrificial material assembly to prepare a high-throughput biomimetic in vitro tumor drug screening model. The mold of this invention can be used to quickly and in batches prepare high-throughput biomimetic in vitro tumor drug screening models.

[0034] Furthermore, the bottom surface of the receiving cavity is any one of a polycarbonate membrane, a polyester membrane, or a polytetrafluoroethylene membrane, which allows the culture medium to pass through, facilitating the exchange of components between the substances inside and outside the receiving cavity.

[0035] Furthermore, the first side and the second side are also provided with windows opposite each other, with one or more windows on each side, to facilitate the flow of culture medium and the exchange of material components on both sides of the window.

[0036] Furthermore, the number of baskets is 96 or 384, the number of the first groove and the second groove is equal to the number of baskets, and the sacrificial material component is a row of needles, which can mass-produce biomimetic microchannels and facilitate the simultaneous setting of multiple sets of experiments for the prepared high-throughput biomimetic in vitro tumor drug screening model, thereby achieving high-throughput screening of tumor drugs and improving experimental efficiency.

[0037] In addition, the method for preparing a high-throughput biomimetic in vitro tumor drug screening model provided in this embodiment of the invention can prepare biomimetic microchannels in batches and quickly, and the high-throughput biomimetic in vitro tumor drug screening model prepared requires less sample size during drug screening, which greatly shortens the drug screening cycle.

[0038] Furthermore, the method for preparing the biomimetic in vitro tumor drug screening model provided in this embodiment of the invention uses a cell-adhesive, reversible, temperature-sensitive hydrogel. Utilizing its temperature-sensitive properties (liquid below 35°C and gel above 35°C), the hydrogel is mixed with tumor cells at temperatures ranging from 4°C to 35°C and injected into pre-fabricated microchannel lumens. At 37°C, tumor cells migrate from the temperature-sensitive hydrogel to methacryloyl gelatin to form tumor lumen structures, adhere, and grow, forming physiological lumens. The components are then placed at 4°C. After 5 minutes, the temperature-sensitive hydrogel changes from a gel state back to a liquid state and flows out of the microchannel. Thus, a biomimetic in vitro tumor drug screening model can be quickly and conveniently formed.

[0039] In addition, the high-throughput biomimetic in vitro tumor drug screening model provided in this embodiment of the invention allows for the combination or separation of its components, facilitating high-throughput combined culture of different cells. The combination and separation methods are simple, and no sample transfer is required, allowing for direct detection using traditional instruments, which is convenient and fast.

[0040] Furthermore, the high-throughput biomimetic in vitro tumor drug screening model described in this embodiment of the invention can also be applied to high-throughput screening of anti-intraluminal tumor drugs and nano-anti-tumor drugs. The high-throughput biomimetic in vitro tumor drug screening model provided by this embodiment of the invention has advantages in anti-tumor drug screening, including fast modeling speed, low testing cost (high-throughput detection results can be obtained using traditional equipment such as ELISA readers), and small sample requirement (the model with 96 baskets requires as little as 1×10⁻⁶ tumor cell samples). 5 It has advantages such as high accuracy and can achieve rapid, high-throughput drug screening (from the preparation of a high-throughput biomimetic in vitro tumor drug screening model to the end of drug screening, it only takes three days). Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 This is an exploded structural diagram of the mold used to prepare a high-throughput biomimetic in vitro tumor drug screening model in one embodiment of the present invention.

[0043] Figure 2 This is a schematic diagram of the assembly structure of the suspended basket assembly, the first base, and the sacrificial material assembly in one embodiment of the present invention.

[0044] Figure 3 This is a schematic diagram of the assembly structure of the suspended basket assembly and the second base in one embodiment of the present invention.

[0045] Figure 4 This is a schematic diagram of the structure of the suspended basket in one embodiment of the present invention.

[0046] Figure 5 This is a schematic diagram of the structure of the first groove in one embodiment of the present invention.

[0047] Figure 6 This is a schematic diagram of the structure of the second groove in one embodiment of the present invention.

[0048] Figure 7 This is a flowchart illustrating the process of preparing a high-throughput biomimetic in vitro tumor drug screening model, using a single basket in a mold as an example, in one embodiment of the present invention.

[0049] Figure 8 This is a schematic diagram of drug addition in Example 1 of the present invention.

[0050] Figure 9 The results of the fluorescence diffusion experiment of the nanomedicine in the model in Example 2 of this invention are shown.

[0051] Figure 10 This is a schematic diagram of tumor cell addition in Example 2 of the present invention.

[0052] Among them, 10- is the mold for preparing a high-throughput biomimetic in vitro tumor drug screening model;

[0053] 100-Suspended basket assembly; 101-Suspended basket; 102-Suspended frame; 103-Top frame; 104-Receiving cavity; 105-Connecting rod; 106-First side; 107-Second side; 108a, 108b-Perforations; 109a, 109b-Window;

[0054] 200 - First base; 201 - First groove; 202 - First sidewall; 203 - Second sidewall; 204a, 204b - Through holes;

[0055] 300 - Second base; 301 - Second groove;

[0056] 400 - Sacrificial Material Component. Detailed Implementation

[0057] To enable those skilled in the art to better understand the present invention, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] As can be seen from the background technology, the existing technologies for anti-tumor drug screening models still have the following problems: (1) There is no high-throughput 3D bionic sample preparation technology; (2) There is no rapid preparation and personalized tumor drug screening model; (3) It is difficult to prepare bionic tumor chips for micro-samples; (4) The bionic 3D printed chips on the market cannot be matched with traditional equipment and require sample transfer, and cannot be directly detected by traditional instruments such as enzyme readers; (5) The bionic 3D printed chips on the market cannot be batch assembled or separated into tumor drug screening components; (6) The 3D bionic lumen preparation process is complex and has a long cycle.

