CN119410587A - Rectal tumor organoid chip drug sensitivity detection kit and application method thereof - Google Patents
Rectal tumor organoid chip drug sensitivity detection kit and application method thereof Download PDFInfo
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Abstract
本发明涉及一种直肠肿瘤类器官芯片药敏检测试剂盒及其使用方法，其中试剂盒包括：微孔点阵类器官芯片1块、类器官培养基1瓶200ml、组织消化酶1瓶20ml、接种液1瓶50ml；其使用方法将新鲜获取的肿瘤样本剪碎，加入组织消化酶将组织打散成单细胞；加入接种液，制成单细胞悬液；将单细胞悬液通过类器官芯片接种孔接种在芯片上；吸去接种液，加入类器官培养基进行培养；待培养形成类器官后，按下药敏板，使药物作用类器官；读取荧光信号，进行数据分析。本发明的有益效果在于，提供了一种基于穿刺样本即可在短时间构建满足药敏检测用的直肠肿瘤类器官的试剂盒。
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
The invention relates to the technical field of drug sensitivity detection of primary rectal tumor cells, in particular to a drug sensitivity detection kit of a rectal tumor organoid chip and a use method thereof.
Background
The primary tumor cell drug sensitivity test is mainly used for detecting the condition of primary cancer cells which react to treatment, and providing information about whether the tumor is likely to spread and where the tumor is spread. The sensitization test is effective in detecting and determining which drugs, therapies or nutritional supplements a patient exhibits an optimal response to. In order to perform targeted drug treatment on patients in time, it is necessary to perform organoid culture with as few focal tumor tissue samples as possible, so as to achieve rapid construction of organoids meeting the requirements of drug sensitivity detection tests in a shorter time. However, the known primary rectal tumor cell drug sensitivity test kit has large focal tumor tissue sample amount and long test time, and the invention of the kit for satisfying the primary rectal tumor cell drug sensitivity test is necessary to be constructed in a shorter time (not more than 3 days) based on a puncture sample (less than 0.5 g).
Disclosure of Invention
In order to achieve the purpose, the invention provides a rectal tumor organoid chip drug sensitive detection kit, which comprises the following specific technical scheme:
Each kit comprises 1 micropore lattice organoid chip, 1 bottle of 200ml organoid culture medium, 1 bottle of 20ml tissue digestive enzyme and 1 bottle of 50ml inoculating liquid;
The 200mL organoid culture medium is prepared from the following raw materials of penicillin-streptomycin with a final concentration of 50-150×, B27 serum substitute with a final concentration of 70-150×, glutamine I with a final concentration of 1-4 mM, gastrin I with a final concentration of 8-15 nM, wnt3a with a final concentration of 50-150 ng/mL, R-spondin with a final concentration of 50-150 ng/mL, noggin with a final concentration of 70-150 ng/mL, epidermal growth factor EGF with a final concentration of 30-80 ng/mL, A83-01 with a final concentration of 400-600 nM, N-acetylcysteine with a final concentration of 1-3 mM, niacinamide with a final concentration of 8-15 mM, HEPES with a final concentration of 8-15 mM, SB 202190 with a final concentration of 8-15 mu M and ADVANCED DMEM/F12 buffer basic culture medium with a final concentration of up to 200mL;
the 20mL tissue digestive enzyme is prepared from the following raw materials of collagenase with a final concentration of 0.1-0.5 mg/mL, dispase with a final concentration of 0.1-0.5 mg/mL, DNase I with a final concentration of 0.1-0.5 mg/mL, penicillin-streptomycin with a final concentration of 50-150 x, HEPES with a final concentration of 8-15 mM, Y27632, and buffer basic culture medium with a final concentration of 8-15 uM and ADVANCED DMEM/F12, wherein the basic culture medium is supplemented to 20mL;
The 50ml inoculation liquid is prepared from the following raw materials of Y27632 with the final concentration of 8-15 uM, albumin with the final concentration of 1-3 g/L and PBS with the final concentration of up to 50ml.
