A rectal tumor organoid chip drug sensitivity detection kit and its use method

Abstract

The present invention relates to a drug sensitivity detection kit for a rectal tumor organoid chip and a method for using the kit, wherein the kit comprises: a microporous lattice organoid chip, a 200ml bottle of organoid culture medium, a 20ml bottle of tissue digestion enzyme, and a 50ml bottle of inoculation liquid; the method for using the kit is to cut up a freshly obtained tumor sample, add tissue digestion enzyme to break up the tissue into single cells; add inoculation liquid to make a single cell suspension; inoculate the single cell suspension on the chip through the inoculation hole of the organoid chip; absorb the inoculation liquid, add the organoid culture medium for culturing; after the organoid is formed by culturing, press the drug sensitivity plate to allow the drug to act on the organoid; read the fluorescent signal and perform data analysis. The beneficial effect of the present invention is that a kit for rectal tumor organoids that can be constructed in a short time based on puncture samples to meet drug sensitivity detection is provided.

Description

Technical Field

[0001] The present invention relates to the technical field of drug sensitivity detection of primary rectal tumor cells, and in particular to a rectal tumor organoid chip drug sensitivity detection kit and a method of using the same. Background Art

[0002] The purpose of primary tumor cell drug sensitivity testing is mainly to detect under what circumstances primary cancer cells respond to treatment and provide information about whether the tumor is likely to spread and where it may spread. This drug sensitivity test can effectively detect and determine which drugs, therapies or nutritional supplements patients respond best to. In order to provide targeted drug treatment to patients in a timely manner, it is necessary to use as few lesion tumor tissue samples as possible for organoid culture, so that organoids that meet the requirements of drug sensitivity testing can be quickly constructed in a shorter time. However, the known primary rectal tumor cell drug sensitivity test kits have large lesion tumor tissue sample size and long test time. It is necessary to invent a kit based on puncture samples (less than 0.5g) that can be constructed in a shorter time (no more than 3 days) to meet the requirements of primary rectal tumor cell drug sensitivity testing. Summary of the invention

[0003] To achieve the above purpose, the present invention provides a rectal tumor organoid chip drug sensitivity detection kit, the specific technical solution is:

[0004] Each kit includes 1 microwell array organoid chip, 1 bottle of 200 ml organoid culture medium, 1 bottle of 20 ml tissue digestion enzyme and 1 bottle of 50 ml inoculation solution;

[0005] The 200 ml organoid culture medium is made of the following raw materials: penicillin-streptomycin, final concentration of 50-150×; B27 serum replacement, final concentration of 70-150×; glutamine I, final concentration of 1-4 mM; gastrin I, final concentration of 8-15 nM; Wnt3a, final concentration of 50-150 ng / ml; R-spondin, final concentration of 50-150 ng / ml; noggin, final concentration of 70-150 ng / ml; epidermal growth factor EGF, final concentration of 30-80 ng / mL; A83-01, final concentration of 400-600 nM; N-acetylcysteine, final concentration of 1-3 mM; nicotinamide, final concentration of 8-15 mM; HEPES, final concentration of 8-15 mM; SB 202190, final concentration of 8-15 μM and Advanced DMEM / F12 buffer basal medium is added to 200 ml;

[0006] The 20 ml tissue digestion enzyme is made of the following raw materials: collagenase, final concentration is 0.1-0.5 mg / mL; dispase, final concentration is 0.1-0.5 mg / mL; DNase I, final concentration is 0.1-0.5 mg / mL; penicillin-streptomycin, final concentration is 50-150×; HEPES, final concentration is 8-15 mM; Y27632, final concentration is 8-15 uM and Advanced DMEM / F12 buffer basal culture medium is supplemented to 20 ml;

[0007] The 50 ml inoculation solution is made of the following raw materials: Y27632, with a final concentration of 8 to 15 uM; albumin, with a final concentration of 1 to 3 g / L and PBS to make up to 50 ml.

[0008] As a further improvement, the 200 ml organoid culture medium is made of the following raw materials: penicillin-streptomycin, final concentration of 55-65×; B27 serum replacement, final concentration of 70-75×; glutamine I, final concentration of 1-2 mM; gastrin I, final concentration of 8-10 nM; Wnt3a, final concentration of 80-85 ng / ml; R-spondin, final concentration of 55-65 ng / ml; noggin, final concentration of 75-85 ng / ml; epidermal growth factor EGF, final concentration of 30-40 ng / mL; A83-01, final concentration of 400-450 nM; N-acetylcysteine, final concentration of 1-2 mM; nicotinamide, final concentration of 8-9 mM; HEPES, final concentration of 8-9 mM; SB 202190, final concentration of 9-11 μM and Advanced DMEM / F12 buffer basal medium is added to 200 ml;

[0009] The 20 ml tissue digestion enzyme is made of the following raw materials: collagenase, final concentration is 0.1-0.15 mg / mL; dispase, final concentration is 0.1-0.15 mg / mL; DNase I, final concentration is 0.1-0.5 mg / mL; penicillin-streptomycin, final concentration is 50-55×; HEPES, final concentration is 8-9 mM; Y27632, final concentration is 8-9 uM and Advanced DMEM / F12 buffer basal culture medium is supplemented to 20 ml;

[0010] The 50 ml inoculation solution is made of the following raw materials: Y27632, with a final concentration of 8 to 9 uM; albumin, with a final concentration of 1 to 1.5 g / L and PBS to make up to 50 ml.

