High-throughput living tissue chip kit for in-vitro drug reaction detection

Abstract

The invention provides a high-flux living tissue chip kit for in-vitro drug reaction detection, and solves the technical problem of low flux for PDEs culture in the prior art. The invention provides a high-throughput living tissue chip kit for in-vitro drug reaction detection. The high-throughput living tissue chip kit comprises a tissue specimen cleaning aqueous solution, a tissue specimen auxiliary forming reagent, a PDEs preparation buffer aqueous solution, a PDE culture aqueous solution and a chip. A tissue specimen cleaning aqueous solution, a tissue specimen auxiliary forming reagent, a PDEs preparation buffer aqueous solution and a PDEs culture aqueous solution provided by the invention are matched with the chip provided by the invention, so that a plurality of PDEs can be subjected to independently controlled high-throughput culture, a comprehensive nutrition environment and possible signal molecules are provided for drug reaction detection, and the detection sensitivity is improved. The kit allows simultaneous detection of a plurality of samples, and can adapt to convenient and efficient preparation requirements of PDEs of living tissues and high-throughput and high-activity culture and detection requirements.

Description

Technical Field

[0001] The invention belongs to the technical field of kits, and in particular relates to a high-throughput living tissue chip kit for in vitro drug reaction detection. Background Art

[0002] Human tissue slices (PDEs) are thin slices obtained using biopsy techniques, which maintain cell activity and tissue integrity, including microenvironmental components such as stromal cells and tumor-infiltrating lymphocytes (TiLs), as well as spatial structural characteristics within the tissue. This PDE model can accurately replicate the layout of tissues in vivo, providing a highly potential platform for pathological and physiological research and drug screening, especially immune function and drug sensitivity testing.

[0003] Tissue chip technology is a new method of molecular biology based on morphology. It extracts tissue samples from conventional tissue wax blocks and rearranges them to achieve parallel analysis of multiple cases or different tissues. This method ensures that samples are treated consistently in experiments and allows for rapid, high-throughput comparative observations. However, due to the lack of activity of these samples, they are not suitable for experiments that require in vivo responses, such as drug response detection or electrophysiological testing.

[0004] However, most of the culture equipment in the existing literature only supports the culture of single PDEs, with extremely low throughput, which cannot meet the actual needs of applications such as large-scale in vitro drug screening, and cannot meet the needs of physiological and pathological research and in vitro drug sensitivity testing, especially in application scenarios that require maintaining tissue activity and conducting large-scale screening. Summary of the invention

[0005] The present invention provides a high-throughput living tissue chip kit for in vitro drug reaction detection, aiming to solve the technical problem of low throughput for PDEs culture in the prior art.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a high-throughput living tissue chip kit for in vitro drug reaction detection, comprising a tissue specimen cleaning aqueous solution, a tissue specimen auxiliary shaping reagent, a PDEs preparation buffer aqueous solution, a PDE culture aqueous solution and a chip;

[0008] The tissue specimen cleaning aqueous solution includes a balanced salt buffer solution PBS and a multi-effect anti-biological contamination reagent;

[0009] The tissue specimen auxiliary molding reagent includes a composite hydrogel reagent and 2,4,6-trimethylbenzoyl;

[0010] The PDEs for preparing the buffered aqueous solution includes a buffer base reagent and a multi-effect anti-biological contamination reagent;

[0011] The PDEs culture aqueous solution includes a PDEs culture basic reagent, fetal bovine serum, a basic anti-biological contamination reagent and a PDEs culture additive;

[0012] The chip includes a cover plate and a base, wherein a plurality of culture chambers are arranged on the base, wherein the cover plate is arranged on the culture chambers, wherein a plurality of injection ports are arranged on the cover plate, wherein each culture chamber is connected with a plurality of liquid inlet branches, wherein one end of the liquid inlet branch is connected with the culture chamber, and the other end of the liquid inlet branch is arranged through the injection port, and wherein a valve for controlling the on-off of the liquid inlet branch is arranged in the liquid inlet branch.

[0013] In a preferred embodiment, the multi-effect anti-biological contamination reagent includes 2500-10000 IU / mL penicillin, 2000-10000 ug / ml streptomycin, 25-250 ug / mL amphotericin B and 1000-5000 ug / mL gentamicin sulfate; the basic anti-biological contamination reagent includes 2500-10000 IU / mL penicillin and 2000-10000 ug / ml streptomycin.

[0014] Based on the above scheme, penicillin has a broad-spectrum bactericidal effect, can inhibit the synthesis of a variety of bacterial and fungal cell walls, and prevent bacterial growth and reproduction; streptomycin mainly has an antibacterial effect on Gram-positive bacteria, while gentamicin sulfate has a strong inhibitory effect on Gram-negative bacteria. The combination of streptomycin and gentamicin sulfate can inhibit the growth of a variety of bacteria. Among them, the amphotericin B contained in the multi-effect anti-biological contamination reagent can bind to ergosterol on the fungal cell membrane, destroy the integrity of the cell membrane, and decompose and remove the biofilm when cleaning the tissue specimen, thereby reducing the attachment and growth of microorganisms on the tissue specimen. The multi-effect anti-biological contamination reagent can not only remove contamination, but also help cells better adapt to the culture environment and promote cell adhesion. Basic anti-biological contamination reagents can prevent bacterial contamination, maintain the sterile state of the culture environment, inhibit possible invading microorganisms, and help maintain the purity of cell culture, thereby protecting cells from pathogens and maintaining cell health and activity. At the same time, antibiotics in the multi-effect anti-biological contamination reagent and the basic anti-biological contamination reagent are maintained at an appropriate concentration to prevent excessive concentrations from causing bacteria to develop drug resistance.