[0059] To address the aforementioned issues, the present invention provides a mold for preparing high-throughput biomimetic in vitro tumor drug screening models, comprising a basket assembly, a first base, a second base, and a sacrificial material assembly. The basket assembly can be used in conjunction with the first base, the second base, and the sacrificial material assembly to prepare high-throughput biomimetic in vitro tumor drug screening models. The mold of the present invention enables rapid and mass production of high-throughput biomimetic in vitro tumor drug screening models.

[0060] In addition, the method for preparing a high-throughput biomimetic in vitro tumor drug screening model provided in this embodiment of the invention can prepare biomimetic microchannels in batches and quickly, and the high-throughput biomimetic in vitro tumor drug screening model prepared requires less sample size during drug screening, which greatly shortens the drug screening cycle.

[0061] In addition, the biomimetic in vitro tumor drug screening model provided in this embodiment of the invention allows for the combination or separation of its components, facilitating high-throughput combined culture of different cells. The combination and separation methods are simple, and no sample transfer is required, allowing for direct detection using traditional instruments, which is convenient and fast.

[0062] Furthermore, the high-throughput biomimetic in vitro tumor drug screening model described in this embodiment of the invention can also be applied to high-throughput screening of anti-intraluminal tumor drugs and nano-anti-tumor drugs. The high-throughput biomimetic in vitro tumor drug screening model provided by this embodiment of the invention has advantages in anti-tumor drug screening, including fast modeling speed, low testing cost (high-throughput detection results can be obtained using traditional equipment such as ELISA readers), and small sample requirement (the model with 96 baskets requires as little as 1×10⁻⁶ tumor cell samples). 5 It has advantages such as high accuracy and can achieve rapid, high-throughput drug screening (from the preparation of a high-throughput biomimetic in vitro tumor drug screening model to the end of drug screening, it only takes three days).

[0063] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0064] Unless otherwise specified, this invention generally does not impose special restrictions on the source of reagents and cell lines used; for example, commercially available reagents can be used. For example, esophageal squamous cell carcinoma TE-1 was purchased from the Chinese Academy of Sciences Cell Bank; hepatocytes were purchased from the Chinese Academy of Sciences Cell Bank; vascular endothelial cells (HUVECs) were purchased from the Chinese Academy of Sciences Cell Bank; HepG2 was purchased from the Chinese Academy of Sciences Cell Bank; MCF-7 was purchased from the Chinese Academy of Sciences Cell Bank; 3AO was purchased from the Chinese Academy of Sciences Cell Bank; SKMES1 was purchased from the Chinese Academy of Sciences Cell Bank; MDA-MB-231 was purchased from the Chinese Academy of Sciences Cell Bank; and primary human lymphatic endothelial cells (LECs) were purchased from Wuhan Yunclone Technology Co., Ltd.

[0065] Example 1

[0066] As described below, this embodiment provides a mold for preparing a high-throughput biomimetic in vitro tumor drug screening model.

[0067] Please refer to Figures 1 to 6 , Figure 1 This is an exploded structural diagram of the mold used to prepare a high-throughput biomimetic in vitro tumor drug screening model in one embodiment of the present invention. Figure 2 This is a schematic diagram of the assembly structure of the suspended platform assembly, the first base, and the sacrificial material assembly in one embodiment of the present invention. Figure 3 This is a schematic diagram of the assembly structure of the suspended platform assembly and the second base in one embodiment of the present invention. Figure 4 This is a schematic diagram of the structure of the suspended basket in one embodiment of the present invention. Figure 5 This is a schematic diagram of the structure of the first groove in one embodiment of the present invention. Figure 6 This is a schematic diagram of the structure of the second groove in one embodiment of the present invention.

[0068] This embodiment provides a mold 10 for preparing a high-throughput biomimetic in vitro tumor drug screening model, comprising: a basket assembly 100, the basket assembly 100 including multiple baskets 101, each basket 101 including a frame 102 and a top frame 103, each frame 102 including a receiving cavity 104 and a connecting rod 105, each receiving cavity 104 including a first side 106 and a second side 107 disposed opposite to each other, the first side 106 and the second side 107 having perforations 108a and 108b disposed opposite to each other, one end of the connecting rod 105 connected to the upper edge of the opening of the receiving cavity 104, the other end of the connecting rod 105 connected to the lower edge of the opening of the top frame 103, the opening of the top frame 103 communicating with the opening of the receiving cavity 104; and a first base 200, the first base 200 including multiple first grooves 201, each first groove 201 including a first sidewall 202 and a second sidewall 203 disposed opposite to each other. The first base 203 is provided with through holes 204a and 204b. The first groove 201 matches the hanging basket 101. When the hanging basket assembly 100 is close to the first base 200, the hanging frame 102 can penetrate into the first groove 201, and the top frame 103 can cover the first base 200. The through holes 108a and 108b can communicate with the through holes 204a and 204b. The second base 300 includes a plurality of second grooves 301. The second grooves 301 match the hanging basket 101. When the hanging basket assembly 100 is close to the second base 300, the hanging frame 102 can penetrate into the second groove 301, and the top frame 103 can cover the second base 300. The sacrificial material assembly 400 is adapted to the through holes 108a and 108b and the through holes 204a and 204b.