[0011] As a further improvement, the 200 ml organoid culture medium is made of the following raw materials: penicillin-streptomycin, final concentration of 95-105×; B27 serum replacement, final concentration of 100-105×; glutamine I, final concentration of 1.5-2.5 mM; gastrin I, final concentration of 10-11 nM; Wnt3a, final concentration of 95-105 ng / ml; R-spondin, final concentration of 95-105 ng / ml; noggin, final concentration of 95-105 ng / ml; epidermal growth factor EGF, final concentration of 45-55 ng / mL; A83-01, final concentration of 480-520 nM; N-acetylcysteine, final concentration of 1-1.5 mM; niacinamide, final concentration of 10-11 mM; HEPES, final concentration of 10-11 mM; SB 202190, final concentration 10-11 μM and Advanced DMEM / F12 buffer basal medium made up to 200 ml;

[0012] The 20 ml tissue digestion enzyme is made of the following raw materials: collagenase, final concentration is 0.15-0.25 mg / mL; dispase, final concentration is 0.1-0.15 mg / mL; DNase I, final concentration is 0.1-0.15 mg / mL; penicillin-streptomycin, final concentration is 95-100×; HEPES, final concentration is 9-10 mM; Y27632, final concentration is 9-10 uM and Advanced DMEM / F12 buffer basal culture medium is supplemented to 20 ml;

[0013] The 50 ml inoculation solution is made of the following raw materials: Y27632, with a final concentration of 9-10 uM; albumin, with a final concentration of 1.5-2 g / L and PBS to make up to 50 ml.

[0014] As a further improvement, the 200 ml organoid culture medium is made of the following raw materials: penicillin-streptomycin, final concentration of 115-125×; B27 serum replacement, final concentration of 115-125×; glutamine I, final concentration of 3.5-4 mM; gastrin I, final concentration of 12-14 nM; Wnt3a, final concentration of 115-120 ng / ml; R-spondin, final concentration of 115-125 ng / ml; noggin, final concentration of 125-135 ng / ml; epidermal growth factor EGF, final concentration of 65-75 ng / mL; A83-01, final concentration of 550-600 nM; N-acetylcysteine, final concentration of 2.5-3 mM; niacinamide, final concentration of 12-13 mM; HEPES, final concentration of 13-14 mM; SB 202190, final concentration 12-13 μM and Advanced DMEM / F12 buffer basal medium made up to 200 ml;

[0015] The 20 ml tissue digestion enzyme is prepared from the following raw materials: collagenase, final concentration is 0.3-0.4 mg / mL; dispase, final concentration is 0.3-0.4 mg / mL; DNase I, final concentration is 0.3-0.4 mg / mL; penicillin-streptomycin, final concentration is 125-135×; HEPES, final concentration is 12-13 mM; Y27632, final concentration is 13-14 uM and Advanced DMEM / F12 buffer basal culture medium is supplemented to 20 ml;

[0016] The 50 ml inoculation solution is made of the following raw materials: Y27632, with a final concentration of 12-13 uM; albumin, with a final concentration of 2-2.5 g / L and PBS to make up to 50 ml.

[0017] As a further improvement, the specifications of the microwell array organoid chip include 5×5, 5×10, 10×5 or 10×10.

[0018] As a further improvement, the invention also includes a bottle of 20 ml enzymatic hydrolysate, wherein the 20 ml enzymatic hydrolysate is made of the following raw materials: Y27632, with a final concentration of 9 to 11 uM; Trypsin, with a final concentration of 0.1 to 0.2 mg / mL; EDTA, with a final concentration of 0.01 to 0.03% and Advanced DMEM / F12 buffer basal culture medium supplemented to 20 ml.

[0019] A method for using the above-mentioned rectal tumor organoid chip drug sensitivity detection kit, each box of the kit comprises the following steps when used:

[0020] S1: rectal tumor tissue was obtained to prepare rectal tumor single cells based on the 1 bottle of 20 ml tissue digestion enzyme;

[0021] S2: preparing a rectal tumor single cell suspension based on the 50 ml inoculation solution in the bottle, and inoculating the rectal tumor single cell suspension on the microporous lattice organoid chip;

[0022] S3: Continue to add the 200 ml organoid culture medium to the microwell array organoid chip to prepare rectal tumor organoids;

[0023] S4: The rectal tumor organoids are made into rectal tumor organoid single cells based on the 1 bottle of 20 ml enzymatic hydrolysate, and the rectal tumor organoid single cells are used for drug sensitivity detection.

[0024] As a further improvement, in step S1: the rectal tumor tissue is 0.2-0.3 g.

[0025] As a further improvement, in step S2: the amount of the inoculation solution is 5 to 10 ml.

[0026] As a further improvement, in step S3: the specifications of the microwell array organoid chip include 5×5 or 5×10.