[0015] In a preferred embodiment, the buffer base reagent is HEPES buffer, which has no permeability to cell membranes and thus does not affect the pH balance inside cells. The composite hydrogel reagent comprises 5-10% w / v gelatin hydrogel, 2-5% w / v gelatin and 4-8% w / v sodium alginate hydrogel, and the solvent of the composite hydrogel reagent and 2,4,6-trimethylbenzoyl is physiological saline or balanced salt buffer PBS.

[0016] Based on the above scheme, 2,4,6-trimethylbenzoyl is a photoinitiator, and the composite hydrogel reagent is prepared with 2,4,6-trimethylbenzoyl to prepare a tissue specimen auxiliary molding reagent, which can quickly gel under ultraviolet light after the tissue block is immersed to form a hydrogel block with appropriate strength and toughness. It can ensure that the entire structure has a certain hardness and toughness, so that the tissue block does not deform excessively during the tissue slice preparation process, and can also avoid excessive strength causing additional damage to cells or even death.

[0017] In a preferred embodiment, the PDEs culture basic reagent is DMEM / F12 culture medium, and the PDEs culture additives include 10-25 ug / mL epidermal growth factor, 35-65 mg / mL vitamin C, 100-200 mM L-glutamine and 100000-200000 U / mL interleukin 2.

[0018] Based on the above scheme, 10-25ug / mL of epidermal growth factor can effectively promote cell proliferation and repair; 35-65mg / mL of vitamin C acts as an antioxidant to protect cells from damage by reactive oxygen species and promote collagen synthesis; 100-200mM of L-glutamine can meet the high demand of cells for glutamine and support cell metabolism and growth; 100000-200000U / mL of interleukin 2 can regulate cell differentiation and function.

[0019] In a preferred embodiment, the PDEs culture aqueous solution comprises 77-93 parts by volume of DMEM / F12 culture medium, 5-20 parts by volume of fetal bovine serum, 0.5-1.0 parts by volume of basic anti-biological contamination reagent and 0.1-1.0 parts by volume of PDEs culture additive.

[0020] Based on the above scheme, in the culture of PDEs, DMEM / F12 medium provides the basic nutrients required by cells, including amino acids, vitamins, inorganic salts and glucose; fetal bovine serum contains a variety of growth factors, hormones and nutrients to promote cell growth and proliferation; basic anti-biological contamination reagents contain antibiotics, which can effectively inhibit the growth of bacteria and other microorganisms and protect cell culture from contamination; PDEs culture additives can promote specific functions and metabolic processes of cells.

[0021] In a preferred embodiment, the culture chamber comprises an upper cavity and a lower cavity, a porous PET membrane is provided between the upper cavity and the lower cavity, a plurality of liquid inlet branches are connected to the lower cavity, and the upper cavity of each culture chamber is connected to a plurality of liquid outlet branches.

[0022] Based on the above scheme, the porous PET membrane allows cells to grow on the upper and lower surfaces of the membrane, forming a three-dimensional culture environment, which is closer to in vivo conditions than traditional two-dimensional culture. The liquid inlet branch can transport culture medium and nutrients to the lower cavity, while the liquid outlet branch can remove metabolic waste and excess culture medium to maintain the stability of the culture environment. It can also facilitate the simultaneous culture and comparison of multiple samples, which is suitable for high-throughput experiments such as drug screening and toxicity testing.

[0023] In a preferred embodiment, the cover plate is provided with a liquid outlet main pipe, a liquid inlet main pipe and a side branch pipe, a plurality of the injection interfaces are connected to the liquid inlet main pipe, a plurality of the liquid outlet branch pipes are connected to the liquid outlet main pipe, and the liquid inlet main pipe is connected to a plurality of liquid inlet branch pipes through a side branch pipe.

[0024] Based on the above scheme, the cover plate can accurately control the flow direction of liquid entering and leaving the culture chamber, and the side branch can evenly distribute the liquid in the main inlet pipe to each inlet branch pipe, ensuring that the cells in the culture chamber receive uniform nutrient supply, and can reduce the shear force caused by the fluid on the cells, protecting the cells from physical damage. Multiple injection interfaces and outlet branches make the chip suitable for high-throughput drug screening and cytotoxicity testing.

[0025] In a preferred solution, the side branch pipe is rotatably connected to the liquid inlet main pipe, and the rotation range of the included angle between the side branch pipe and the liquid inlet main pipe is 30°-145°.

[0026] Based on the above scheme, by adjusting the angle of the side branch, the pressure of the inlet and outlet liquids at each injection interface can be kept consistent, and the filling can be done at the same time to avoid uneven fluid distribution caused by pressure differences, thereby achieving uniform filling in the culture chamber. This design not only helps to reduce the formation of bubbles and reduce the potential damage to cells by fluid shear force, but also improves the utilization rate of the culture medium and the efficiency of cell culture by optimizing the fluid path.

[0027] In a preferred solution, the cover plate is further provided with a plurality of liquid collection ports, each of which is connected to a liquid outlet branch pipe.

[0028] Based on the above scheme, each liquid collection port is connected to a liquid outlet branch, allowing liquid samples to be collected from multiple culture chambers at the same time, and the independence of each collection port ensures accurate collection of samples and avoids cross contamination, which is suitable for large-scale drug screening, cytotoxicity testing or other tests that require a large number of repetitive tests.

[0029] A preferred solution further comprises a shelf, wherein the base and the shelf are both provided with a plurality of culture chambers, and the cover plate is provided on the base and the cover is provided on the shelf.

