System and method suitable for circulation culture of organs / organoids and organoids

Abstract

The invention discloses a system and method suitable for organ / organoid circulation culture and an organoid, the system comprises a chip with a cavity and a micro-channel of a simulation circulation system connected with the cavity, in the culture process, matrigel with sample cells is contained in the cavity, and an organ culture medium is provided for the cavity through the micro-channel; the culture medium comprises amino acids, vitamins, inorganic salts, proteins, trace elements and the like. The basic culture medium provided by the invention not only can be used for static culture, but also can be used for circulating culture, so that diversified culture modes are realized, and culture models are provided for subsequent different application scenes.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a system, method and organoid suitable for cyclic culture of organs / organoids. Background Art

[0002] Organoid culture is a hot technology field. It is differentiated from adult stem cells in normal or tumor tissues. Under in vitro three-dimensional culture conditions, the three-dimensional in vitro cell structure is formed. It has the ability to self-organize to form "micro-organs" similar to the original tissues, and can better express the characteristics and cell heterogeneity of the source tissues. As a preclinical model for tumor research, organoids can be used for drug screening, predicting patients' response to treatment, and selecting the most appropriate treatment plan for patients. Gene editing technology can also provide a research basis for the occurrence, development and metastasis mechanism of tumors.

[0003] In the existing organoid culture technology, in order to overcome the accumulation of harmful substances such as toxic ammonia in the culture environment, it is necessary to maintain the carbon environment concentration of carbon dioxide and add additional neutralizing substances to the culture medium. This can meet the requirements of static culture, but cannot achieve large-scale and efficient dynamic culture. For example, the nutrient composition ratio cannot meet the requirements of cyclic culture, especially it is difficult to meet the real-time adjustment of protein content and trace element content, and it is difficult to meet the development needs of modern medicine.

[0004] Therefore, in response to the above technical problems, it is necessary to provide a system, method and organoid suitable for organ / organoid cyclic culture.

[0005] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0006] The object of the present invention is to provide a system, method and organoid suitable for organ / organoid cyclic culture.

[0007] In order to achieve the above object, a technical solution provided by a specific embodiment of the present invention is as follows: A system suitable for organ / organoid circulation culture, comprising a chip having a chamber and a microfluidic channel connecting the chambers to simulate a circulation system, wherein during the culture process, the chamber contains a matrix gel with sample cells and the microfluidic channel is used as a chamber to provide an organ culture medium, wherein the organ culture medium comprises: a basic culture medium for multiple cancer types, comprising amino acids, vitamins, inorganic salts, proteins and trace elements; and in order to adapt to the culture of different types of cancer cells, the following are also added, all in final concentration: For lung cancer sample culture, add: 100ng / mL FGF10 (fibroblast growth factor 10), 250ng / mL Noggin, 50ng / mL FGF2 (fibroblast growth factor 2), 30nM SB202190, 100ng / mL Rspondin-1, 1XB27, 3μM CHIR99021; For gastric cancer sample culture, add: 50ng / mL EGF (epidermal growth factor), 5nM Gastrin, 100ng / mL Rspondin-1, 250ng / mL Noggin, 1X B27, 20nM A83-01, 50μg / mL Primocin; For colorectal cancer sample culture, add: 100ng / mL Rspondin-1, 250ng / mL Noggin, 1X B27, 20nM A83-01, 50μg / mL Primocin, 30nM SB202190; For liver cancer sample culture, add: 300ng / mL Rspondin-1, 500ng / mL Noggin, 3X B27, 10nM A83-01, 50μg / mL Primocin, 3nM SB202190, 100ng / mL HGF (hepatocyte growth factor).

[0008] In one or more embodiments of the present invention, the amino acid is selected from Glycine, L-Alanine, L-Alanyl-L-Glutamine, L-Arginine hydrochloride, L-Asparagine, L-Aspartic acid, L-Cystine2HCl, L-Glutamic Acid, L-Histidine hydrochloride-H2O, L-Isoleucine, L-Leucine, L-Methionine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine disodiμm salt dihydrate(L-Tyrosine disodium salt dihydrate), L-Valine(L-Valine), L-Cysteine ​​hydrochloride-H2O(L-Cysteine ​​hydrochloride-H2O(L-Cysteine ​​hydrochloride), L-Asparagine-H2O(L-Asparagine-hydrate), L-Lysine hydrochloride(L-Lysine hydrochloride), L-Phenylalanine(L-Phenylalanine).