[0027] The above technical solution of the present invention has the following beneficial effects: The present invention provides a kit for constructing rectal tumor organoids for drug sensitivity detection based on a puncture sample of less than 0.5 g in a short time of 3 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is an exploded view of the structure of a culture chip based on open chamber patterned array microfluidics as exemplified in Chinese patent CN118440822A;

[0029] Figure 2 The structural diagram and partial enlarged schematic diagram of the culture chip based on open chamber patterned array microfluidics exemplified in Chinese patent CN118440822A;

[0030] Figure 3 This is a diagram showing the effect of the organoids of Example 4 of the present invention after being cultured for one day;

[0031] Figure 4 This is a diagram showing the effect of the organoids of Example 4 of the present invention after 3 days of culture;

[0032] Figure 5 This is a rendering of the uniform size of the devices formed in Example 4 of the present invention at three days;

[0033] Figure 6 This is a diagram showing the effect of the organoids of Example 4 of the present invention after being cultured for 5 days.

[0034] exist Figure 1 and Figure 2 In the figure, the same reference numerals are used to represent the same elements or structures, wherein: 1-inlet, 2-outlet, 31-first flow channel, 32-second flow channel, 41-first flow rate stabilization chamber, 42-second flow rate stabilization chamber, 5-open channel, 6-culture fluid storage chamber, 7-hole array layer, 8-patterned array;

[0035] exist Figure 3 to Figure 6 In the figure, the magnification is 10X. DETAILED DESCRIPTION

[0036] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0037] Reagents and sources:

[0038] Penicillin-streptomycin (100×, Shanghai Yuanpei Biotechnology Co., Ltd.); PBS (phosphate buffered saline, Wuhan Sevier Biotechnology Co., Ltd.); trypan blue staining solution (Wuhan Pronosai Life Science Co., Ltd.); fresh rectal tumor tissue (cooperated with a tertiary hospital, and the cooperation has passed the formal medical ethics review. The attending physician selected the patients for inclusion in the group according to the eighth edition of the TNM staging of malignant tumors published by the International Union Against Cancer (UICC), and selected appropriate puncture samples for in vitro culture according to the intraoperative clinical indications); advanced DMEM / F12 (Gibco™, Dulbecco's modified Eagle medium / Ham's F-12); DNase I (deoxyribonuclease I); HEPES (4-hydroxyethylpiperazineethanesulfonic acid); Y27632 (trans-4-[(R)-1-aminoethyl]-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride); B27 supplement (Gibco™ B-27™ supplement, Thermo Fisher); GlutaMAX supplement (Gibco™ GlutaMAX™ Supplement, L-alanyl-L-glutamine dipeptide, Glutamine I, Thermo Fisher); gastrin I; Wnt-3A (Wnt3a protein, Human (His), P56704-1 (S19-C351), MCE); FGF10 (fibroblast growth factor 10); bFGF (basic fibroblast growth factor); R-Spondin 1 (R-spondin 1, Recombinant R-Spondin 1 (RSPO1), Shanghai Kanglang Biotechnology Co., Ltd.); Noggin protein (MCE); EGF (epidermal growth factor); A83–01 (3-(6-methyl-2-pyridyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-thiocarboxamide); N-acetylcysteine; nicotinamide; SB 202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridinyl)-1H-imidazole); Trypsin; Ethylenediaminetetraacetic acid (EDTA).

[0039] In some embodiments, a rectal tumor organoid chip drug sensitivity detection kit is provided, wherein the kit is used for enzymatic digestion of rectal tumor tissue to obtain single cell suspension, cell inoculation organoid chip, organoid culture, drug treatment, and drug sensitivity detection reagent, specifically including 1 microporous dot array organoid chip (5×5 or 5×10), 1 bottle of organoid culture medium (200 ml), 1 bottle of tissue digestion enzyme (20 ml), 1 bottle of enzymatic hydrolysis solution (20 ml), 1 bottle of inoculation solution (50 ml), and 1 drug concentration gradient generation plate. The specific structure of the microporous dot array organoid chip in the present application is not limited, and it can contain multiple specifications, including but not limited to 5×5, 5×10, 10×5, or 10×10 microporous dot array organoid chips, which can perform drug sensitivity detection of 5 schemes and 5 concentrations, 5 schemes and 10 concentrations, 10 schemes and 5 concentrations, and 10 schemes and 10 concentrations, respectively.

[0040] In some embodiments, the microwell array organoid chip of the present invention preferably adopts: Chinese patent CN118440822 A; Patent name: A culture chip based on open chamber patterned array microfluidics, preferably, combined with Figure 1 and Figure 2 As shown, the specific structure of the microporous lattice organoid chip is as follows: recorded in the embodiment section [0040-0051] of the specification, and the method of use can be found in section

[0053] of the specification. Specifically: The microporous lattice organoid chip: comprises an open chamber cover plate and an array channel layer stacked in sequence from top to bottom.

[0041] Flow channel: the array channel layer, which includes: an open channel area 5 composed of multiple flow channels arranged side by side and a culture liquid storage chamber 6 connected to the outlet of the open channel area 5;

[0042] Seeding holes: are the hole array layer 7 and the pattern array 8 bonded in sequence below the open channel area 5; wherein, the hole row in the hole array layer 7 and the pattern row in the pattern array 8 correspond directly below the flow channel, and the holes in the hole array layer 8 and the patterns of the pattern array 8 correspond one to one to form an isolated chamber array;

[0043] Inoculation tank: It is a culture solution storage chamber 6. The middle part of the open chamber cover has a hollow opening. Both ends of the open chamber cover have an inlet 1 connected to the open channel area 5 and an outlet 2 connected to the culture solution storage chamber 6. The array channel layer also includes a second shunt channel 32 at the entrance of the outlet 2, which is used to shunt and transport the liquid in the culture solution storage chamber 6 to the outlet 2 through the second shunt channel 32 so that the fluid is evenly converged to avoid overall imbalance caused by local poor circulation during the flow process. The second flow rate stabilization chamber 42 is a channel with a reduced spatial height along the outlet direction of the culture solution storage chamber 6, such as Figure 2As shown, a height baffle is provided near the second flow rate stabilizing chamber 42 , so that the culture fluid in the culture fluid storage chamber 6 flows out slowly and steadily through the second flow rate stabilizing chamber 42 ; wherein the hollow opening is the same size as the open channel area 5 .