[0030] Beneficial effects of the present invention:

[0031] The present invention effectively reduces sample contamination and ensures the accuracy of the results by adding anti-biological contamination reagents during tissue specimen cleaning, PDEs preparation and culture, and uses composite hydrogel reagents to simulate the in vivo environment in tissue specimen auxiliary molding, providing a suitable three-dimensional structure, thereby obtaining results closer to the physiological state. At the same time, the culture basic reagents, fetal bovine serum and culture additives contained in the PDEs culture aqueous solution provide comprehensive nutrition and signal molecules for drug reaction detection, simulating the response of cells in the body to drugs. In addition, the chip design of the present invention realizes precise control of samples and reagents through independent liquid inlet branches and control valves, ensuring high-throughput screening and dynamic monitoring. At the same time, the use of valves allows the stability of other culture chamber environments to be maintained when replacing reagents or operating to avoid interference, and realizes instant mixing of different culture components and independent control of multiple PDEs, meeting the requirements of efficient preparation of living tissue PDEs and high-throughput, high-activity culture detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 It is the first schematic diagram of the high-throughput living tissue chip box structure of the present invention.

[0034] Figure 2 It is the second schematic diagram of the high-throughput living tissue chip box structure of the present invention.

[0035] Figure 3 It is a schematic diagram of the structure of the culture chamber in the high-throughput living tissue chip box of the present invention.

[0036] Figure 4 It is a structural cross-sectional view of the culture chamber in the present invention.

[0037] Figure 5 It is a schematic diagram of the bottom structure of the cover plate in the present invention.

[0038] Figure 6 It is a schematic diagram of the structural explosion state of the valve in the present invention.

[0039] Figure 7These are the gross images of PDEs in gastric cancer tissues derived from patients in the embodiments of the present invention, HE staining images of frozen sections, and Ki67 staining images.

[0040] Figure 8 1 and 2. The hydrogel block image (A), the PDEs light microscopy image (B), and the frozen section HE staining image (C) of the mouse colorectal tissue in the embodiment of the present invention are shown.

[0041] Fig. 9 It is a comparison of HE staining of frozen sections of mouse colorectal PDEs cultured in static culture and high-throughput gas-liquid culture after 7 days of culture in the embodiments of the present invention.

[0042] Notes on the numbers:

[0043] 1-cover plate; 2-liquid collection port; 3-culture chamber; 4-liquid inlet branch pipe; 5-liquid inlet main pipe; 6-liquid outlet branch pipe; 7-liquid outlet main pipe; 8-side branch pipe; 9-injection interface; 10-upper cavity; 11-PET porous membrane; 12-lower cavity; 13-base; 14-bracket; 15-valve; 151-sealing rubber plug; 152-rubber ring. DETAILED DESCRIPTION

[0044] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0045] See also Figure 1-Figure 9 , this embodiment provides a high-throughput living tissue chip kit for in vitro drug reaction detection, including a tissue specimen cleaning aqueous solution, a tissue specimen auxiliary shaping reagent, a PDEs preparation buffer aqueous solution, a PDE culture aqueous solution and a chip;

[0046] The tissue specimen cleaning aqueous solution includes a balanced salt buffer solution PBS and a multi-effect anti-biological contamination reagent;

[0047] The tissue specimen auxiliary molding reagent includes a composite hydrogel reagent and 2,4,6-trimethylbenzoyl;

[0048] The PDEs for preparing the buffered aqueous solution includes a buffer base reagent and a multi-effect anti-biological contamination reagent;

[0049] The PDEs culture aqueous solution includes a PDEs culture basic reagent, fetal bovine serum, a basic anti-biological contamination reagent and a PDEs culture additive;

[0050] The chip includes a cover plate 1 and a base 13, wherein the base 13 is provided with a plurality of culture chambers 3, the cover plate 1 is covered on the culture chambers 3, the cover plate 1 is provided with a plurality of injection ports 9, each culture chamber 3 is connected with a plurality of liquid inlet branches 4, one end of the liquid inlet branch 4 is connected with the culture chamber 3, the other end of the liquid inlet branch 4 is passed through the injection port 9, and a valve 15 for controlling the on and off of the liquid inlet branch 4 is provided in the liquid inlet branch 4.

[0051] Wherein, the multi-effect anti-biological contamination reagent includes 2500-10000IU / mL penicillin, 2000-10000ug / ml streptomycin, 25-250ug / mL amphotericin B and 1000-5000ug / mL gentamicin sulfate; the basic anti-biological contamination reagent includes 2500-10000IU / mL penicillin and 2000-10000ug / ml streptomycin.

[0052] Specifically, different microorganisms have different sensitivities to antibiotics, and it is necessary to select an appropriate concentration based on the target source of contamination. When selecting the antibiotic concentration, it is necessary to ensure that the selected concentration can effectively inhibit or kill possible contaminating microorganisms, while ensuring that the selected antibiotic is compatible with other reagents or culture medium components and will not cause adverse reactions or precipitation.

[0053] This embodiment provides the following solutions:

[0054] Multi-effect anti-biological contamination reagent solution 1: penicillin 8000IU / mL, streptomycin 8000ug / mL, amphotericin B 100ug / mL, gentamicin sulfate 3000ug / mL. This solution focuses on a broad antimicrobial spectrum and is suitable for a variety of possible sources of contamination, with a higher gentamicin concentration targeting Gram-negative bacteria.

[0055] Alternatively, multi-effect anti-biological contamination reagent solution 2: penicillin 2500IU / mL, streptomycin 2000ug / mL, amphotericin B 25ug / mL, gentamicin sulfate 1000ug / mL. This solution reduces the amount of antibiotics used and is suitable for samples that are sensitive to antibiotics or when the risk of specific contamination is known to be low.

[0056] Alternatively, Multi-effect Anti-biological Contamination Reagent Solution 3: Penicillin 5000IU / mL, Streptomycin 5000ug / mL, Amphotericin B 50ug / mL, Gentamycin Sulfate 2000ug / mL. This solution provides a moderate level of antimicrobial activity and is suitable for most common laboratory contamination microorganisms.

[0057] Basic anti-biological contamination reagent solution 1: penicillin 2500IU / mL, streptomycin 2000ug / mL, for basic contamination protection.

[0058] Basic anti-biological contamination reagent solution 2: Penicillin 10000IU / mL, streptomycin 10000ug / mL, for use when a higher level of protection is required.