As a further improvement, the 200mL organoid culture medium is prepared from penicillin-streptomycin with a final concentration of 55-65×, B27 serum substitute with a final concentration of 70-75×, glutamine I with a final concentration of 1-2 mM, gastrin I with a final concentration of 8-10 nM, wnt3a with a final concentration of 80-85 ng/mL, R-spondin with a final concentration of 55-65 ng/mL, noggin with a final concentration of 75-85 ng/mL, epidermal growth factor EGF with a final concentration of 30-40 ng/mL, A83-01 with a final concentration of 400-450 nM, N-acetylcysteine with a final concentration of 1-2 mM, niacinamide with a final concentration of 8-9 mM, HEPES with a final concentration of 8-9 mM, SB 202190 with a final concentration of 9-11 mu M and ADVANCED DMEM/F12 buffer basal culture medium with a final concentration of 200mL;
The 20mL tissue digestive enzyme is prepared from the following raw materials of collagenase with a final concentration of 0.1-0.15 mg/mL, dispase with a final concentration of 0.1-0.15 mg/mL, DNase I with a final concentration of 0.1-0.5 mg/mL, penicillin-streptomycin with a final concentration of 50-55 x, HEPES with a final concentration of 8-9 mM, Y27632, and buffer basic culture medium with a final concentration of 8-9 uM and ADVANCED DMEM/F12, wherein the basic culture medium is supplemented to 20mL;
The 50ml inoculation liquid is prepared from the following raw materials of Y27632 with the final concentration of 8-9 uM, albumin with the final concentration of 1-1.5 g/L and PBS with the final concentration of being complemented to 50ml.
As a further improvement, the 200mL organoid culture medium is prepared from penicillin-streptomycin with a final concentration of 95-105X, B27 serum substitute with a final concentration of 100-105X, glutamine I with a final concentration of 1.5-2.5 mM, gastrin I with a final concentration of 10-11 nM, wnt3a with a final concentration of 95-105 ng/mL, R-spondin with a final concentration of 95-105 ng/mL, noggin with a final concentration of 95-105 ng/mL, epidermal growth factor EGF with a final concentration of 45-55ng/mL, A83-01 with a final concentration of 480-520 nM N-acetylcysteine with a final concentration of 1-1.5 mM, niacinamide with a final concentration of 10-11 mM, HEPES with a final concentration of 10~11mM;SB 202190, a final concentration of 10-11 mu M and ADVANCED DMEM/F12 buffer basal culture medium to 200mL;
the 20mL tissue digestive enzyme is prepared from the following raw materials of collagenase with a final concentration of 0.15-0.25 mg/mL, dispase with a final concentration of 0.1-0.15 mg/mL, DNase I with a final concentration of 0.1-0.15 mg/mL, penicillin-streptomycin with a final concentration of 95-100 x, HEPES with a final concentration of 9-10 mM, Y27632, and buffer basic culture medium with a final concentration of 9-10 uM and ADVANCED DMEM/F12, wherein the basic culture medium is supplemented to 20mL;
the 50ml inoculation liquid is prepared from the following raw materials of Y27632 with the final concentration of 9-10 uM, albumin with the final concentration of 1.5-2 g/L and PBS with the final concentration of being complemented to 50ml.
As a further improvement, the 200mL organoid culture medium is prepared from the following raw materials of penicillin-streptomycin with a final concentration of 115-125×, B27 serum substitute with a final concentration of 115-125×, glutamine I with a final concentration of 3.5-4 mM, gastrin I with a final concentration of 12-14 nM, wnt3a with a final concentration of 115-120 ng/mL, R-spondin with a final concentration of 115-125 ng/mL, noggin with a final concentration of 125-135 ng/mL, epidermal growth factor EGF with a final concentration of 65-75 ng/mL, A83-01 with a final concentration of 550-600 nM, N-acetylcysteine with a final concentration of 2.5-3 mM, niacinamide with a final concentration of 12-13 mM, HEPES with a final concentration of 13~14mM;SB 202190, a final concentration of 12-13 mu M and ADVANCED DMEM/F12 buffer basic culture medium with a final concentration of 200mL;
The 20mL tissue digestive enzyme is prepared from the following raw materials of collagenase with a final concentration of 0.3-0.4 mg/mL, dispase with a final concentration of 0.3-0.4 mg/mL, DNase I with a final concentration of 0.3-0.4 mg/mL, penicillin-streptomycin with a final concentration of 125-135 x, HEPES with a final concentration of 12-13 mM, Y27632, and buffer basic culture medium with a final concentration of 13-14 uM and ADVANCED DMEM/F12, wherein the basic culture medium is supplemented to 20mL;
the 50ml inoculation liquid is prepared from the following raw materials of Y27632 with the final concentration of 12-13 uM, albumin with the final concentration of 2-2.5 g/L and PBS with the final concentration of up to 50ml.