[0044] When in use, the culture suspension is directly added from the injection port 1, and the culture enters the open channel area 5 evenly under the action of the first diversion channel 31 and the first stabilization chamber 41 including but not limited to the overflow, but does not reach the culture fluid storage chamber 6. Then, the culture suspension is refluxed from the injection port 1, and the culture is evenly divided under the action of the hole array layer 7. After settling in the open channel area 5, the culture enters the isolation chamber 8 to form a cell sphere.

[0045] The microporous dot matrix organoid chip achieves highly uniform and rapid inoculation of tumor organoid culture, forming tumor organoids of uniform size; when used in conjunction with a drug concentration gradient generation plate, it enables rapid dosing of tumor treatment drugs; and together with the self-developed high-throughput, automated drug detection all-in-one machine, it enables perfusion culture of tumor organoids and automatic drug sensitivity data collection and analysis.

[0046] In some embodiments, the drug sensitivity detection device in the present invention preferably adopts: Chinese patent CN 118956573 A; Patent name: An integrated and modular drug concentration gradient generation and drug delivery device.

[0047] Tissue digestion enzymes are used to break up tumor tissue and release rectal tumor cells to form a single cell suspension. Freshly obtained rectal tumor tissue is chopped into a paste, and 2-10 times the volume of the sample is added with tissue digestion enzymes. Incubate at 37°C for 20-60 minutes; gently blow 5-10 times to disperse the cells, filter through a filter, and collect the cells by centrifugation for later use.

[0048] The enzymatic hydrolysate is used to perform drug sensitivity testing on cultured organoids. Organoids are broken up into single cells by enzymatic hydrolysis, centrifuged to remove the supernatant, and the cell pellet is used for later use.

[0049] The inoculation solution is used to prepare a rectal tumor cell suspension, which is inoculated onto a microporous lattice organoid chip. The above-mentioned tumor single cell precipitation from tumor tissue or organoid is mixed with the inoculation solution to prepare a single cell suspension.

[0050] Using the tissue digestion solution provided in this application to enzymatically hydrolyze tumor tissue can improve the acquisition rate and activity of rectal tumor cells, thereby greatly improving the formation rate of organoids, reducing the amount of tumor tissue used, and increasing the applicable sample types of the kit. For example, a small amount of tumor tissue obtained by puncture, approximately 0.23 g, can also complete the culture and drug sensitivity testing of organoids.

[0051] The organoid culture medium is used for organoid culture on an organoid chip. The organoid culture medium of the present application can also be used for solid tumor organoid culture including but not limited to hepatocellular carcinoma organoid culture, bile duct carcinoma organoid culture, colorectal tumor organoid culture medium, gastric cancer organoid culture medium, and pancreatic cancer organoid culture medium.

[0052] In some embodiments, the method of using the rectal tumor organoid chip drug sensitivity detection kit includes: 0.20-0.50g of freshly obtained rectal tumor tissue is chopped with scissors, and 2-10 times the sample volume of tissue digestion enzyme is added, and the mixture is placed in a 37°C, 5% CO2 incubator for incubation and digestion for 20-60 minutes. After the incubation, the loose tissue is gently blown 5-10 times with a pipette to further disintegrate the loose tissue and free the cells therein. The cells are collected by centrifugation, and the parameters are 200-300g centrifugation for 3-5 minutes. The supernatant is removed, 5-10 ml of inoculation solution is added, and the cells are gently blown 2-4 times to resuspend the cells. The cell suspension is inoculated onto the microporous lattice organoid chip, and placed in a 37°C, 5% CO2 incubator for 5-10 minutes to allow the cells to fall evenly into the micropores of the microporous lattice organoid chip. Gently aspirate the inoculation solution outside the microwells of the organoid chip, add 1mL-1.5mL (5×5 microwell dot matrix organoid chip) of organoid culture medium, overlay the drug concentration gradient generation plate, cover it, and place it in a 37°C, 5% CO2 incubator or an automated drug sensitivity detection machine for 1 to 5 days. After the culture is completed, the organoid is well formed. Press the drug concentration gradient generation plate manually or operate the automated drug sensitivity detection machine to allow the drugs to be tested and the living cell fluorescent dye to quickly enter the microwells with organoids. Return the organoid chip to the incubator or automated drug sensitivity detection machine for incubation and culture for 2 to 3 days. After the incubation is completed, collect the fluorescence signal, analyze the drug sensitivity data, establish the concentration-cell activity fitting curve, and generate the IC50 value or inhibition rate.