[0009] In one or more embodiments of the present invention, the vitamin is selected from Ascorbic Acid phosphate, Choline chloride, D-Calciμm pantothenate, FolicAcid, Niacinamide, Pyridoxine hydrochloride, Riboflavin, Thiamine hydrochloride, and i-Inositol.

[0010] In one or more embodiments of the present invention, the inorganic salt is selected from Calciμm Chloride (CaCl2) (anhyd.) (calcium chloride), Ferric Nitrate (Fe(NO3)3"9H2O) (ferric nitrate), Magnesiμm Sulfate(MgSO4) (anhyd.) (magnesium sulfate), Potassiμm Chloride (KCl) (potassium chloride), Sodiμm Bicarbonate(NaHCO3) (sodium bicarbonate), Sodiμm Chloride (NaCl) (sodium chloride), Sodiμm Phosphate dibasic(Na2HPO4-H2O) (disodium hydrogen phosphate).

[0011] In one or more embodiments of the present invention, the protein is selected from AlbμmAX® II (lipid-rich BSA), Hμman Transferrin (Holo), and Insulin Recombinant Full Chain (recombinant human insulin).

[0012] In one or more embodiments of the present invention, the trace elements are selected from Ammoniμm Metavanadate (ammonium metavanadate), Cupric Sulfate (copper sulfate), Manganous Chloride (manganous chloride), and Sodiμm Selenite (sodium selenite).

[0013] In one or more embodiments of the present invention, other components are also included, which are selected from D-Glucose (Dextrose) (anhydrous glucose), Ethanolamine (ethanolamine), Glutathione (reduced) (reduced glutathione), Phenol Red (phenol red), Sodiμm Pyruvate (sodium pyruvate), Linoleic Acid (linoleic acid), Lipoic Acid (lipoic acid), Hypoxanthine Na (hypoxanthine), Putrescine 2HCl (putrescine dihydrochloride), Thymidine (thymidine nucleoside), and HEPES (4-hydroxyethylpiperazineethanesulfonic acid).

[0014] In one or more embodiments of the present invention, the basal medium is specifically:

[0015] In one or more embodiments of the present invention, a method for using a system suitable for organ / organoid circulatory culture comprises: Prepare samples and prepare basal culture medium matching the cancer type; The sample is washed, tissue digested, cell precipitated, and matrix gel solidified, and the solidified sample and organ culture medium are added to the microfluidic system for culture.

[0016] In one or more embodiments of the present invention, the culture is a circulation culture based on a microfluidic chip.

[0017] In one or more embodiments of the present invention, the organoid at least includes the organoid tissue cultured by the aforementioned system, including but not limited to the three-dimensional organoid tissue cultured itself, and also includes commodities or medical supplies or experimental supplies containing the organoid tissue.

[0018] In one or more embodiments of the present invention, the cyclic culture is to inoculate the sample into a circulatory closed chip with a flow channel, and connect a matching basal culture medium into the flow channel for culture.

[0019] In one or more embodiments of the present invention, the washing is performed by soaking the target tumor tissue sample in a washing solution composed of a penicillin / streptomycin dual antibody solution for 1 minute.

[0020] In one or more embodiments of the present invention, tissue digestion is as follows: the sample is added with tissue digestion solution, shaken and digested in a shaker at 37° C. and 50 rpm, and serum is added after digestion to terminate digestion.

[0021] In one or more embodiments of the present invention, harvesting the cell precipitate includes: filtering the cell suspension obtained in the tissue digestion process with a 100 μm filter and centrifuging at 1000 rpm and 4°C for 5 minutes; after centrifugation, discarding the supernatant, resuspending the cells, and centrifuging again at 1000 rpm and 4°C for 5 minutes.

[0022] In one or more embodiments of the present invention, the matrix gel solidification specifically includes: mixing the matrix gel and the basal culture medium in a ratio of 7:3, then spotting the matrix gel in the cell culture plate, and then placing the cell culture plate in an incubator at 37°C for 30 minutes to wait for the matrix gel to solidify.