As a further improvement, the specifications of the microporous lattice organoid chip include 5×5, 5×10, 10×5, or 10×10.
As a further improvement, the method further comprises 1 bottle of 20mL of enzymolysis liquid, wherein the 20mL of enzymolysis liquid is prepared from the following raw materials of Y27632 with the final concentration of 9-11 uM, trypsin with the final concentration of 0.1-0.2 mg/mL, EDTA with the final concentration of 0.01-0.03% and ADVANCED DMEM/F12 buffer basic culture medium which are complemented to 20mL.
The application method of the rectal tumor organoid chip drug sensitive detection kit comprises the following steps of:
s1, taking a rectocele tumor tissue, and preparing a single cell of the rectum tumor based on the 1 bottle of 20ml tissue digestive enzyme;
S2, preparing single-cell suspension of the rectal tumor based on 50ml of the 1-bottle inoculation liquid, and inoculating the single-cell suspension of the rectal tumor on the 1-piece micropore lattice organoid chip;
s3, continuously adding the 1 bottle of 200ml organoid culture medium into the 1 piece of microporous lattice organoid chip to prepare a rectal tumor organoid;
S4, preparing the rectal tumor organoid into rectal tumor organoid single cells based on the 1 bottle of 20ml enzymolysis liquid, and performing drug sensitivity detection by using the rectal tumor organoid single cells.
As a further improvement, in the step S1, the amount of the rectal tumor tissue is 0.2-0.3 g.
As a further improvement, in the step S2, the dosage of the inoculation liquid is 5-10 ml.
As a further improvement, in the step S3, the specification of the micro-porous lattice organoid chip comprises 5×5 or 5×10.
The technical scheme of the invention has the beneficial effects that the kit for constructing the rectal tumor organoid for drug sensitivity detection in a short time and 3 days based on the puncture sample of less than 0.5g is provided.
Drawings
FIG. 1 is an exploded view of an open chamber patterned array microfluidic based culture chip illustrated in China patent CN 118440822A;
FIG. 2 is a schematic diagram of a structure and a partial enlarged view of an open chamber patterned array microfluidic based culture chip exemplified in China patent CN 118440822A;
FIG. 3 is a graph showing the effect of organoids according to example 4 of the present invention after 1 day of culture;
FIG. 4 is a graph showing the effect of organoids of example 4 of the present invention after 3 days of culture;
FIG. 5 is a graph showing the effect of forming a classifier with uniform size over three days in accordance with example 4 of the present invention;
FIG. 6 is a graph showing the effect of organoids according to example 4 of the present invention after 5 days of culture.
In FIGS. 1 and 2, the same reference numerals are used to designate the same elements or structures, wherein 1-sample inlet, 2-sample outlet, 31-first flow path, 32-second flow path, 41-first flow stabilization chamber, 42-second flow stabilization chamber, 5-open channel, 6-culture fluid storage chamber, 7-well array layer, 8-patterned array;
in fig. 3 to 6, the magnification is 10X.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
Reagent and source:
Penicillin-streptomycin (100×, shanghai source culture Biotech Co., ltd.); PBS (phosphate buffer, wuhansai wili biotechnology limited); trypan blue staining solution (marsupenario life technologies limited); fresh rectal tumor tissue (in cooperation with the three hospitals, the collaborative development passed through formal medical ethical examination. The attending physician selects patients into groups according to TNM staging of the eighth edition of malignancy issued by the International anticancer Union (UICC) and selects appropriate puncture samples for in vitro culture according to clinical indications in surgery), ADVANCED DMEM/F12 (GibcoTM, dulbecco 'S modified Eagle Medium/Ham' S F-12), DNase I (deoxyribonuclease I), HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), Y27632 (trans-4- [ (R) -1-aminoethyl ] -N- (4-pyridinyl) cyclohexane carboxamide dihydrochloride), B27 additive (GibcoTM B-27TM additive, siemet), glutaMAX additive (GibcoTM GlutaMAXTM additive, L-alanyl-L-glutamine dipeptide, glutamine I, siemet), gastrin I (gastrin I), wnt-3A (Wnt 3A protein, hum (His), P56704-1 (S19-C351)), MCE (10-FGF 1, spinfibroblast growth factor (Sponer 1), growth factor (Sponer 1) growth factor (10-spinal growth factor 1) Recombinant R-Spondin 1 (RSPO 1), shanghai Kangwang Biotechnology Co., ltd.), noggin protein (MCE Co., ltd.), EGF (epidermal growth factor), A83-01 (3- (6-methyl-2-pyridyl) -N-phenyl-4- (4-quinolyl) -1H-pyrazole-1-thiocarboxamide), N-acetylcysteine (N-ACETYLCYSTEINE), nicotinamide (nicotinamide), SB 202190 (4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole), trypsin (Trypsin), ethylenediamine tetraacetic acid (EDTA).