[0053] There are two ways to inoculate the cell suspension onto the organoid chip: manual inoculation or automatic inoculation. When inoculating manually, drop the cell suspension in the channel as evenly as possible, and gently shake the organoid chip plate to evenly disperse the cells into the microwells of the chip. When inoculating automatically, first add the cells into the inoculation tank through the inoculation hole at one end of the organoid chip, then connect to the perfusion culture system, turn on the liquid circulation, and set the parameters to evenly disperse the cells in the inoculation tank into each microwell.

[0054] The above-mentioned manual or automated drug sensitivity detection integrated machine presses the drug concentration gradient generation plate in the following manner: manually pressing the drug concentration gradient generation plate requires first removing the cover of the organoid chip in the biosafety cabinet, and then lightly pressing the drug concentration gradient generation plate to make it contact with the culture liquid surface to release the drug.

[0055] The above-mentioned drug concentration gradient generation plate is used for drug loading and fluorescent dye loading during drug treatment. The drug concentration gradient generation plate is pre-fabricated with the drugs to be tested and the fluorescent dyes for detecting active cells. When performing drug sensitivity testing, the drug concentration gradient generation plate is pressed down, and the pre-fabricated drugs at the corresponding sites contact the culture wells at the corresponding sites of the organoid chip plate. The drugs and fluorescent dyes enter the wells and penetrate into the organoids to act on rectal tumor cells. Fluorescent staining marks living cells; the relevant fluorescent signals are captured by the detection machine.

[0056] The kit provided by the present invention is convenient and quick. The kit contains all reagents for quickly constructing highly uniform tumor organoids and accurately completing drug sensitivity testing without the need to prepare additional reagents. Moreover, these reagents and culture devices have been repeatedly optimized and can form better organoids in a shorter time.

[0057] The organoid culture medium provided by the present invention is a reagent that has been repeatedly optimized and is fully compatible with the other components of the entire kit. The culture medium is suitable for different tumor subtypes and gene mutations. When used in combination with the organoid chip, tissue enzymatic solution, and drug concentration gradient generation plate in the kit, the formation efficiency and speed of the organoid can be significantly improved, and the speed of drug sensitivity detection can be accelerated.

[0058] The inoculation solution provided by the present invention can significantly improve the efficiency and viability of cell inoculation. When used in combination with the organoid chip, culture medium, drug concentration gradient generation plate, etc. in the kit, it can effectively improve the efficiency of cell inoculation, increase the organoid formation rate, and accelerate the detection speed of drug sensitivity.

[0059] This kit greatly simplifies the steps of organoid drug sensitivity testing, improves the speed and accuracy of drug sensitivity testing, and makes the originally complex and technically demanding organoid detection technology easy to operate. Ordinary technicians can complete the entire operation after simple training.

[0060] The convenience of this kit also enables organoid drug sensitivity testing technology to be widely carried out in scientific research institutes, hospital medical technology departments, medical testing institutes, etc. Example 1

[0061] The rectal tumor organoid chip drug sensitivity detection kit includes the following components. The components and dosages are shown in Table 1:

[0062] Table 1: Components of the kit and their final concentrations

[0063]

[0064] The above kit was used to culture rectal tumor organoids. The culture method was to culture 0.42 g (equivalent to 1 mm 3About 100 μl), cut the rectal tumor tissue into pieces with scissors, add 5 times the volume of the rectal tumor tissue to the tissue digestion enzyme, and place it in a 37°C, 5% CO2 incubator for 30 minutes to form rectal tumor single cells. After the incubation, gently blow with a pipette to further disintegrate the loose tissue and free the cells. Collect the cells by centrifugation, the parameter is 200g centrifugation for 3 minutes. Remove the supernatant, add 7 ml of inoculation solution to the rectal tumor single cell to form a rectal tumor single cell suspension, and gently blow the rectal tumor single cell suspension. Inoculate the rectal tumor single cell suspension onto the microporous lattice organoid chip and place it in a 37°C, 5% CO2 incubator for 5 minutes to allow the cells to fall evenly into the micropores of the microporous lattice organoid chip. Gently aspirate the inoculum outside the microwells of the microwell array organoid chip, add 1 ml of organoid culture medium to the microwells of the microwell array organoid chip, and place it in a 37°C, 5% CO2 incubator or an automated drug sensitivity detection machine for 3 days. After the culture is completed, the organoid is formed.

[0065] Example 2

[0066] The same organoid culture method as in Example 1 was used, except that 0.43 g of rectal tumor tissue was taken. The kit includes the following components, and the components and amounts are shown in Table 2:

[0067] Table 2: Components and dosage of the kit

[0068]

[0069] Example 3

[0070] The same organoid culture method as in Example 1 was used, except that 0.41 g of rectal tumor tissue was collected. The kit includes the following components, and the components and amounts are shown in Table 3:

[0071] Table 3: Components and dosage of the kit

[0072]

[0073] Example 4

[0074] The kit in Example 2 was used, except for the specific culture method: 0.23 g of rectal tumor tissue was taken, and tissue digestion enzymes were added in an amount 3 times the volume of the tumor tissue; 5 ml of inoculation solution was added; and 1.2 ml of organoid culture medium was added.

[0075] Example 5

[0076] The kit in Example 2 was used, except that the specific method of use was as follows: 0.45 g of rectal tumor tissue was taken, and tissue digestion enzymes were added in an amount 10 times the volume of the tumor tissue; 10 ml of inoculation solution was added; and 1.5 ml of organoid culture medium was added.