[0069] It should be noted that the basket assembly 100 can be used in conjunction with the first base 200, the second base 300, and the sacrificial material assembly 400 to prepare the first biochip. The second base 300 can be used to prepare the second biochip. After the first biochip and the second biochip are assembled, a high-throughput biomimetic in vitro tumor drug screening model can be obtained. The mold described in this invention can be used to quickly and in batches prepare high-throughput biomimetic in vitro tumor drug screening models.

[0070] Optionally, the bottom surface of the receiving cavity 104 can be any one of a polycarbonate (PC) membrane, a polyester (PET) membrane, and a polytetrafluoroethylene (PTFE) membrane. This membrane allows the culture medium to permeate, facilitating component exchange between substances inside and outside the receiving cavity. It should be noted that, in addition to the aforementioned membrane materials, any material with a pore size of 0.1-16 μm and cell compatibility can also be used for the bottom surface of the receiving cavity 104.

[0071] Optionally, the first side 106 and the second side 107 are also provided with windows 109a and 109b opposite to each other. The number of windows 109a on the first side 106 and windows 109b on the second side 107 is one or more. For example, in one embodiment, the number of windows 109a and 109b on the first side 106 and the second side 107 is four, which facilitates the flow of culture medium and the exchange of material components on both sides of the window.

[0072] Optionally, the number of the first groove 201 and the second groove 202 is equal to the number of the basket 101, and the sacrificial material assembly 400 is a pin array.

[0073] Optionally, the suspended basket 101 is arranged along the axial direction and has multiple rows evenly distributed. The row of needles can pass through the perforations on one side of the suspended basket 101 in sequence and through the perforations on the opposite side to reach the opposite side of the suspended basket.

[0074] Optionally, the number of hanging baskets 101 is 96 or 384. It can be understood that when the number of hanging baskets 101 is 96, the number of the first groove 201 and the second groove 301 is also 96; when the number of hanging baskets 101 is 384, the number of the first groove 201 and the second groove 301 is also 384.

[0075] In one embodiment, there are 96 hanging baskets 101, 96 first grooves 201, and 96 second grooves 301. The hanging baskets 101 are arranged in 8 rows and 12 columns. The arrangement of the first grooves 201 and the second grooves 301 is the same as the arrangement of the hanging baskets 101, which facilitates the adaptation of the hanging basket assembly 100 to the first base 200 and the second base 300. In another embodiment, there are 384 hanging baskets, 384 first grooves 201, and 384 second grooves 301. The hanging baskets 101 are arranged in 16 rows and 24 columns. The arrangement of the first grooves 201 and the second grooves 301 is the same as the arrangement of the hanging baskets 101, which facilitates the adaptation of the hanging basket assembly 100 to the first base 200 and the second base 300.

[0076] It should be noted that the number of baskets 101 is 96 or 384, the number of the first groove 201 and the second groove 301 is equal to the number of baskets 101, and the sacrificial material component is a row of needles, which can mass-produce biomimetic microchannels and facilitate the simultaneous setting of multiple sets of experiments on the prepared high-throughput biomimetic in vitro tumor drug screening model, thereby achieving high-throughput screening of tumor drugs and improving experimental efficiency.

[0077] It should also be noted that the aforementioned basket assembly with 96 baskets is compatible with traditional 96-well cell culture plates, facilitating direct detection using conventional instruments. Similarly, the first base with 96 first grooves and the second base with 96 second grooves are also compatible with traditional 96-well cell culture plates, facilitating direct detection using conventional instruments. Likewise, the aforementioned basket assembly with 384 baskets is compatible with traditional 384-well cell culture plates, facilitating direct detection using conventional instruments. The first base with 384 first grooves and the second base with 384 second grooves are also compatible with traditional 384-well cell culture plates, facilitating direct detection using conventional instruments.

[0078] Example 2

[0079] As described below, this embodiment provides a method for preparing a high-throughput biomimetic in vitro tumor drug screening model. The method uses the mold described in Embodiment 1 for preparing the high-throughput biomimetic in vitro tumor drug screening model to prepare the high-throughput biomimetic in vitro tumor drug screening model.

[0080] Please refer to Figure 7 , Figure 7 This is a flowchart illustrating the process of preparing a high-throughput biomimetic in vitro tumor drug screening model, using a single basket in a mold as an example, in one embodiment of the present invention.