In some embodiments, a kit for detecting drug sensitivity of a rectal tumor organoid chip is provided, wherein the kit is used for obtaining single cell suspension, cell inoculation organoid chip, organoid culture, drug treatment and drug sensitivity detection reagent based on enzymolysis digestion of rectal tumor tissue, and specifically comprises a microporous lattice organoid chip 1 block (5×5 or 5×10), an organoid culture medium 1 bottle (200 ml), a tissue digestive enzyme 1 bottle (20 ml), an enzymolysis solution 1 bottle (20 ml), an inoculation solution 1 bottle (50 ml) and a drug concentration gradient generation plate 1 block, the specific structure of the microporous lattice organoid chip in the application is not limited, and the microporous lattice organoid chip can contain various specifications including but not limited to a microporous lattice of 5×5, 5×10, 10×5 or 10×10, and can respectively carry out 5 schemes of 5 concentrations, a 5 schemes of 10 concentrations, a 10 schemes of 5 concentrations and a 10 schemes of 10 concentrations of drug sensitivity detection.
In some embodiments, the micro-porous lattice organoid chip in the invention is preferably Chinese patent CN 118440822A, and the patent name is a culture chip based on an open cavity patterned array micro-fluid, and preferably, the micro-porous lattice organoid chip is specifically structured as shown in the combination of fig. 1 and fig. 2, and is described in the embodiment section [ 0040-0051 ] of the specification, and the use method is described in the section [ 0053 ] of the specification, and specifically, the micro-porous lattice organoid chip comprises an open cavity cover plate and an array channel layer which are sequentially laminated from top to bottom.
The flow channel is the array channel layer and comprises an open channel area 5 formed by a plurality of flow channels in parallel and a culture solution storage chamber 6 connected with an outlet of the open channel area 5;
The inoculation holes are hole array layers 7 and patterning arrays 8 which are sequentially bonded below the open channel region 5, wherein the right lower part of the flow channel corresponds to hole rows in the hole array layers 7 and pattern rows in the patterning arrays 8, and the holes in the hole array layers 8 and the patterns of the patterning arrays 8 are in one-to-one correspondence with each other to form an isolation cavity array;
The inoculation groove is a culture solution storage chamber 6, a hollowed opening is formed in the middle of the open chamber cover plate, a sample inlet 1 connected with the open channel area 5 and a sample outlet 2 connected with the culture solution storage chamber 6 are formed at two ends of the open chamber cover plate, the array channel layer further comprises a second shunt channel 32 for shunting and conveying liquid in the culture solution storage chamber 6 to the sample outlet 2 through the second shunt channel 32 so as to enable the liquid to be uniformly converged, the integral unbalance caused by partial unsmooth circulation in the flowing process is avoided, the second flow rate stabilizing chamber 42 is a channel with reduced space height along the outlet direction of the culture solution storage chamber 6, as shown in fig. 2, a baffle with a height is arranged near the second flow rate stabilizing chamber 42, so that the culture solution in the culture solution storage chamber 6 slowly and stably flows out through the second flow rate stabilizing chamber 42, and the hollowed opening is the same as the size of the open channel area 5.
In use, the culture suspension is directly added from the sample inlet 1, the culture uniformly enters the open channel region 5 under the action of the first diversion channel 31 and the first stable chamber 41 including but not limited to overflow, but does not reach the culture solution storage chamber 6, then the culture suspension is refluxed from the sample inlet 1, the culture is uniformly divided under the action of the hole array layer 7, and the culture enters the isolation chamber 8 to form cell spheres after settling in the open channel region 5.
The micropore lattice organoid chip realizes the high uniformity and quick inoculation of the culture of the tumor organoid to form the tumor organoid with uniform size, is matched with a drug concentration gradient generation plate to be used for realizing the quick drug adding of tumor treatment drugs, and realizes the perfusion culture of the tumor organoid and automatic drug sensitive data acquisition and analysis with a self-grinding high-flux automatic drug detection integrated machine.