[0077] Example 6

[0078] The same rectal tumor organoid culture method as in Example 1 was used, except that the obtained rectal tumor organoid was broken into single cells by the enzymatic hydrolysis solution, the supernatant was removed by centrifugation, the cell sediment was set aside, the drug concentration gradient generation plate was pressed to allow the drug to be detected and the living cell fluorescent dye to quickly enter the microwells where the organoids grew, and the incubation and culture were continued for 2 days. After the incubation, the fluorescent signal was collected. The kit used in this method includes the following components, and the components and amounts are shown in Table 4:

[0079] Table 4: Components and dosage of the kit

[0080]

[0081] Comparative Example 1

[0082] The same rectal tumor organoid culture method as in Example 1 was used, except that the kit used comprised the following components, the components and amounts are shown in Table 5, and the unshown parts are the same as in Example 1;

[0083] Table 5: Components and dosage of the kit

[0084]

[0085] Comparative Example 2

[0086] The same rectal tumor organoid culture method as in Example 1 was used, except that the kit used included the following components, the components and amounts are shown in Table 6, and the unshown parts are the same as in Example 1;

[0087] Table 6: Kit components and final concentrations

[0088]

[0089] Comparative Example 3

[0090] The same rectal tumor organoid culture method as in Example 1 was used, except that the kit used comprised the following components, and the components and amounts are shown in Table 7;

[0091] Table 7: Components and dosage of the kit

[0092]

[0093] The present invention is further described below through test examples.

[0094] Experimental Example 1 Acquisition and Activity Test of Rectal Tumor Cells

[0095] Samples: The tissue digestion enzymes of Examples 1-5 were used as Test Examples 1-5, respectively; the tissue digestion enzymes of Comparative Example 1 were used as Test Example 6.

[0096] Experimental method: Fresh rectal tumor tissue was washed with PBS containing antibiotics, blood stains and non-tumor tissue were removed, and then weighed. 3 Size, add sample 1-6 tissue digestion enzymes respectively, incubate at 37℃ for 20 minutes. After digestion, add 8 volumes of PBS to dilute the digestion solution, and gently blow with a pipette to completely disperse the rectal tumor tissue and release the cells. Filter with a strainer to remove undigested impurities. Then centrifuge at a centrifugal force of 300g for 5 minutes and remove the supernatant. Add 10mL of PBS to resuspend the cells to obtain a cell suspension. Take 200ul of the cell suspension and stain it with trypan blue dye. Count the number of dead and live cells, calculate the rectal tumor cell acquisition rate and rectal tumor cell activity, and the test results are detailed in Table 8.

[0097] Rectal tumor cell acquisition rate (cells / gram) = total number of rectal tumor cells (cells) / tumor tissue (gram)

[0098] Rectal tumor cell activity (%) = number of live rectal tumor cells (cells) / total number of rectal tumor cells (cells) × 100%

[0099] Table 8: Acquisition and activity test results of rectal tumor cells

[0100]

[0101] Analysis of test results: The tissue digestion enzymes of Examples 1-5 and Comparative Example 1 were used to perform cell acquisition and activity tests. The results showed that the acquisition rate of rectal tumor cells in Examples 1-5 was significantly higher than that in Example 6, and the activity of rectal tumor cells also reached more than 85%, especially the activity of rectal tumor cells in Example 2 reached more than 90%, so that the formation rate of organoids can be greatly improved in the subsequent organoid culture process. In Example 6, the dosage of each component of the tissue digestion enzyme was changed, and the results showed that the acquisition rate and activity of rectal tumor cells were significantly reduced, which can explain that the components and dosage of the tissue digestion enzyme can affect the acquisition rate and activity of rectal tumor cells.

[0102] Experimental Example 2 Inoculation test of rectal tumor cells

[0103] Vaccinator (Model: SH-2090A, Manufacturer: Beijing Jinsangte Medical Instrument Co., Ltd.)

[0104] Incubator (RKJ3DOMI-01-03, manufacturer: Nanjing Ruikangjian Biomedical Technology Co., Ltd.)

[0105] Test samples: using the cell inoculation solution with the components and dosages in Examples 1-5 as Test Examples 7-11, using the cell inoculation solution with the components and dosages in Comparative Example 2 as Test Example 12, trypan blue dye, microwell lattice organoid chip (5×5);

[0106] Experimental method: Take the number of rectal tumor single cells in Table 9 and resuspend them in the inoculation solution to form a rectal tumor single cell suspension. Evenly drip the cell suspension into the entire flow channel or add it to the inoculation tank through the inoculation hole (5×5 microwell dot matrix organoid chip, 5 flow channels, 5 inoculation holes in each flow channel), and inoculate 1mL in each flow channel. After inoculation, place it in an incubator and let it stand for 30 minutes. Aspirate the flow channel inoculation solution, add trypan blue dye, and count the total number of cells and the number of live cells in each small hole under a microscope. Calculate the cell inoculation efficiency and cell inoculation viability.