[0059] Basic anti-biological contamination reagent solution 3: penicillin 7500IU / mL, streptomycin 7500ug / mL; medium protection level.

[0060] In order to better simulate the in vivo environment and provide a more suitable three-dimensional structure for tissue samples, based on any of the above schemes, the buffer base reagent is HEPES buffer, the composite hydrogel reagent includes 5-10% w / v gelatin hydrogel, 2-5% w / v gelatin and 4-8% w / v sodium alginate hydrogel, and the solvent for the composite hydrogel reagent and 2,4,6-trimethylbenzoyl is physiological saline or balanced salt buffer PBS.

[0061] Specifically, when selecting the concentration of the composite hydrogel reagent and solvent, the concentration of the hydrogel affects its mechanical strength, and the degree of cross-linking of the hydrogel affects its stability and degradation rate. It is necessary to select according to the duration of the experiment and the ability of cells to remodel the matrix. It is necessary to ensure that the selected concentration is non-toxic to cells and supports cell growth and tissue structure maintenance.

[0062] This embodiment provides the following solution: the composite hydrogel reagent includes 5% w / v gelatin hydrogel, 2% w / v gelatin and 4% w / v sodium alginate hydrogel, providing a relatively soft matrix environment, which is suitable for the type that requires a relatively flexible support.

[0063] Alternatively, the composite hydrogel agent includes 10% w / v gelatin hydrogel, 5% w / v gelatin and 8% w / v sodium alginate hydrogel, providing a relatively rigid matrix environment suitable for simulating relatively rigid tissues (such as cartilage or certain types of tumors).

[0064] Alternatively, the composite hydrogel agent includes 7.5% w / v gelatin hydrogel, 4% w / v gelatin and 6% w / v sodium alginate hydrogel, provides moderate mechanical strength and pore structure, and is suitable for most types.

[0065] The solvent of the composite hydrogel reagent and 2,4,6-trimethylbenzoyl is physiological saline or balanced salt buffer PBS. Physiological saline is suitable for experiments requiring osmotic pressure balance; balanced salt buffer PBS is suitable for experiments requiring specific ion concentration or pH value control.

[0066] The PDEs culture basic reagent is DMEM / F12 culture medium, and the PDEs culture additives include 10-25ug / mL epidermal growth factor, 35-65mg / mL vitamin C, 100-200mM L-glutamine and 100000-200000U / mL interleukin 2.

[0067] Specifically, different cells have different requirements for growth factors, vitamins, amino acids and cytokines. The corresponding additive concentration is selected based on whether the requirement is to promote cell proliferation, differentiation or other specific functions.

[0068] This embodiment provides the following scheme: the PDEs culture additive includes 10ug / mL of epidermal growth factor, 65mg / mL of vitamin C, 200mM of L-glutamine and 100000U / mL of interleukin 2. This scheme provides a high concentration of vitamin C, which is particularly useful for cell culture requiring high antioxidant capacity, while a lower concentration of epidermal growth factor is used to maintain cell health rather than promote rapid proliferation.

[0069] Alternatively, the PDEs culture additive includes 20ug / mL of epidermal growth factor, 40mg / mL of vitamin C, 100mM of L-glutamine and 200000U / mL of interleukin 2, which provides a high concentration of epidermal growth factor to promote cell proliferation, while the high concentration of interleukin 2 is used to enhance immune response or activation of specific types of cells.

[0070] Alternatively, the PDEs culture additive includes 25ug / mL of epidermal growth factor, 35mg / mL of vitamin C, 120mM of L-glutamine and 180000U / mL of interleukin 2. This solution provides a high concentration of epidermal growth factor and moderate concentrations of other additives, which is suitable for conditions that require special promotion of cell proliferation or migration. At the same time, the concentration of interleukin 2 is also high and can be used for the culture of immune cells.

[0071] Alternatively, the PDEs culture additive includes 15ug / mL of epidermal growth factor, 50mg / mL of vitamin C, 150mM of L-glutamine and 150000U / mL of interleukin 2. This scheme is suitable for most cell types that require epidermal growth factor and interleukin 2, and the concentrations of vitamin C and L-glutamine are also suitable for the metabolic needs of cells.

[0072] See also Figure 1-3In order to be closer to the in vivo conditions, on the basis of any of the above schemes, the culture chamber 3 includes an upper cavity 10 and a lower cavity 12, a porous PET membrane is provided between the upper cavity 10 and the lower cavity 12, a plurality of liquid inlet branches 4 are connected to the lower cavity 12, and the upper cavity 10 of each culture chamber 3 is connected to a plurality of liquid outlet branches 6.

[0073] Specifically, the culture chamber 3 has an inner diameter of 12-24 mm, a wall thickness of 1.5-2.5 mm, a height of 4-7 mm, and is divided into an upper cavity 10 and a lower cavity 12, separated by a porous PET membrane with a pore size of 0.1-8 mm in the middle, and a height of 1.5-2 mm for the lower cavity 12. The high-throughput living tissue chip can be stacked by multiple layers of culture chambers 3, and the inner diameter of the culture chamber 3 of the lower layer is 3-5 mm longer than that of the upper layer.

[0074] A better solution: Each culture chamber 3 is equipped with one liquid outlet branch 6 and two liquid inlet branches 4, wherein the inner diameter of the channel is between 0.4-0.8 mm, the outer diameter is within the range of 0.5-1.0 mm, and the length is set at 8-21 mm as required, ensuring that the upper ends of each liquid inlet and outlet branch 6 are flush, and these channels pass through the wall of the culture chamber 3 and accurately lead to the corresponding positions of each culture chamber 3. The two liquid inlet branches 4 are specially designed to lead to the lower cavity 12 of the culture chamber 3, so that they can independently input different liquids to meet the dual needs of cell culture and drug treatment. At the same time, the liquid outlet branch 6 leads to the upper cavity 10 of the culture chamber 3 and opens at 1-1.5 mm above the porous PET membrane, ensuring the precise control of liquid flow.