[0023] In one or more embodiments of the present invention, adding culture medium specifically includes: adding a certain volume of basal culture medium to a cell culture vessel placed in a 37°C incubator for culturing: dynamic culture is carried out in a recyclable microfluidic chip, but both require that the recyclable chip be placed in a cell culture incubator for culturing.

[0024] In one or more embodiments of the present invention, the concentration of HEPES is 595.8 mg / L.

[0025] In one or more embodiments of the present invention, the concentration of L-alanyl-L-glutamine dipeptide is 151.3 mg / L.

[0026] In one or more embodiments of the present invention, the osmotic pressure of L-alanyl-L-glutamine dipeptide is 440-500 mOsm / kg.

[0027] Compared with the prior art, the system, method and organoid of the present invention are suitable for organ / organoid cyclic culture.

[0028] Compared with the existing basal culture medium, the present invention provides a culture medium with clear ingredients by optimizing the culture medium technology, and does not require the additional addition of HEPES and L-alanyl-L-glutamine dipeptide. It can effectively overcome the problems of toxic ammonia accumulation in the existing system, thereby meeting the needs of cyclic dynamic culture.

[0029] The basal culture medium provided by the present invention contains a dual buffer system, sodium bicarbonate and HEPES buffer system, which can meet the needs of traditional static culture and chip circulation culture; it has better energy supply and does not form accumulation of toxic ammonia. Different types of factors can be added according to the different types of cancer cultured. Organoids of multiple cancer types can be cultured using the basal culture medium of the present invention, which can show the activity characteristics of inherent tissues both statically and dynamically, and achieve comprehensive characteristics similar to those of tumor tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 A schematic diagram of a culture process of a system suitable for cyclic culture of an organ / organoid in one embodiment of the present invention; Figure 2 This is a comparative diagram of lung cancer organoids cultured in a culture medium in an embodiment of the present invention: A is cultured in the basic culture medium of the present invention, and B is cultured in the commercial basic culture medium Gibco / 12634010; Figure 3 Organoids of multiple cancer types statically cultured using a basic culture medium in one embodiment of the present invention; Figure 4 This is an organoid cultured in a cycle using a basic culture medium in one embodiment of the present invention. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0033] As an embodiment, the system for organ / organoid cyclic culture involved in the present invention may specifically include the following steps in its culture process: Step S1, first prepare a basal culture medium, then prepare an organoid culture medium and a tissue digestion solution; Step S2, taking ex vivo human tumor tissue (taking ex vivo tumor tissue, separating muscle connective tissue with surgical forceps, and chopping the tissue with surgical scissors) and placing it in the basal culture medium containing step S1, and adding type I collagenase; Step S3, transferring the centrifuge tube to a shaking incubator for shaking and digestion (the condition may be placing it on a constant temperature shaker at 37°C, digesting at 50 rpm for 10-30 minutes, and observing the degree of digestion under a microscope during digestion). After digestion is completed, the digested tumor tissue is obtained; Step S4: Filter the digested tumor tissue and centrifuge the filtered supernatant to obtain a cell pellet, and observe the red blood cell content in the cell pellet. If there are a lot of red blood cells, add 2 mL of red blood cell lysis buffer, lyse at room temperature for 5 minutes, then add PBS to resuspend, and then centrifuge; if there are not many red blood cells, there is no need to add red blood cell lysis buffer; add the organoid culture medium in step S1 to resuspend the cell pellet, then mix it with matrix gel, and then add it to the cell culture plate, place it in a carbon dioxide incubator at 37°C for 30 minutes; Step S5, adding the cell precipitate without red blood cells collected in step S4 mixed with the matrix gel with the culture medium to the circulation chip, and then placing the chip in a cell culture incubator at 37° C. for 30 minutes; Step S6: After the standing is completed, a circulating culture medium is added into the culture medium flow channel at a flow rate of 10 μL / min, and the medium is returned to the carbon dioxide incubator for circulating culture.

[0034] Of course, in order to achieve static culture, step S5 can also be replaced by step S5-1. After the static culture is completed, add the corresponding cancer organoid culture medium and put it back into the carbon dioxide incubator for static culture, and change the liquid every two days.

[0035] Preferably, the culture medium in step S1 is configured to have a clear composition, a dual buffer system of HEPES and sodium bicarbonate, and the addition of L-alanyl-L-glutamine dipeptide as the basal culture medium, and to add cytokines required for different cancer types, such as FGF10, Noggin, FGF2, Rspondin-1, EGF, HGF, etc., to allow tumor stem cells to differentiate into tumor organoids of various cancer types.