In some embodiments, the drug sensitive detection device of the present invention is preferably chinese patent CN 118956573A, entitled integrated and modular drug concentration gradient generation and administration device.
Tissue digestive enzymes are used to break up tumor tissue and release the rectal tumor cells therein to form a single cell suspension. Cutting freshly obtained rectal tumor tissue into paste, adding tissue digestive enzyme 2-10 times the sample volume, incubating at 37deg.C for digestion for 20-60 min, gently beating for 5-10 times to disperse cells, filtering with a filter screen, and centrifuging to collect cells for use.
The enzymatic hydrolysate was used for drug sensitive detection starting from cultured organoids. The organoids are broken into single cells by enzymolysis, and the supernatant is removed by centrifugation, and the cells are precipitated for standby.
The inoculation liquid is used for preparing rectal tumor cell suspension, and is inoculated onto a micropore lattice organoid chip for use. The above-mentioned tumor single cell pellet from tumor tissue or organoid is mixed with an inoculation liquid to prepare a single cell suspension.
The tissue digestive juice provided by the application is used for enzymolysis of tumor tissues, so that the acquisition rate and activity of rectal tumor cells can be improved, the formation rate of organoids can be greatly improved, the dosage of the tumor tissues can be reduced, the sample application type of the kit can be improved, for example, about 0.23g of a small amount of tumor tissues obtained by puncture can be increased, and the cultivation of organoids and drug sensitivity detection can be completed.
Organoid media for organoid culture on organoid chips, organoid media of the present application may also be used for solid tumor organoid culture including, but not limited to, hepatocellular carcinoma organoid culture, cholangiocarcinoma organoid culture, colorectal tumor organoid culture, gastric carcinoma organoid culture, pancreatic carcinoma organoid culture.
In some embodiments, the method for using the rectal tumor organoid chip drug sensitive detection kit comprises the steps of cutting 0.20-0.50 g of freshly obtained rectal tumor tissue with scissors, adding tissue digestive enzyme with a volume which is 2-10 times that of the sample, and placing the tissue digestive enzyme in a 37 ℃ and 5% CO 2 incubator for incubation and digestion for 20-60 minutes. After the incubation is finished, lightly blowing the mixture by a pipette for 5-10 times to further disintegrate loose tissues and free cells in the loose tissues. And centrifuging to collect cells, wherein the parameters are 200-300g for 3-5 minutes. Removing the supernatant, adding 5-10 ml of the inoculation liquid, and gently blowing 2-4 lower cells for resuspension. And inoculating the cell suspension onto a micropore lattice organoid chip, and standing in a 37 ℃ and 5% CO 2 incubator for 5-10 minutes so that the cells uniformly fall into micropores of the micropore lattice organoid chip. Gently sucking the inoculating liquid outside the micro-pores of the organoid chip, adding 1mL-1.5mL (5X 5 micro-pore lattice organoid chip) organoid culture medium, superposing a drug concentration gradient generation plate, covering a cover, and culturing in a 37 ℃ and 5% CO 2 incubator or an automatic drug sensitivity detection integrated machine for 1-5 days. After the culture is finished, the organoid is well formed, and a manual or operation automatic drug sensitive detection integrated machine presses down a drug concentration gradient generating plate, so that the drug to be detected and the living cell fluorescent dye quickly enter the micropores with the organoid. And (5) returning the organoid chip to the incubator or the automatic drug sensitive detection integrated machine for incubation and culture for 2-3 days. After incubation, fluorescence signal collection is carried out, drug sensitive data are analyzed, a concentration-cell activity fitting curve is established, and an IC50 value or inhibition rate is generated.
The seeding of the cell suspension onto the organoid chip can be done in two ways, manual seeding or automatic seeding. When in manual inoculation, the cell suspension is dripped into a channel as uniformly as possible, and the organoid chip plate is gently shaken, so that the cells are uniformly dispersed into the micropores of the chip. When in automatic inoculation, cells are added into an inoculation groove through an inoculation hole at one end of the organoid chip, then the inoculation groove is connected into a perfusion culture system, and the liquid circulation is opened, wherein parameters are that the cells in the inoculation groove are uniformly dispersed into each micropore.
The manual or operation automatic drug sensitive detection integrated machine presses down the drug concentration gradient generation plate in a mode that the manual pressing down of the drug concentration gradient generation plate needs to firstly take off the cap of the organoid chip in the biosafety cabinet, and then lightly press the drug concentration gradient generation plate to enable the organoid chip to be in contact with the culture liquid level, so that the drug is released.