[0075] In addition, the high-throughput biopsy chip kit for in vitro drug reaction detection also includes a shelf, and a plurality of culture chambers 3 are provided on the base 13 and the shelf, and the cover plate 1 is covered on the base 13 and covered on the shelf.

[0076] Specifically, the culture chambers 3 are stacked up and down on the shelf, each layer is provided with a plurality of culture chambers 3, and the base 13 is provided with a plurality of shelves. A preferred solution is: each layer is provided with 8 culture layers, and the shelf has a total of 3 layers.

[0077] See also Figure 4 The cover plate 1 is provided with a liquid outlet main pipe 7, a liquid inlet main pipe 5 and a side branch pipe 8, a plurality of the injection interfaces 9 are connected to the liquid inlet main pipe 5, a plurality of the liquid outlet branch pipes 6 are connected to the liquid outlet main pipe 7, and the liquid inlet main pipe 5 is connected to a plurality of liquid inlet branch pipes 4 through the side branch pipe 8.

[0078] The side branch pipe 8 is rotatably connected to the liquid inlet main pipe 5, and the rotation range of the angle between the side branch pipe 8 and the liquid inlet main pipe 5 is 30°-145°.

[0079] Specifically, the chip cover plate 1 is provided with an injection port 9 which matches the inlet and outlet branches 6 in the culture chamber 3. The injection port 9 has an inner diameter of 0.5-1.0 mm and is equipped with a circle of 0.1-0.3 mm thick sealing strip to ensure that after the chip is assembled, the channel in the culture chamber 3 and the injection port 9 on the cover plate 1 can be sealed and connected, which not only ensures the smoothness of the liquid flow, but also ensures the sealing of the entire system, effectively preventing the occurrence of leakage. In addition, the inner diameter of the liquid inlet main pipe 5 is 0.8-1.2mm, the inner diameter of its side branch pipe 8 is 0.3-0.8mm, the angle between the side branch pipe 8 and the liquid inlet main pipe 5 is 30°-145°, and the inner diameter of the liquid outlet main pipe 7 is 0.8-1.2mm, and the inner diameter of the side branch pipe 8 is 0.3-0.8mm, so that the inlet and outlet pressures at each injection interface 9 on the same passage are consistent, ensuring uniform filling of the liquid, while the culture waste liquid can be effectively discharged through the liquid outlet main pipe 7 by the suction force of the microfluidic pump, and the fresh culture medium is introduced into the chip through the two liquid inlet main pipes 5 under the precise control of the microfluidic pump, realizing efficient and precise liquid management.

[0080] This embodiment uses a sealing rubber plug 151 with a diameter of 0.5-1.0mm and a thickness of 0.9-1.2mm as the valve 15, and a rubber ring 152 is provided in the liquid inlet main pipe, which can completely close the opening of the liquid inlet main pipe at the liquid inlet interface, effectively preventing unnecessary liquid from flowing in, and realizing the instant mixing of different culture components. For example, one liquid inlet pipeline transports basic culture reagents, and the other transports additive components that are easily ineffective. It also allows the use of up to three different culture reagents to perform high-throughput gas-liquid culture on multiple PDEs. At the same time, this design also allows up to three different dosing treatments for multiple PDEs, which improves the flexibility and diversity of the experiment. In order to further enhance the real-time monitoring capability of the experiment, a number of rubber sampling channels with a diameter of about 0.3-0.5mm are specially designed on the chip cover 1 to support manual and separate real-time collection of culture supernatants from different culture chambers 3, which is convenient for researchers to conduct fast and accurate real-time detection.

[0081] A method for using a high-throughput biopsy chip kit for in vitro drug response detection:

[0082] 1. PDEs pretreatment

[0083] (1) Add 1 part by volume of the multi-effect anti-biological contamination reagent to 99 parts by volume of the tissue specimen cleaning basic reagent and mix well to prepare a tissue specimen cleaning aqueous solution.

[0084] (2) Then use the tissue specimen cleaning solution to clean the living human tissue 5-15 times to remove the microorganisms that may exist on the surface of the tissue specimen as much as possible, thereby reducing the risk of microbial contamination during the subsequent culture process. Each time the tissue is cleaned, 5-10 times the volume of the specimen tissue should be used for cleaning. If the required cleaning reagent is less than 5 mL, add 5 mL of tissue specimen cleaning solution.

[0085] (3) Under light-proof conditions, 1 volume part of the composite hydrogel reagent and 1 volume part of 2,4,6-trimethylbenzoyl are mixed and uniformly prepared to obtain a tissue specimen auxiliary molding reagent.

[0086] (4) Transfer the cleaned tissue specimen to a sterile culture dish, remove the tissue specimen cleaning solution on the surface as much as possible, and then add 20-100uL of tissue specimen auxiliary molding reagent according to the size of the specimen to fully contact the surface of the tissue specimen.

[0087] (5) According to the size of the specimen and the size of the selected pretreatment mold, add 100uL-500uL of tissue specimen auxiliary molding reagent into the pretreatment mold, then put the tissue block, and then add an appropriate amount of tissue specimen auxiliary molding reagent into the mold until the tissue block is covered and immersed. After adjusting the position of the tissue block, irradiate it with a UV light source with an intensity of 20-40mW / cm2 for 30-180s. Finally, push the mold push rod to make a fixed-shape tissue-containing hydrogel block that can be used for subsequent slicing.

[0088] 2. Preparation of PDEs

[0089] (1) Add 1 volume of the multi-effect anti-biological contamination reagent to 99 volumes of the PDEs preparation buffer base reagent, and then mix by inversion to obtain the PDEs preparation buffer aqueous solution.

[0090] (2) The prepared hydrogel block is fixed on the stage of a vibrating slicer, and then 150-500 mL of PDEs is added to prepare a buffer solution according to the size of the vibrating slicer until the tissue gel block is completely immersed in the buffer solution.