[0107] Cell seeding efficiency (%) = average number of cells per well × number of wells (cells) / total number of cells seeded (cells)

[0108] Cell seeding viability (%) = number of live cells per well / total number of cells per well × 100%

[0109] The test results are shown in Table 9:

[0110] Table 9: Results of rectal tumor cell inoculation test (5×5 chip)

[0111]

[0112] Analysis of test results:

[0113] The inoculation solutions of Examples 1-5 and Comparative Example 2 were respectively used to carry out inoculation tests of rectal tumor cells. From the comparison of the results, it can be seen that the cell inoculation efficiency of Examples 7-11 is significantly higher than that of Example 12, and the cell inoculation viability is more than 90%, which can greatly improve the formation rate of organoids in the subsequent organoid culture process. In Example 12, the dosage of each component of the inoculation solution was changed, indicating that the cell inoculation efficiency and viability were reduced, which can explain that the composition and dosage of the inoculation solution can better affect the cell inoculation efficiency and cell inoculation viability.

[0114] Experimental Example 3 Organoid Culture Experiment

[0115] Experimental method: The organoid culture experiment was carried out using the methods of Examples 1-5 and Comparative Examples 1-3, respectively, and the single tumor cells (freshly isolated or organoid source) were resuspended with the inoculation solution to form a single cell suspension. The cell suspension was evenly dripped into the entire flow channel or added to the inoculation tank through the inoculation hole (5×5 chip plate, 5 flow channels, 5 large inoculation holes in each flow channel), and 1 mL was inoculated in each flow channel. After inoculation, it was placed in an incubator for 30 minutes. The flow channel inoculation solution was aspirated and the organoid culture medium was connected. On the second day, fourth day, and 6-8 days after inoculation, the number of organoid formation holes and the diameter of the organoid were counted, and the organoid formation rate was calculated. The experimental results are shown in Table 10:

[0116] Organoid formation rate (%) = number of organoid-forming wells / total number of wells × 100%

[0117] Table 10: Comparison of organoid culture test results

[0118]

[0119] Analysis of experimental results: The experimental results show that when the method and kit of Examples 1-5 are used for organoid culture, the resulting organoid formation rate reaches more than 95%, the formation days are shorter, and the diameter reaches about 200 μm after 3 days of culture, and the diameter can reach about 220 μm after 5-7 days of culture, with uniform morphology and size. However, Comparative Examples 1-3 changed the components and dosage of the kit on the basis of the embodiments, which ultimately led to not only a decrease in the formation rate of organoids, but also unsatisfactory size and morphology. Therefore, it can be explained that the various components of tissue digestion enzymes, cell inoculation solution and culture medium and their dosage can affect the formation rate, size and uniformity of the final organoids.

[0120] First combine Figure 3 As shown, in Example 4, organoids were formed after 1 day of culture. Figure 4 , Figure 5 As shown, Example 4 only used 0.23 g of rectal tumor tissue, but still after 3 days of culture, the diameter of the organoid was 200 μm ( Figure 4 ), and the shape and size are uniform ( Figure 5 ), which meets the requirements for drug sensitivity testing. Finally, combined Figure 6 As shown, after 5 days of culture, the diameter of Example 4 can reach about 220 μm, so Example 4 is superior to Comparative Examples 1-3 and other examples.

[0121] The above-described embodiments are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A rectal tumor organoid chip drug sensitivity detection kit, characterized in that: Each kit includes 1 microwell array organoid chip, 1 bottle of 200 ml organoid culture medium, 1 bottle of 20 ml tissue digestion enzyme and 1 bottle of 50 ml inoculation solution; The 200 ml organoid culture medium is made of the following raw materials: penicillin-streptomycin, final concentration of 50-150×; B27 serum replacement, final concentration of 70-150×; glutamine I, final concentration of 1-4 mM; gastrin I, final concentration of 8-15 nM; Wnt3a, final concentration of 50-150 ng / ml; R-spondin, final concentration of 50-150 ng / ml; noggin, final concentration of 70~150ng / ml; epidermal growth factor EGF, final concentration is 30~80ng / mL; A83-01, final concentration is 400~600nM; FGF10, final concentration is 80~120ng / mL; bFGF, final concentration is 8~12ng / mL; N-acetylcysteine, final concentration is 1~3mM; nicotinamide, final concentration is 8~15mM; HEPES, final concentration is 8~15mM; SB 202190, final concentration is 8~15μM and Advanced DMEM / F12 buffer basal medium is supplemented to 200ml; The 20 ml tissue digestion enzyme is made of the following raw materials: collagenase, final concentration is 0.1-0.5 mg / mL; dispase, final concentration is 0.1-0.5 mg / mL; DNase I, final concentration is 0.1-0.5 mg / mL; penicillin-streptomycin, final concentration is 50-150×; HEPES, final concentration is 8-15 mM; Y27632, final concentration is 8-15 uM and Advanced DMEM / F12 buffer basal culture medium is supplemented to 20 ml; The 50 ml inoculation solution is made of the following raw materials: Y27632, with a final concentration of 8 to 15 uM; albumin, with a final concentration of 1 to 3 g / L and PBS to make up to 50 ml.