[0036] Preferably, step S2 specifically includes the following steps: Step S21, soaking the ex vivo human tumor tissue in a penicillin / streptomycin dual antibody solution (wherein the concentration of penicillin is 100 U / mL and the concentration of streptomycin is 0.1 mg / mL) for 1 min; peeling off fat, connective tissue and the like, discarding the dual antibody-containing liquid, and continuing to wash for 2-3 times; Step S22, cut the soaked ex vivo human tumor tissue into pieces with surgical scissors (e.g., cut into 1 mm 3 Cube), place the cut tissue in a centrifuge tube containing 5 mL of tissue digestion solution; Step S23, adding 1 mg / unit tissue of type I collagenase; Preferably, step S4 specifically includes the following steps: Step S41, filtering the digested tumor tissue with a 100 μm filter, collecting the filtrate and completing centrifugation, the centrifugation steps are: performing a first centrifugation on the collected filtrate, the first centrifugation is centrifuged at a centrifugal rate of 1000 rpm for 5 minutes; then discarding the supernatant, adding 2 mL of red blood cell lysis solution to the precipitate, resuspending the cells, incubating at room temperature for 5 minutes, and continuing to perform a second centrifugation, the second centrifugation is centrifuged at a centrifugal rate of 1000 rpm for 5 minutes; discarding the supernatant, adding 5 mL of PBS buffer to the precipitate for washing, and then performing a third centrifugation, the third centrifugation is centrifuged at a centrifugal rate of 1000 rpm for 5 minutes, and taking the precipitate to obtain a cell precipitate, wherein the third centrifugation (i.e., adding 5 mL of PBS buffer to the precipitate for washing, and then performing a third centrifugation, the third centrifugation is centrifuged at a centrifugal rate of 1000 rpm for 5 minutes) is optional; Step S42, resuspend the cell precipitate in S41 with the basal culture medium of the corresponding cancer type, then add matrix gel to fully mix the culture medium and cell precipitate, apply the gel on the cell culture plate, and then let it stand in a carbon dioxide incubator for 30 minutes; Preferably, in step S41, the concentration of the PBS buffer is 0.01 mol / L, the PBS buffer comprises 8 mM Na2HPO4, 136 mM NaCl, 2 mM KH2PO4, and 2.6 mM KCL, and the pH value is 7.2-7.4.

[0037] In order to avoid affecting the gel drop structure when adding culture medium, add culture medium slowly along the side wall of the cell culture plate.

[0038] Example 1

[0039] The culture medium preparation method of this embodiment comprises the following steps: Step S1: prepare a basic culture medium, and mix amino acids, vitamins, inorganic salts, proteins, trace elements and other substances into a basic culture medium with clear ingredients, as shown in Table 1. ; Step S2, prepare lung cancer organoid culture medium, add 100ng / mL FGF10, 250ng / mL Noggin, 50ng / mL FGF2, 30nM SB202190, 100ng / mL Rspondin-1, 1X B27, and 3μM CHIR99021 to the prepared basal culture medium, the aforementioned concentrations are all final concentrations when added and can be used for circulation culture; prepare gastric cancer organoid culture medium, add 50ng / mL EGF, 5nM Gastrin, 100ng / mL Rspondin-1, 250ng / mL Noggin, 1X B27, 20nM A83-01, and 50μg / mL Primocin to the prepared basal culture medium, the aforementioned concentrations are all final concentrations when added and can be used for circulation culture; prepare intestinal cancer organoid culture medium, add 100ng / mL Rspondin-1, 250ng / mL Noggin, 1X B27, 20nM A83-01, 50μg / mL Primocin, 30nM SSB202190, the above concentrations are the final concentrations when added and can be used for circulation culture; 300ng / mL Rspondin-1, 500ng / mL Noggin, 3X B27, 10nM A83-01, 50μg / mL Primocin, 3nM SSB202190, 100ng / mL HGF were added to treat liver cancer tumor samples. The above concentrations are the final concentrations when added and can be used for circulation culture. Of course, the above culture medium can also be used for static culture.