[0091] (3) Turn on the vibratome and perform serial sectioning to obtain PDEs.

[0092] 3. High-throughput gas-liquid culture of PDEs

[0093] (1) Add 5-20 parts by volume of fetal bovine serum, 0.5-1 parts by volume of basic anti-biological contamination reagent and 0.1-1 parts by volume of each PDEs culture additive to 77-93 parts by volume of PDEs culture basic reagent, and then mix well to obtain a PDEs culture aqueous solution.

[0094] (2) Layers 1 to 3 of the chip are laid out separately, and then 300-500 uL of PDEs culture aqueous solution is added to each culture chamber to be used.

[0095] (3) The PDEs were transferred to each culture chamber of the chip, and then the upper surface of the PET porous membrane was contacted with a pipette to suck out the excess culture solution in each well. The sucked out PDEs culture solution was temporarily stored in a sterile container for later use.

[0096] (4) After all the PDEs are transferred and the excess PDEs culture solution is absorbed, the high-throughput chip is assembled by stacking and placing it according to the size of each layer of culture chambers and the size of each layer of external supports, and then the cover of the high-throughput biopsy chip is covered.

[0097] (5) According to the experimental requirements, place the valve into the corresponding liquid inlet interface, and then cover the cover of the high-throughput living tissue chip.

[0098] (6) According to the experimental requirements, the excess PDEs culture solution sucked out in step (4) and the fresh PDEs culture solution are added to the culture reservoir, and then the inlet and outlet pipes on both sides of the chip cover are connected to the corresponding channels in the microfluidic liquid control device to perform high-throughput gas-liquid culture of PDEs.

[0099] (7) According to the experimental requirements, remove the chip cover, insert the valve into the corresponding interface, and then replace the chip cover. Finally, add the corresponding drug treatment reagent to the culture effluent pool, or directly replace the aqueous solution in the reservoir with the drug treatment working solution to perform drug treatment of PDEs.

[0100] (8) When real-time supernatant detection is required, wipe the outer surface of the sampling channel on the chip cover with 75% alcohol, and then use a sterile syringe to perform real-time sampling of the culture supernatant through the sampling channel.

[0101] The present invention is further explained below in conjunction with specific experiments:

[0102] 1. Preparation of reagents and aqueous solutions

[0103] Required materials: Turn on the ultraviolet light on the clean bench for 30 minutes in advance; preheat the water bath in advance; ice box; ice; pipette; pipette tip; 15mL centrifuge tube; 1.5mL EP tube; 250mL culture medium bottle; 500mL culture medium bottle; basic reagent for tissue specimen cleaning; composite hydrogel reagent; 2,4,6-trimethylbenzoyl; basic reagent for PDEs preparation buffer; multi-effect anti-biological contamination reagent; basic anti-biological contamination reagent; basic reagent for PDEs culture; fetal bovine serum; EGF; vitamin C; L-glutamine and IL-2.

[0104] The specific steps for preparing each reagent and aqueous solution are as follows:

[0105] A1. Preparation of tissue specimen cleaning aqueous solution: 173.25 mL of tissue specimen cleaning basic reagent and 1.75 mL of multi-effect anti-biological contamination reagent are added to a 250 mL culture medium bottle in sequence. After the bottle cap is closed, the mixture is inverted and mixed evenly to obtain the tissue specimen cleaning aqueous solution.

[0106] A2. Preparation of tissue specimen auxiliary molding reagent: Heat the composite hydrogel reagent in a 45°C water bath to convert it into a highly fluid liquid. Then, in a dark environment, mix 2 mL of the composite hydrogel reagent and 2 mL of 2,4,6-trimethylbenzoyl to obtain 4 mL of tissue specimen auxiliary molding reagent.

[0107] A3. Preparation of PDEs preparation buffer solution: Add 495 mL of PDEs preparation buffer base reagent and 5 mL of multi-effect anti-biological contamination reagent to a 500 mL culture medium bottle in sequence, cover the bottle cap and invert to mix evenly to obtain 500 mL of tissue specimen cleaning aqueous solution.

[0108] A4. Preparation of patient-derived gastric cancer tissue PDEs culture aqueous solution: 8.19 mL of PDEs culture basic reagent, 1.5 mL of fetal bovine serum, 0.1 mL of basic anti-biological contamination reagent, 0.03 mL of EGF, 0.01 mL of vitamin C, 0.15 mL of L-glutamine and 0.02 mL of IL-2 were added into a 15 mL centrifuge tube in sequence, and then mixed evenly to obtain the patient-derived gastric cancer tissue PDEs culture aqueous solution.

[0109] A5. Preparation of mouse colorectal tissue PDEs culture aqueous solution: 9.26 mL of PDEs culture basic reagent, 0.5 mL of fetal bovine serum, 0.1 mL of basic anti-biological contamination reagent, 0.02 mL of EGF, 0.01 mL of vitamin C, 0.1 mL of L-glutamine and 0.01 mL of IL-2 were added into a 15 mL centrifuge tube in sequence, and then mixed evenly to obtain the mouse colorectal tissue PDEs culture aqueous solution.

[0110] 2. Tissue specimen cleaning

[0111] Required materials: Ophthalmic scissors and ophthalmic tweezers sterilized in high temperature and high pressure in advance; ultraviolet light on the clean bench for 30 minutes in advance; ice box; ice; 60mm culture dish; 15mL centrifuge tube; 3mL Pasteur tube; tissue specimen cleaning water solution.