The drug concentration gradient generating plate is used for drug loading and fluorescent dye loading during drug treatment. The medicine concentration gradient generating plate is prefabricated with medicine to be detected and fluorescent dye for detecting active cells. In the drug sensitivity detection, the drug concentration gradient generation plate is pressed down, the prefabricated drugs at the corresponding sites are contacted with the culture holes at the corresponding sites of the organoid chip plate, and the drugs and the fluorescent dye enter the holes and permeate into organoids to act on rectal tumor cells. Fluorescent staining marks living cells, and the related fluorescent signals are captured by a detection machine.
The kit provided by the invention is convenient and quick, the kit comprises all reagents for rapidly constructing highly uniform tumor organoids and accurately finishing drug sensitivity detection, no additional reagent is required, and the reagents and the culture device are obtained through repeated optimization, so that better organoids can be formed in a shorter time.
The organoid culture medium provided by the invention is a reagent which is repeatedly optimized and fully adapted to other components of the whole kit. The culture medium is suitable for different tumor subtypes and gene mutant types. The kit can be used in combination with the organoid chip, the tissue enzymolysis liquid and the drug concentration gradient generation plate, so that the organoid formation efficiency and the organoid formation speed can be obviously improved, and the drug sensitivity detection speed can be accelerated.
The inoculation liquid provided by the invention can obviously improve the efficiency and the activity rate of cell inoculation. The kit can be used in combination with an organoid chip, a culture medium, a drug concentration gradient generation plate and the like in the kit, so that the cell inoculation efficiency can be effectively improved, the organoid formation rate can be improved, and the detection speed of drug sensitivity can be accelerated.
The kit greatly simplifies the steps of detecting the drug sensitivity of the organoid, improves the speed and the precision of detecting the drug sensitivity, makes the organoid detection technology which is originally complicated and has high technical requirements easy to operate, and can complete the whole operation after simple training by common technicians.
The convenience of the kit also enables the organoid drug sensitivity detection technology to be widely developed in places such as scientific research institutions, medical technical departments of hospitals, medical examination institutions and the like.
Example 1
The rectal tumor organoid chip drug sensitive detection kit comprises the following components in parts by weight, see table 1:
TABLE 1 Each component of the kit and final concentration
The kit is used for culturing the rectum tumor organoid, and the culturing method comprises the steps of cutting up 0.42g (equivalent to about 1mm 3) of freshly obtained rectum tumor tissue by scissors, adding tissue digestive enzyme with the volume of 5 times of the rectum tumor tissue into the cut-up rectum tumor tissue, and incubating and digesting the rectum tumor tissue in a 37 ℃ and 5% CO 2 incubator for 30 time to form single rectum tumor cells. After the incubation, the loose tissue was further disintegrated by gentle pipetting to free the cells therein. Cells were collected by centrifugation at 200g for 3 minutes. Removing the supernatant, adding 7ml of inoculation liquid into the single cells of the rectal tumor to form single cell suspension of the rectal tumor, and lightly blowing the single cell suspension of the rectal tumor. Inoculating the single cell suspension of the rectal tumor to a micropore lattice organoid chip, and standing in a 37 ℃ and 5% CO 2 incubator for 5 minutes so that the cells uniformly fall into micropores of the micropore lattice organoid chip. The inoculation liquid outside the micro-pore of the micro-pore lattice organoid chip is gently sucked, 1ml of organoid culture medium is added into the micro-pore of the micro-pore lattice organoid chip, and the culture is carried out for 3 days in a 37 ℃ and 5% CO 2 incubator or an automatic drug sensitive detection integrated machine. Organoids are formed after the culture is completed.
Example 2
The same organoid culture procedure as in example 1 was used, except that 0.43g of straight intestine tumor tissue was taken and the kit included the following ingredients in the composition and amounts as shown in Table 2:
TABLE 2 Each component and the amount of the kit
Example 3
The same organoid culture procedure as in example 1 was used, except that 0.41g of straight intestine tumor tissue was taken and the kit included the following components in composition and amounts as shown in Table 3:
TABLE 3 Each component and the amount of the kit
Example 4
The kit of example 2 was used, except that 0.23g of the rectocele tumor tissue was taken, 3 times the tumor tissue volume of tissue digestive enzyme was added, 5ml of the inoculum was added, and 1.2ml of the organoid medium was added.