[0081] The method for preparing the high-throughput biomimetic in vitro tumor drug screening model provided in this embodiment includes:

[0082] S100, assemble the basket assembly with the first base, insert the sacrificial material assembly into the perforation and through hole in sequence, inject the photosensitive material and photoinitiator into the receiving cavity of the basket to solidify and shape it, add culture medium to the basket to immerse the photosensitive material, place it in a 37°C cell culture incubator to fill the photosensitive material with culture medium, remove the sacrificial material assembly to form a microchannel;

[0083] S200, under conditions of 4℃ to 35℃, a thermosensitive hydrogel cell suspension containing tumor cells is injected into the microchannel using a syringe, and then placed in a cell culture incubator for culture. When the thermosensitive hydrogel changes from a liquid state to a gel state, the basket assembly is removed from the first base, and the removed basket assembly is placed in the second base and cultured in a cell culture incubator for 8h-12h. After confirming that the tumor cells have adhered to the photosensitive material, it is then placed at 4℃ to allow the thermosensitive hydrogel to change from a gel state to a liquid state and flow out. The basket assembly is then removed to obtain the first biochip.

[0084] S300, take another new second base, add a photosensitive material containing cells and a photoinitiator into the second groove, and solidify it to obtain a second biochip;

[0085] S400, continue to add fresh culture medium to the second groove of the second biochip and culture it in a cell culture incubator. Place the first biochip into the second groove of the second biochip to obtain the high-throughput biomimetic in vitro tumor drug screening model.

[0086] The method for preparing a high-throughput biomimetic in vitro tumor drug screening model provided in this embodiment can prepare biomimetic microchannels in batches and quickly. Moreover, the high-throughput biomimetic in vitro tumor drug screening model prepared requires less sample size during drug screening, which greatly shortens the drug screening cycle.

[0087] In some embodiments, the preparation method includes:

[0088] S1000, assemble the basket assembly with the first base, insert the sacrificial material assembly into the perforation and through hole in sequence, inject methacrylamide gelatin and photoinitiator into the basket cavity, irradiate the methacrylamide gelatin with blue-violet light with a wavelength of 405nm to solidify it, add culture medium to the basket to immerse the methacrylamide gelatin, place it in a cell culture incubator at 37°C for 2 hours to fill the culture medium with methacrylamide gelatin, remove the sacrificial material assembly to form a microchannel;

[0089] S2000, under conditions of 4℃ to 35℃, a thermosensitive hydrogel cell suspension containing tumor cells is injected into the microchannel using a syringe, and then placed in a 37℃ cell culture incubator for 5 minutes. When the thermosensitive hydrogel changes from liquid to gel state, the basket assembly is removed from the first base, and the removed basket assembly is placed in the second base and cultured in a cell culture incubator for 8h-12h. After confirming that the tumor cells have adhered to the photosensitive material, it is then placed at 4℃ for 5 minutes to allow the thermosensitive hydrogel to change from gel state to liquid state and flow out. The basket assembly is then removed to obtain the first biochip.

[0090] S3000, take another new second base, add methacrylamide gelatin containing cells and photoinitiator into the second groove, irradiate the methacrylamide gelatin with 405nm blue-violet light to solidify it and obtain the second biochip;

[0091] S4000, continue to add fresh culture medium to the second groove of the second biochip, and culture in a cell culture incubator at 37°C for 24 hours. Place the first biochip into the second groove of the second biochip to obtain the high-throughput biomimetic in vitro tumor drug screening model.

[0092] The method for preparing the biomimetic in vitro tumor drug screening model provided in this embodiment uses a cell-adhesive, reversible, temperature-sensitive hydrogel. Utilizing its temperature-sensitive properties (liquid below 35°C and gel above 35°C), the hydrogel is mixed with tumor cells at temperatures between 4°C and 35°C and injected into pre-fabricated microchannel lumens. At 37°C, tumor cells migrate from the temperature-sensitive hydrogel to methacryloyl gelatin to form tumor lumen structures, adhere, and grow, forming physiological lumens. After placing the components at 4°C for 5 minutes, the temperature-sensitive hydrogel changes from a gel state back to a liquid state and flows out of the microchannels. Thus, a biomimetic in vitro tumor drug screening model can be quickly and conveniently formed.

[0093] In addition, this embodiment of the invention also provides a high-throughput biomimetic in vitro tumor drug screening model, which is prepared by the preparation method described in Example 2.

[0094] In addition, embodiments of the present invention also provide the application of the high-throughput biomimetic in vitro tumor drug screening model described above in high-throughput screening of anti-intraluminal tumor drugs.

[0095] It should be noted that the high-throughput biomimetic in vitro tumor drug screening model described in this embodiment of the invention can be applied to the screening of various anti-intraluminal tumor drugs. Optionally, the tumor can be any one of esophageal cancer, intestinal cancer, ureteral cancer, bile duct cancer, gastric cancer, fallopian tube cancer, uterine cancer, vas deferens cancer, or nasopharyngeal carcinoma.

[0096] In addition, embodiments of the present invention also provide the application of the high-throughput biomimetic in vitro tumor drug screening model described above in the screening of nano-anti-tumor drugs.

[0097] The high-throughput biomimetic in vitro tumor drug screening model provided in this invention has the advantages of fast modeling speed, low testing cost (high-throughput detection results can be obtained through traditional equipment, such as ELISA readers), and small sample requirement (the model with 96 baskets requires as little as 1×10⁻⁶ tumor cell samples). 5 It has advantages such as high accuracy and can achieve rapid, high-throughput drug screening (from the preparation of a high-throughput biomimetic in vitro tumor drug screening model to the end of drug screening, it only takes three days).