[0040] Example 2

[0041] The culture method of this embodiment comprises the following steps: Step S1, taking ex vivo human lung cancer tumor tissue, separating muscle connective tissue with surgical forceps, chopping the tissue with surgical scissors, and placing it in a tissue digestion solution containing type I collagenase; Step S2, transferring the centrifuge tube to a shaking incubator for shaking and digestion, and after the digestion is completed, obtaining the digested tumor tissue; Step S3: Filter the digested lung cancer tumor tissue and centrifuge the filtered supernatant to obtain a cell pellet, and observe the red blood cell content in the cell pellet. If there are many red blood cells, add 2 mL of red blood cell lysis buffer, lyse at room temperature for 5 minutes, then add PBS to resuspend, and then centrifuge; if there are not many red blood cells, there is no need to add red blood cell lysis buffer; add the organoid culture medium in step S1 to resuspend the cell pellet, then mix it with matrix gel, and then add it to the cell culture plate, place it in a carbon dioxide incubator at 37°C for 30 minutes; Step S4: After the static standing is completed, the corresponding cancer organoid culture medium obtained in Example 1 is added, and the culture medium is returned to the carbon dioxide incubator for static culture. The medium is changed every two days. Figure 2 As shown in A.

[0042] Under the same culture conditions, commercially available culture medium was used to culture organoids statically. Figure 2 As shown in B.

[0043] Example 3

[0044] The culture method of this embodiment comprises the following steps: Step S1, taking in vitro human gastric cancer, intestinal cancer, and liver cancer tumor tissues, separating muscle connective tissues with surgical forceps, chopping the tissues with surgical scissors, and placing them in a tissue digestion solution containing type I collagenase; Step S2, transferring the centrifuge tube to a shaking incubator for shaking and digestion, and after the digestion is completed, obtaining the digested tumor tissue; Step S3: Filter the digested tumor tissue and centrifuge the filtered supernatant to obtain a cell pellet, and observe the red blood cell content in the cell pellet. If there are a lot of red blood cells, add 2 mL of red blood cell lysis buffer, lyse at room temperature for 5 minutes, then add PBS to resuspend, and then centrifuge; if there are not many red blood cells, there is no need to add red blood cell lysis buffer; add the organoid culture medium in step S1 to resuspend the cell pellet, then mix it with matrix gel, and then add it to the cell culture plate, place it in a carbon dioxide incubator at 37°C for 30 minutes; Step S4, the cell precipitate without red blood cells collected in step S3 is mixed with culture medium and matrix gel (which can be a product of Model Biotechnology; item number: 082755) and added dropwise to the circulation chip (the circulation chip can be: the circulation chip is a transparent rectangular structure with a length of 85.5 mm and a width of 37.5 mm as a whole, and has a chamber with a length of 15 mm, a width of 15 mm, and a height of 6 mm inside, and the chamber is used to circulate the culture medium through microfluidic channels with a diameter of 1 mm at both ends). The circulation chip is a transparent rectangular structure with a length of 85.5 mm and a width of 37.5 mm as a whole, and has a chamber with a length of 15 mm, a width of 15 mm, and a height of 6 mm inside, and the chamber is used to circulate the culture medium through microfluidic channels with a diameter of 1 mm at both ends. Then put the chip into a cell culture incubator at 37° C. in a carbon dioxide incubator for 30 minutes; Step S5: After the standing is completed, the culture medium obtained in Example 1 is added into the culture medium flow channel at a flow rate of 10 μL / min, and the medium is returned to the carbon dioxide incubator for cyclic culture. Figure 3 The liver cancer organoids and colorectal cancer organoids shown and Figure 4 Liver cancer organoids shown.

[0045] Example 4

[0046] The culture method of this embodiment comprises the following steps: Step S1: Collect the cultured tumor organoids and transfer 2×10 4 The cell spheres were evenly mixed in 50 μL of culture medium containing 60% Matrigel, and then the Matrigel was dropped into the center of the cavity of the dynamic chip. The chip was then placed in a cell culture incubator at 37°C for 30 min to allow the Matrigel to gel.

[0047] Step S2: After the matrix gel is formed, the chip is taken out and fixed in the corresponding position of the culture chamber. The two ends of the culture chamber are connected to the syringe filled with tumor organoid culture medium through a hose. The syringe is fixed on a peristaltic pump. Two peristaltic pumps are set to extract and perfuse respectively. The culture medium is perfused into the chip at a flow rate of 10 μL / min. The entire culture chamber is placed in a 37°C cell culture incubator for cyclic culture. The tumor organoid culture medium is replaced every 3 days.