[0112] The specific steps for tissue specimen cleaning are as follows:

[0113] B1. Human gastric cancer tissue was obtained from the lesion site of clinical gastric cancer patients. The sampling method was surgical sampling. The tissue was placed in a tissue preservation tube 7 minutes after sampling and transported to the laboratory at 2-8°C for subsequent processing. Mouse colon tissue was obtained from the colorectal tissue of 6-8 week healthy KM mice. Use ophthalmic forceps to remove the patient-derived gastric cancer tissue specimen or mouse colon tissue from the transportation and storage container, transfer it to a 60mm culture dish on ice, and then use ophthalmic scissors to trim and remove excess fat tissue on the tissue specimen.

[0114] B2. Transfer the trimmed gastric cancer tissue specimens from patients or colon tissues from mice to 15 mL centrifuge tubes, add about 5 mL of complete tissue specimen cleaning reagent using a Pasteur tube, cover the tube cap, invert 5-10 times, and then discard the waste liquid.

[0115] B3. Repeat the above cleaning operation 15 times to complete the cleaning of the tissue specimen.

[0116] 4. Preparation of PDEs

[0117] Required preparations: Ophthalmic forceps sterilized at high temperature and high pressure in advance; biological safety cabinet turned on UV for 30 minutes in advance; water bath turned on and preheated (37°C); pipette; pipette tip; ice box and ice; tissue specimen auxiliary molding reagent; PDEs preparation buffer solution precooled to 4°C in advance; pre-treated mold and vibration slicer.

[0118] The specific steps for preparing PDEs are as follows:

[0119] C1. Use ophthalmic forceps to transfer the cleaned patient-derived gastric cancer tissue specimen or mouse colorectal tissue specimen to a sterile culture dish, remove the tissue specimen cleaning reagent on the surface as much as possible, and then add about 50uL of tissue specimen auxiliary molding reagent to the patient-derived gastric cancer tissue specimen, and add about 20uL of tissue specimen auxiliary molding reagent to the mouse colorectal tissue specimen. Make sure that the tissue specimen auxiliary molding reagent is in full contact with the surface of the tissue specimen.

[0120] C2. Add 200uL of tissue specimen auxiliary molding reagent into the pre-treated mold, then put in the tissue block that has completed the previous step, and then add an appropriate amount of tissue specimen auxiliary molding reagent into the mold until the tissue block is covered and immersed. After adjusting the position of the tissue block, use a UV light source with an intensity of 20-40mW / cm2 to irradiate for 30s-120s. Among them, the curing time of the patient-derived gastric cancer tissue specimen block is 120s, and the curing time of the mouse colorectal tissue specimen block is 30s.

[0121] C3. Push the pre-treated mold push rod to obtain a tissue-containing hydrogel block with a fixed shape that can be used for subsequent slicing.

[0122] C4. Fix the prepared patient-derived gastric cancer tissue hydrogel block on the stage of the vibration slicer; then inject 250 mL of 4°C pre-cooled PDEs preparation buffer reagent into the slice groove. Turn on the vibration slicer and perform continuous slicing to obtain patient-derived gastric cancer tissue PDEs.

[0123] C5. Fix the prepared mouse colorectal tissue hydrogel block on the stage of the vibrating slicer; then inject 250 mL of 4°C precooled PDEs preparation buffer reagent into the slice groove. Turn on the vibrating slicer and perform continuous slicing to obtain mouse colorectal tissue PDEs.

[0124] 5. High-throughput gas-liquid culture of PDEs

[0125] Required preparations: Ophthalmic forceps sterilized in high temperature and high pressure in advance; biological safety cabinet turned on UV for 30 minutes in advance; chip; pipette; pipette tip; microfluidic control system; PDEs culture solution.

[0126] The specific steps of high-throughput gas-liquid culture of PDEs are as follows:

[0127] D1. Lay out the 1st to 3rd layers of the chip separately and add 400uL of the corresponding PDEs culture aqueous solution into each culture chamber to be used.

[0128] D2. Transfer the PDEs from gastric cancer tissues of patients or PDEs from colorectal tissues of mice to the PET porous membranes in each culture chamber of the chip.

[0129] D3. Then place the pipette tip on the PET porous membrane and slowly absorb the excess PDEs culture solution and place it in a 15mL centrifuge tube for later use.

[0130] D4. After all the PDEs are transferred and the excess culture medium is absorbed, the assembled high-throughput chips are stacked and covered with a cover plate of the high-throughput living tissue chip according to the size of each layer of culture chambers and the size of each layer of external supports.

[0131] D5. Add the excess patient-derived gastric cancer tissue PDEs culture solution or mouse colorectal tissue PDEs culture solution to the corresponding storage bottle in the microfluidic control device, and add 10 mL of fresh culture solution of the same solution to the storage bottle.

[0132] D6. Finally, according to the experimental design, each inlet and outlet liquid main pipe was connected to the corresponding channel in the microfluidic liquid control device, and the parameters of the microfluidic pump were adjusted to perform high-throughput gas-liquid culture of PDEs at a flow rate of 80uL / h.

[0133] 6. Static culture of PDEs

[0134] Required preparations: Ophthalmic forceps sterilized in high temperature and high pressure in advance; biological safety cabinet with UV light turned on for 30 minutes in advance; 24-well cell culture plate; pipette; pipette tip; PDEs culture solution; carbon dioxide cell culture incubator.

[0135] The specific steps of static culture of PDEs are as follows:

[0136] E1. Add 350uL of the corresponding PDEs culture aqueous solution to each culture well to be used.

[0137] E2. Transfer PDEs from patient-derived gastric cancer tissue or mouse colorectal tissue to each culture well plate.

[0138] E3. Place the culture plate in a 37°C carbon dioxide incubator for culture and change the medium every 24-48 hours.