[0098] The following specific examples will further illustrate the features and advantages of this invention.

[0099] Example 1: High-throughput screening of drugs for intraluminal tumors

[0100] 1. Mold for preparing a high-throughput biomimetic in vitro tumor drug screening model

[0101] The number of baskets in the basket assembly of the mold is 96, arranged in 8 rows and 12 columns; the number of first grooves in the first base is 96, arranged in 8 rows and 12 columns; the number of second grooves in the second base is 96, arranged in 8 rows and 12 columns; the sacrificial material assembly is a pin array, with 8 pins.

[0102] In addition, the basket assembly, first base, and second base in this example are all compatible with traditional 96-well cell culture plates. Furthermore, the basket assembly, first base, and second base are all covered with lids during use to ensure sterility during culture.

[0103] 2. Specific drug screening steps and results

[0104] (1) Insert the basket assembly into the first base, ensuring that the perforation on the basket assembly is connected to the through hole on the first base. Insert the pins into the perforation and through hole in sequence, inject 50 μL of liquid methacrylamide gelatin into the basket, and add 0.1% w / w phenyl-2,4,6-trimethylbenzoyl lithium phosphite. Irradiate it under blue-violet light at a wavelength of 405 nm for 40 s to allow it to solidify.

[0105] (2) After soaking in culture medium (RPMI 1640 + 10% w / w FBS), the needle is pulled out to form a microchannel.

[0106] (3) Place 10 5 One esophageal squamous cell carcinoma cell, TE-1, was thoroughly mixed with 110 μL of thermosensitive hydrogel (PLGA-PEG-PLGA, gelling above 35°C, injectable liquid below 35°C) at 4°C to obtain a tumor cell-hydrogel (liquid) mixture, which was then aspirated into a microsyringe.

[0107] (4) Inject the tumor cell-hydrogel (liquid) mixture in the microsyringe into the microchannel at a rate of 10 μL / channel.

[0108] (5) Incubate in a 37°C cell culture incubator for 5 min until the tumor cell-hydrogel (liquid) mixture forms a gel. Remove the basket assembly and place it in the second base. Add 100 μL of cell culture medium (RPMI 1640 + 10% w / w FBS) and continue culturing in a 37°C incubator for 12 h.

[0109] (6) After observing that the cells adhered to the inner wall of the microchannel, the cell culture medium was replaced and the cell culture medium was replaced. The cell culture medium was then replaced and the cell culture medium was replaced for 24 hours. The basket assembly was then removed to obtain the esophageal cancer biochip.

[0110] (7) Take 1×10 6 One hepatocyte, 1×105 One vascular endothelial cell was mixed thoroughly with 1 ml of photosensitive bio-ink (5% w / w GelMA-0.1% w / w LAP) and added to a new second substrate. 100 μL of the cell-bio-ink mixture was added to each second groove. The second substrate was then irradiated with blue-violet light at a wavelength of 405 nm for 40 seconds. After photocuring, 100 μL of cell culture medium (RPMI 1640 + 10% w / w FBS) was added, and the substrate was incubated at 37°C for 24 hours to obtain vascular and liver biochips.

[0111] (8) Assemble the esophageal cancer biochip by placing it into the second groove of the vascular and liver biochips, add 150 μL of cell culture medium containing chemotherapy drugs (RPMI 1640 + 10% w / w FBS + different concentrations of chemotherapy drugs), and continue culturing in a 37℃ incubator for 24 h. (Please refer to...) Figure 8 , Figure 8 (This is a schematic diagram of drug addition in Example 1 of the present invention)

[0112] (9) Place the tumor biochip into the new second base, add 150 μL of culture medium and 15 μL of CCK-8, mix thoroughly, and continue to culture in a 37℃ incubator for 4h. Read the 450 nm light absorbance value (A) with an ELISA reader and calculate the cell viability.

[0113] (10) Preparation and detection of 2D control samples: 1×10 3 One TE-1 esophageal cancer cell line was seeded into a 96-well plate. After adhesion, 100 μL of cell culture medium (RPMI 1640 + 10% w / w FBS) and different concentrations of chemotherapy drugs were added, and the plate was cultured for 24 h. On the second day, the supernatant was discarded, and 100 μL of cell culture medium and 10 μL of CCK8 were added. The plate was incubated at 37°C for 4 h, and the absorbance at 450 nm was read using a microplate reader to calculate cell viability. Cell viability (%) = [A(drug-treated) - A(blank)] / [A(0-drug-treated) - A(blank)] × 100%. The results are shown in Table 1.

[0114] The results show that the esophageal cancer biochip, through hepatic drug metabolism, exhibits higher sensitivity to the pro-tumor drug cyclophosphamide than 2D screening; higher cytotoxicity to the metabolically enhanced chemotherapy drugs cisplatin and teniposide compared to 2D screening; and lower cytotoxicity to the metabolically degraded drug paclitaxel compared to 2D screening, which is similar to the in vivo dose-toxicity results. Furthermore, parallel drug action analysis, such as cisplatin 1 versus cisplatin 2, demonstrates that this high-throughput drug screening model has excellent reproducibility.