[0048] In this dynamic circulation culture device, the delivery of nutrients and growth factors can be more effectively regulated to ensure the normal growth of cells and improve cell survival rate. The entire device is the size of a glass slide, which saves chip loading space and achieves the advantage of imaging. Compared with other complex culture systems, our device may be easier to operate and reduce the technical threshold for users.

[0049] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0050] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A system suitable for organ / organoid circulatory culture, comprising a chip having a chamber and a microfluidic channel connecting the chambers to simulate a circulatory system, wherein during the culture process, the chamber contains a matrix gel with sample cells and the microfluidic channel is used as the chamber to provide an organ culture medium, wherein the organ culture medium comprises: The basic culture medium used for multiple cancer types includes amino acids, vitamins, inorganic salts, proteins and trace elements; In order to adapt to the culture of different types of cancer cells, the following are also added, all in final concentration: For lung cancer sample culture, the following were added: 100ng / mL fibroblast growth factor 10, 250ng / mL Noggin, 50ng / mL fibroblast growth factor 2, 30nM SB202190, 100ng / mL Rspondin-1, 1X B27, 3μM CHIR99021; For gastric cancer sample culture, add: 50ng / mL epidermal growth factor, 5nM Gastrin, 100ng / mL Rspondin-1, 250ng / mL Noggin, 1X B27, 20nM A83-01, 50μg / mL Primocin; For intestinal cancer sample culture, add: 100ng / mL Rspondin-1, 250ng / mL Noggin, 1X B27, 20nM A83-01, 50μg / mL Primocin, 30nM SB202190; For liver cancer sample culture, add: 300ng / mL Rspondin-1, 500ng / mL Noggin, 3X B27, 10nM A83-01, 50μg / mL Primocin, 3nM SB202190, 100ng / mL Hepatocyte Growth Factor.

2. The system suitable for organ / organoid cyclic culture according to claim 1, characterized in that: The amino acids are selected from glycine, L-alanine, L-alanine-glutamine, L-arginine hydrochloride, L-aspartic acid, L-aspartic acid, L-cysteine ​​dihydrochloride, L-glutamic acid, L-isoleucine, L-leucine, L-methionine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt dihydrate, L-valine, L-cysteine ​​hydrochloride, L-histidine hydrochloride, L-asparagine-hydrate, L-lysine hydrochloride, and L-phenylalanine.

3. The system suitable for organ / organoid cyclic culture according to claim 1, characterized in that: The vitamins are selected from vitamin C phosphate, choline chloride, D-calcium pantothenate, folic acid, niacinamide, vitamin B6 hydrochloride, riboflavin, vitamin B1 hydrochloride, and inositol.

4. The system suitable for organ / organoid cyclic culture according to claim 1, characterized in that: The inorganic salt is selected from calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, and disodium hydrogen phosphate.

5. The system suitable for organ / organoid cyclic culture according to claim 1, characterized in that: The protein is selected from the group consisting of lipid-rich BSA, transferrin, and recombinant human insulin.

6. The system suitable for organ / organoid cyclic culture according to claim 1, characterized in that: The trace elements are selected from ammonium metavanadate, copper sulfate, manganese chloride and sodium selenite.

7. The system suitable for organ / organoid cyclic culture according to any one of claims 1 to 6, characterized in that: The basal medium further comprises other components selected from anhydrous glucose, ethanolamine, reduced glutathione, phenol red, sodium pyruvate, linoleic acid, lipoic acid, hypoxanthine, putrescine dihydrochloride, thymidine, and 4-hydroxyethylpiperazineethanesulfonic acid.

8. A method for using the system suitable for organ / organoid circulatory culture according to any one of claims 1 to 7, comprising: Prepare samples and prepare basal culture medium matching the cancer type; The sample is washed, tissue digested, cell precipitated, and matrix gel solidified, and the solidified sample and organ culture medium are added to the microfluidic system for culture.

9. The method according to claim 8, characterized in that The culture is a cyclic culture based on a microfluidic chip.

10. Organoids, comprising at least organoid tissues cultured by the system according to any one of claims 1 to 7.

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