[0139] Results: See Figure 7-8 , the mouse colorectal tissue hydrogel blocks and gastric cancer tissue PDEs hydrogel blocks showed uniform transparency and consistent color, indicating their uniformity and stability, and there should be no obvious cavities or cracks inside the gel, showing good mechanical properties and integrity. Under the PDEs light microscope, the mouse colorectal tissue blocks maintained their natural structure, the cells were arranged in an orderly manner, the tissue morphology was clearly discernible, and the cell morphology should be complete with clear cell boundaries, showing good cell activity and health. In the frozen section HE staining image, the nuclei of the mouse colorectal tissue and gastric cancer tissue PDEs were blue, the cytoplasm was pink, the staining contrast was sharp, there was no aggregation or loss of dye, and the cell density was moderate, reflecting a healthy growth state, without signs of excessive proliferation or apoptosis.

[0140] See also Fig. 9 After 7 days of high-throughput gas-liquid culture, PDEs from gastric cancer tissues and mouse colorectal tissues showed blue nuclei and pink cytoplasm in frozen sections after HE staining. The cell morphology remained intact, the nuclei were clearly visible, and the cytoplasm was evenly stained, without dye aggregation or loss. PDEs from both tissues maintained their specific tissue structural characteristics, with moderate cell density, no large gaps and no cell accumulation, which reflected healthy cell growth without excessive proliferation. These observations indicate that high-throughput gas-liquid culture technology provides PDEs with superior oxygen and nutrient exchange conditions, so that PDEs under these culture conditions show good cell viability and tissue health, thus verifying the effectiveness of this high-throughput biochip kit for in vitro drug response detection in maintaining tissue characteristics and promoting healthy cell growth.

[0141] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that fall within the scope defined by the claims of the present invention fall within the protection scope of the present invention.

Claims

1. A high-throughput biopsy chip kit for in vitro drug response detection, characterized in that: It includes tissue specimen cleaning aqueous solution, tissue specimen auxiliary molding reagent, PDEs preparation buffer aqueous solution, PDE culture aqueous solution and chip; The tissue specimen cleaning aqueous solution includes a balanced salt buffer solution PBS and a multi-effect anti-biological contamination reagent; The tissue specimen auxiliary molding reagent includes a composite hydrogel reagent and 2,4,6-trimethylbenzoyl; The PDEs for preparing the buffered aqueous solution includes a buffer base reagent and a multi-effect anti-biological contamination reagent; The PDEs culture aqueous solution includes a PDEs culture basic reagent, fetal bovine serum, a basic anti-biological contamination reagent and a PDEs culture additive; The chip includes a cover plate and a base, wherein a plurality of culture chambers are arranged on the base, wherein the cover plate is arranged on the culture chambers, wherein a plurality of injection ports are arranged on the cover plate, wherein each culture chamber is connected with a plurality of liquid inlet branches, wherein one end of the liquid inlet branch is connected with the culture chamber, and the other end of the liquid inlet branch is arranged through the injection port, and wherein a valve for controlling the on-off of the liquid inlet branch is arranged in the liquid inlet branch.

2. A high-throughput biopsy chip kit for in vitro drug response detection according to claim 1, characterized in that: The multi-effect anti-biological contamination reagent includes 2500-10000 IU / mL of penicillin, 2000-10000 ug / ml of streptomycin, 25-250 ug / mL of amphotericin B and 1000-5000 ug / mL of gentamicin sulfate; The basic anti-biological contamination reagent includes 2500-10000 IU / mL penicillin and 2000-10000 ug / ml streptomycin.

3. A high-throughput biopsy chip kit for in vitro drug response detection according to claim 1, characterized in that: The buffer base reagent is HEPES buffer, the composite hydrogel reagent includes 5-10% w / v gelatin hydrogel, 2-5% w / v gelatin and 4-8% w / v sodium alginate hydrogel, and the solvent of the composite hydrogel reagent and 2,4,6-trimethylbenzoyl is physiological saline or balanced salt buffer PBS.

4. A high-throughput biopsy chip kit for in vitro drug response detection according to claim 3, characterized in that: The PDEs culture basic reagent is DMEM / F12 culture medium, and the PDEs culture additives include 10-25ug / mL epidermal growth factor, 35-65mg / mL vitamin C, 100-200mM L-glutamine and 100000-200000U / mL interleukin 2.

5. A high-throughput biopsy chip kit for in vitro drug response detection according to claim 1, characterized in that: The PDEs culture aqueous solution comprises 77-93 parts by volume of DMEM / F12 culture medium, 5-20 parts by volume of fetal bovine serum, 0.5-1.0 parts by volume of basic anti-biological contamination reagent and 0.1-1.0 parts by volume of PDEs culture additive.

6. A high-throughput biopsy chip kit for in vitro drug response detection according to claim 1, characterized in that: The culture chamber comprises an upper cavity and a lower cavity, a porous PET membrane is arranged between the upper cavity and the lower cavity, a plurality of liquid inlet branches are connected to the lower cavity, and the upper cavity of each culture chamber is connected to a plurality of liquid outlet branches.

7. A high-throughput biopsy chip kit for in vitro drug response detection according to claim 6, characterized in that: The cover plate is provided with a liquid outlet main pipe, a liquid inlet main pipe and a side branch pipe, a plurality of the injection interfaces are communicated with the liquid inlet main pipe, a plurality of the liquid outlet branch pipes are communicated with the liquid outlet main pipe, and the liquid inlet main pipe is communicated with a plurality of liquid inlet branch pipes through the side branch pipe.

8. A high-throughput biopsy chip kit for in vitro drug response detection according to claim 7, characterized in that: The side branch pipe is rotatably connected to the liquid inlet main pipe, and the rotation range of the included angle between the side branch pipe and the liquid inlet main pipe is 30°-145°.

9. A high-throughput biopsy chip kit for in vitro drug response detection according to claim 6, characterized in that: The cover plate is also provided with a plurality of liquid collection ports, each of which is connected to a liquid branch pipe.

10. A high-throughput biopsy chip kit for in vitro drug response detection according to claim 1, characterized in that: It also comprises a layer frame, the base and the layer frame are both provided with a plurality of culture chambers, the cover plate is arranged on the base and the cover is arranged on the layer frame.

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