[0115] Table 1 Results of high-throughput screening for intraluminal tumor drugs

[0116]

[0117] Example 2: High-throughput detection of the tumor cell-killing effect of nano-anti-tumor drugs

[0118] Blood vessels are also typical microtubule structures. Existing models cannot visualize the diffusion rate of nano-antitumor drugs from blood vessels to the tumor site, detect the vascular toxicity of nano-drugs, or screen the efficacy against different tumors using high-throughput methods. This example provides a high-throughput nano-antitumor drug screening model that visualizes the diffusion of nano-drugs in a microtubule model and enables high-throughput analysis of the efficacy of nano-antitumor drugs, such as simultaneously analyzing eight types of tumors.

[0119] 1. Mold for preparing a high-throughput biomimetic in vitro tumor drug screening model

[0120] The number of baskets in the basket assembly of the mold is 96, arranged in 8 rows and 12 columns; the number of first grooves in the first base is 96, arranged in 8 rows and 12 columns; the number of second grooves in the second base is 96, arranged in 8 rows and 12 columns; the sacrificial material assembly is a pin array, with 8 pins.

[0121] In addition, the basket assembly, first base, and second base in this example are all compatible with traditional 96-well cell culture plates. Furthermore, the basket assembly, first base, and second base are all covered with lids during use to ensure sterility during culture.

[0122] 2. Visualizing the diffusion of nanomedicines in a microtubule model: specific steps and results.

[0123] (1) Insert the basket assembly into the first base, ensuring that the perforation on the basket assembly is connected to the through hole on the first base. Insert the pins into the perforation and through hole in sequence, inject 50 μL of liquid methacrylamide gelatin into the basket, and add 0.1% w / w phenyl-2,4,6-trimethylbenzoyl lithium phosphite. Irradiate it under blue-violet light at a wavelength of 405 nm for 40 s to allow it to solidify.

[0124] (2) After soaking in PBS, the needle is pulled out to form a microchannel.

[0125] (3) Under light-protected conditions, FITC-gold nanoparticles (10 nm) were mixed with bio-ink (GelMA-LAP) at a concentration of 1 mg / ml to obtain a gold nanoparticle-bio-ink mixture.

[0126] (3) Take 10 μL of the gold nanoparticle-bio-ink mixture, inject it into the microchannel, and immediately perform continuous imaging in a Zeiss live cell workstation. Please refer to the results. Figure 9 , Figure 9 This presents the fluorescence diffusion experiment results of the nanomedicine in the model in Example 2 of this invention. From... Figure 9 The rapid diffusion of nanomedicines within the chip can be dynamically observed.

[0127] 3. High-throughput screening of nano-anti-tumor drugs: specific operation steps and results.

[0128] (1) Insert the basket assembly into the first base, ensuring that the perforation on the basket assembly is connected to the through hole on the first base. Insert the pins into the perforation and through hole in sequence, inject 50 μL of liquid methacrylamide gelatin into the basket, and add 0.1% w / w phenyl-2,4,6-trimethylbenzoyl lithium phosphite. Irradiate it under blue-violet light at a wavelength of 405 nm for 40 s to allow it to solidify.

[0129] (2) Under the premise of soaking in culture medium (DMEM+10% w / w FBS), the needle is pulled out to form a microchannel.

[0130] (3) Place 10 5 One vascular endothelial cell (HUVEC) was thoroughly mixed with 120 μL of thermosensitive hydrogel (PLGA-PEG-PLGA, gelling above 35°C, injectable liquid below 35°C) at 4°C to obtain a vascular endothelial cell-hydrogel (liquid) mixture, which was then aspirated into a microsyringe.

[0131] (4) Inject the vascular endothelial cell-hydrogel (liquid) mixture in the microsyringe into the microchannel at a rate of 10 μL / channel.

[0132] (5) Incubate in a 37°C cell culture incubator for 5 min until the vascular endothelial cell-hydrogel (liquid) mixture forms a gel. Remove the basket assembly and place it in the second base. Add 100 μL of cell culture medium (DMEM + 10% w / w FBS) and continue culturing in a 37°C incubator for 12 h.

[0133] (6) After observing that the cells adhered to the inner wall of the microchannel, the cells were placed in a 4°C refrigerator. After 5 minutes, the temperature-sensitive hydrogel was observed to be in a liquid state. After the temperature-sensitive hydrogel flowed out, it formed a biomimetic blood vessel. The cell culture medium was replaced and cultured for another 24 hours. The basket assembly was then removed to obtain the vascular biochip.

[0134] (7) Take 1×10 4Different tumor cells and 1 mL of photosensitive bio-ink (5% GelMA-0.1% LAP) were mixed thoroughly and added to the second base. From column 2 to column 12, 100 μL of the cell-bio-ink mixture was added to each second groove. The second base was then irradiated with 405 nm blue-violet light for 40 s for photocuring. After photocuring, 100 μL of cell culture medium (DMEM + 10% w / w FBS) was added to each second groove, and the cells were incubated at 37°C for 24 h to obtain the tumor biochip. (Please refer to...) Figure 10 , Figure 10 (This is a schematic diagram of adding tumor cells in Example 2 of the present invention)

[0135] (8) Place the vascular biochip into the new first base, and inject different concentrations of nano-anti-tumor drugs (nano-cyclophosphamide: 5 mM, 10 mM and 20 mM) through the connected microcavities (the microchannels of the vascular biochip are connected to the perforations and through holes of the first base). Then place it at 37 ℃ for 30 min. After the nano-drug diffuses from the vascular cavity into the solidified methacrylamide gelatin matrix, take out the basket assembly and assemble it with the tumor biochip. Add 100 μL of cell culture medium (DMEM+10% w / w FBS) to each basket and incubate in a 37℃ incubator for 24 h. The killing effect of the nano-drug on blood vessels and tumor cells can be detected separately.