2. The rectal tumor organoid chip drug sensitivity detection kit according to claim 1, characterized in that: The 200 ml organoid culture medium is made of the following raw materials: penicillin-streptomycin, final concentration of 55-65×; B27 serum replacement, final concentration of 70-75×; glutamine I, final concentration of 1-2 mM; gastrin I, final concentration of 8-10 nM; Wnt3a, final concentration of 80-85 ng / ml; R-spondin, final concentration of 55-65 ng / ml; noggin, final concentration of 75-85 ng / ml; epidermal growth factor EGF, final concentration of 30-40 ng / mL; A83-01, final concentration of 400-450 nM; FGF10, final concentration of 85-95 ng / mL; bFGF, final concentration of 8-9 ng / mL; N-acetylcysteine, final concentration of 1-2 mM; nicotinamide, final concentration of 8-9 mM; HEPES, final concentration of 8-9 mM; SB 202190, final concentration 9-11 μM and Advanced DMEM / F12 buffer basal medium made up to 200 ml; The 20 ml tissue digestion enzyme is made of the following raw materials: collagenase, final concentration is 0.1-0.15 mg / mL; dispase, final concentration is 0.1-0.15 mg / mL; DNase I, final concentration is 0.1-0.5 mg / mL; penicillin-streptomycin, final concentration is 50-55×; HEPES, final concentration is 8-9 mM; Y27632, final concentration is 8-9 uM and Advanced DMEM / F12 buffer basal culture medium is supplemented to 20 ml; The 50 ml inoculation solution is made of the following raw materials: Y27632, with a final concentration of 8 to 9 uM; albumin, with a final concentration of 1 to 1.5 g / L and PBS to make up to 50 ml.

3. The rectal tumor organoid chip drug sensitivity detection kit according to claim 1, characterized in that: The 200 ml organoid culture medium is made of the following raw materials: penicillin-streptomycin, final concentration is 95-105×; B27 serum replacement, final concentration is 100-105×; glutamine I, final concentration is 1.5-2.5 mM; gastrin I, final concentration is 10-11 nM; Wnt3a, final concentration is 95-105 ng / ml; R-spondin, final concentration is 95-105 ng / ml; noggin, final concentration 95~105ng / ml; epidermal growth factor EGF, final concentration is 45~55ng / mL; A83-01, final concentration is 480~520nM; FGF10, final concentration is 95~105ng / mL; bFGF, final concentration is 9~10ng / mL; N-acetylcysteine, final concentration is 1~1.5mM; niacinamide, final concentration is 10~11mM; HEPES, final concentration is 10~11mM; SB 202190, final concentration is 10~11μM and Advanced DMEM / F12 buffer basal medium is supplemented to 200ml; The 20 ml tissue digestion enzyme is made of the following raw materials: collagenase, final concentration is 0.15-0.25 mg / mL; dispase, final concentration is 0.1-0.15 mg / mL; DNase I, final concentration is 0.1-0.15 mg / mL; penicillin-streptomycin, final concentration is 95-100×; HEPES, final concentration is 9-10 mM; Y27632, final concentration is 9-10 uM and Advanced DMEM / F12 buffer basal culture medium is supplemented to 20 ml; The 50 ml inoculation solution is made of the following raw materials: Y27632, with a final concentration of 9-10 uM; albumin, with a final concentration of 1.5-2 g / L and PBS to make up to 50 ml.

4. The rectal tumor organoid chip drug sensitivity detection kit according to claim 1, characterized in that: The 200 ml organoid culture medium is made of the following raw materials: penicillin-streptomycin, final concentration of 115-125×; B27 serum replacement, final concentration of 115-125×; glutamine I, final concentration of 3.5-4 mM; gastrin I, final concentration of 12-14 nM; Wnt3a, final concentration of 115-120 ng / ml; R-spondin, final concentration of 115-125 ng / ml; noggin, final concentration of 125~135ng / ml; epidermal growth factor EGF, final concentration is 65~75ng / mL; A83-01, final concentration is 550~600nM; FGF10, final concentration is 105~115ng / mL; bFGF, final concentration is 11~12ng / mL; N-acetylcysteine, final concentration is 2.5~3mM; niacinamide, final concentration is 12~13mM; HEPES, final concentration is 13~14mM; SB 202190, final concentration is 12~13μM and Advanced DMEM / F12 buffer basal medium is supplemented to 200ml; The 20 ml tissue digestion enzyme is prepared from the following raw materials: collagenase, final concentration is 0.3-0.4 mg / mL; dispase, final concentration is 0.3-0.4 mg / mL; DNase I, final concentration is 0.3-0.4 mg / mL; penicillin-streptomycin, final concentration is 125-135×; HEPES, final concentration is 12-13 mM; Y27632, final concentration is 13-14 uM and Advanced DMEM / F12 buffer basal culture medium is supplemented to 20 ml; The 50 ml inoculation solution is made of the following raw materials: Y27632, with a final concentration of 12-13 uM; albumin, with a final concentration of 2-2.5 g / L and PBS to make up to 50 ml.

5. The rectal tumor organoid chip drug sensitivity detection kit according to claim 1, characterized in that: The specifications of the microwell array organoid chip include 5×5, 5×10, 10×5 or 10×10.

6. The rectal tumor organoid chip drug sensitivity detection kit according to claim 1, characterized in that: Also included is a bottle of 20 ml enzymatic hydrolysate, wherein the 20 ml enzymatic hydrolysate is made of the following raw materials: Y27632, with a final concentration of 9 to 11 uM; Trypsin, with a final concentration of 0.1 to 0.2 mg / mL; EDTA, final concentration is 0.01-0.03% and Advanced DMEM / F12 buffer basal medium is made up to 20 ml.

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