[0136] (9) The toxic effect of nanomedicine on blood vessels: Take out the basket assembly, place the basket assembly in the new second base, add 100 μL of cell culture medium and 10 μL of CCK8, incubate in a 37℃ incubator for 4 h, read the 450nm light absorption value (A) with an enzyme-linked immunosorbent assay reader, calculate the cell activity, and the toxic effect of nanomedicine on blood vessels can be obtained.

[0137] (10) Killing effect of nanomedicine on tumor cells: After the basket assembly was removed, the co-culture supernatant was discarded, and 100 μL of cell culture medium and 10 μL of CCK8 were added. The cells were incubated in a 37℃ incubator for 4 h. The absorbance value (A) at 450 nm was read using an ELISA reader, and cell viability was calculated to determine the killing effect of nanomedicine on tumor cells. Cell viability (%) = [A(drug-added) - A(blank)] / [A(0-drug-added) - A(blank)] × 100. The results are shown in Table 2.

[0138] The results show that the drug screening chip prepared by this mold can significantly distinguish the drug sensitivity of tumors from different sources. To verify the reliability of the method, we repeatedly tested the same tumor cells (such as tumor 1 and tumor 5) in the experiment. The results confirmed that the results are highly reproducible and reliable.

[0139] Table 2. Results of experiments on the killing effect of nanomedicines on tumor cells.

[0140]

[0141] Example 3: High-throughput screening of targeted drugs to inhibit lymphangiogenesis

[0142] Lymphatic vessels are also typical microtubule structures, and existing models cannot rapidly and in a high-throughput manner analyze the efficacy of targeted drugs that inhibit lymphatic vessel proliferation. This example provides a high-throughput anti-intraluminal tumor drug screening model that can simultaneously analyze eight targeted drugs that inhibit lymphatic vessel proliferation.

[0143] 1. Mold for preparing a high-throughput biomimetic in vitro tumor drug screening model

[0144] The number of baskets in the basket assembly of the mold is 96, arranged in 8 rows and 12 columns; the number of first grooves in the first base is 96, arranged in 8 rows and 12 columns; the number of second grooves in the second base is 96, arranged in 8 rows and 12 columns; the sacrificial material assembly is a pin array, with 8 pins.

[0145] In addition, the basket assembly, first base, and second base in this example are all compatible with traditional 96-well cell culture plates. Furthermore, the basket assembly, first base, and second base are all covered with lids during use to ensure sterility during culture.

[0146] 2. Specific operating steps and results

[0147] (1) Insert the basket assembly into the first base, ensuring that the perforation on the basket assembly is connected to the through hole on the first base. Insert the pins into the perforation and through hole in sequence, inject 50 μL of liquid methacrylamide gelatin into the basket, and add 0.1% w / w phenyl-2,4,6-trimethylbenzoyl lithium phosphite. Irradiate it under blue-violet light at a wavelength of 405 nm for 40 s to allow it to solidify.

[0148] (2) After the needle is removed from the culture medium (RPIM 1640 + 10% w / w FBS), a microchannel is formed.

[0149] (3) Place 10 5 One lymphoepithelial cell (LEC) was thoroughly mixed with 120 μL of thermosensitive hydrogel (PLGA-PEG-PLGA, gelling above 35°C, injectable liquid below 35°C) at 4°C to obtain a lymphoepithelial cell-hydrogel (liquid) mixture, which was then aspirated into a microsyringe.

[0150] (4) Inject the vascular endothelial cell-hydrogel (liquid) mixture in the microsyringe into the microchannel at a rate of 10 μL / channel.

[0151] (5) Incubate at 37°C for 5 min until the vascular endothelial cell-hydrogel (liquid) mixture forms a gel. Remove the basket assembly and place it in the second base. Add 100 μL of cell culture medium (RPIM 1640 + 10% w / w FBS) and continue culturing at 37°C for 12 h.

[0152] (6) After observing that the cells adhered to the inner wall of the microchannel, the cells were placed in a 4°C refrigerator. After 5 minutes, the temperature-sensitive hydrogel was observed to be in a liquid state. After the temperature-sensitive hydrogel flowed out, it formed a biomimetic lymphatic vessel. The cell culture medium was replaced and cultured for another 24 hours. The basket assembly was then removed to obtain the vascular biochip.

[0153] (7) Take 1×10 6 One MDA-MB-231 (breast cancer cell line) was mixed thoroughly with 1 ml of photosensitive bio-ink (5% w / w GelMA-0.1% w / w LAP) and added to a new second substrate. 100 μL of the cell-bio-ink mixture was added to each second groove. The second substrate was then irradiated with light at a wavelength of 405 nm for 40 s. After photocuring, 100 μL of cell culture medium (RPMI 1640 + 10% w / w FBS) was added, and the substrate was incubated at 37°C for 24 h to obtain the breast cancer biochip.

[0154] (8) Assemble the vascular biochip and the breast cancer biochip, add 150 μL of cell culture medium containing growth factors or chemotherapy drugs (RPMI 1640 + 10% w / w FBS), and continue to culture in a 37℃ incubator for 7 days.

[0155] (9) Remove the vascular biochip and place it in a new second base. Add 150 μL of culture medium and 15 μL of CCK-8, mix thoroughly, and continue culturing in a 37℃ incubator for 4 h. Read the absorbance value (A) at 450 nm using a microplate reader, calculate cell viability, and determine the inhibitory effect of the drug on lymphatic vessel proliferation by comparing the drug-treated group with the control group. Cell viability (%) = [A(drug-treated) - A(blank)] / [A(0-drug-treated) - A(blank)] × 100%. The results are shown in Table 3. The results show that VEGF can significantly induce lymphatic vessel growth; while FGF has no obvious effect; at the same time, anti-VEGF antibody inhibited the lymphatic proliferation effect to varying degrees.

[0156] Table 3 Results of high-throughput screening for intraluminal tumor drugs

[0157]

[0158] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A mold for preparing a high-throughput biomimetic in vitro tumor drug screening model, characterized in that, The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof.

2. The mold for preparing a high flux biomimetic in vitro tumor drug screening model according to claim 1, wherein, The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof.

3. The mold for preparing a high flux biomimetic in vitro tumor drug screening model according to claim 2, wherein, The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof.

4. The mold for preparing a high flux biomimetic in vitro tumor drug screening model according to claim 1, wherein, The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof.

5. The mold for preparing a high flux biomimetic in vitro tumor drug screening model according to claim 1, wherein, The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof.

6. The mold for preparing a high flux biomimetic in vitro tumor drug screening model according to claim 1, wherein, The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof.

7. The mold for preparing a high flux biomimetic in vitro tumor drug screening model according to claim 1, wherein, The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof.

8. A method for preparing a high-throughput biomimetic in vitro tumor drug screening model, characterized by, The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof.

9. The method for preparing the high-throughput biomimetic in vitro tumor drug screening model as described in claim 8, characterized in that, The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method thereof. The application relates to a high-throughput biomimetic in-vitro tumor drug screening model and a preparation method injecting a cell suspension of tumor cells in a temperature-sensitive hydrogel into the microchannel at 4-35℃ using a syringe, and then placing it in a cell incubator for culture, and when the temperature-sensitive hydrogel is changed from a liquid state to a gel state, taking the basket assembly out of the first base, placing the taken-out basket assembly into the second base, and culturing it in a cell incubator for 8-12 h to determine that tumor cells have adhered in the photosensitive material, and then placing it at 4℃ to change the temperature-sensitive hydrogel from a gel state to a liquid state, and flowing out, and taking out the basket assembly to obtain a first biochip; taking another new second base, adding a cell-containing photosensitive material and a photoinitiator into the second groove, and curing to form a second biochip; continuing to add fresh culture medium into the second groove of the second biochip, and culturing it in a cell incubator, and placing the first biochip into the second groove of the second biochip to obtain the high-throughput biomimetic in vitro tumor drug screening model.

10. The method for preparing the high-throughput biomimetic in vitro tumor drug screening model as described in claim 8, characterized in that, The preparation method comprises: assembling the basket assembly with the first base, sequentially inserting the sacrificial material assembly into the through hole and the through hole, injecting methacrylated gelatin and a photoinitiator into the basket cavity, irradiating the methacrylated gelatin with blue-violet light with a wavelength of 405 nm to cure and form, adding culture medium into the basket to immerse the methacrylated gelatin, and placing it in a 37℃ cell incubator for 2 h to make the culture medium fill the methacrylated gelatin, and taking out the sacrificial material assembly to form a microchannel; injecting a cell suspension of tumor cells in a temperature-sensitive hydrogel into the microchannel at 4-35℃ using a syringe, and then placing it in a cell incubator for culture, and when the temperature-sensitive hydrogel is changed from a liquid state to a gel state, taking the basket assembly out of the first base, placing the taken-out basket assembly into the second base, and culturing it in a cell incubator for 8-12 h to determine that tumor cells have adhered in the photosensitive material, and then placing it at 4℃ to change the temperature-sensitive hydrogel from a gel state to a liquid state, and flowing out, and taking out the basket assembly to obtain a first biochip; taking another new second base, adding a cell-containing photosensitive material and a photoinitiator into the second groove, and curing to form a second biochip; continuing to add fresh culture medium into the second groove of the second biochip, and culturing it in a cell incubator, and placing the first biochip into the second groove of the second biochip to obtain the high-throughput biomimetic in vitro tumor drug screening model.

11. A high-throughput in vitro biomimetic tumour drug screening model, characterised in that, The preparation method is prepared by any one of claims 9-10.

12. The high-throughput biomimetic in vitro tumor drug screening model of claim 11 is used in high-throughput anti-cavity tumor drug screening.

13. The high-throughput biomimetic in vitro tumor drug screening model of claim 11 is used in nano-anti-tumor drug screening.

Images