Example 5
The kit of example 2 was used, except that 0.45g of the tumor tissue of the rectocele was taken, the tissue digestive enzyme was added at a volume of 10 times the tumor tissue, 10 ml of the inoculum was added, and 1.5ml of the organoid medium was added.
Example 6
The same method for culturing the rectal tumor organoid as in example 1 was adopted, except that the obtained rectal tumor organoid was broken up into single cells by the enzymatic hydrolysate, the supernatant was removed by centrifugation, the cell was precipitated for later use, a plate was produced by pressing down a drug concentration gradient, the drug to be detected and the living cell fluorochrome were rapidly entered into the micropores of the long organoid, incubation was continued for 2 days, and after the incubation was completed, fluorescence signal collection was performed, and the kit used in the method included the following components, compositions and amounts were as shown in table 4:
TABLE 4 Components and amounts of the kit
Comparative example 1
The same rectal neoplasm organoid culture method as in example 1 was used, except that the kit used consisted of the following ingredients, ingredients and amounts as indicated in Table 5, the non-visible parts being the same as in example 1;
TABLE 5 Each component and the amount of the kit
Comparative example 2
The same rectal neoplasm organoid culture method as in example 1 was used, except that the kit used consisted of the following ingredients, ingredients and amounts as indicated in Table 6, the non-visible parts being the same as in example 1;
TABLE 6 Each component and final concentration of the kit
Comparative example 3
The same rectal neoplasm organoid culture method as in example 1 was used, except that the kit used consisted of the following ingredients, ingredients and amounts as set forth in Table 7;
TABLE 7 Each component and the amount of the kit
The invention will be further illustrated by the following test examples.
Test example 1 acquisition and Activity test of rectal tumor cells
Samples were taken as examples 1 to 5 using the tissue digestion enzymes of examples 1 to 5, respectively, and as example 6 using the tissue digestion enzyme of comparative example 1.
Test method fresh rectal tumor tissue, washed with PBS containing antibiotics, and weighed after removing blood and non-tumor tissue. Rectal tumor tissue was cut to a size of 1mm 3, and the tissue digestions of test examples 1-6 were added, respectively, and incubated at 37℃for 20 minutes. After digestion, 8 volumes of PBS was added to dilute the digest, and the rectal tumor tissue was thoroughly dispersed by gentle pipetting to release the cells therein. Filtering with a filter screen to remove undigested impurities. Then, the mixture was centrifuged at 300g for 5 minutes to remove the supernatant. Cells were resuspended by adding 10mL of PBS to give a cell suspension. 200ul of cell suspension was taken and stained with trypan blue dye. The number of dead living cells was counted, the obtaining rate of rectal tumor cells and the activity of rectal tumor cells were calculated, and the test results are shown in Table 8.
Rectal tumor cell acquisition rate (in/gram) =total number of rectal tumor cells (in)/tumor tissue (in gram)
Rectal tumor cell activity (%) =number of viable rectal tumor cells (individual)/total number of rectal tumor cells (individual) ×100%
TABLE 8 acquisition of rectal tumor cells and results of activity tests
The results of the tests show that the obtaining rate of the rectal tumor cells in the test examples 1-5 is obviously higher than that in the test example 6, the activity of the rectal tumor cells in the test example 1-5 is more than 85%, and especially the activity of the rectal tumor cells in the test example 2 is more than 90%, so that the formation rate of organoids can be greatly improved in the subsequent organoid culture process. The results of the test example 6, in which the amounts of the components of the tissue digestion enzyme were changed, show that the obtaining rate and activity of the rectal tumor cells were significantly reduced, and can indicate that the amounts and components of the tissue digestion enzyme were more effective in affecting the obtaining rate and activity of the rectal tumor cells.
Test example 2 inoculation test of rectal tumor cells
Inoculating instrument (model: SH-2090A, manufacturer: beijing Jin Sangte medical instruments Co., ltd.)
Incubator (RKJ DOMI-01-03, manufacturer: nanjing Ruikang biomedical technology Co., ltd.)
Test samples were prepared using the cell inoculum of the compositions and amounts of examples 1 to 5 as examples 7 to 11, the cell inoculum of the composition and amounts of comparative example 2 as example 12, trypan blue dye solution, and a microporous matrix organoid chip (5X 5);
Test methods rectal tumor single cells were resuspended in inoculum to form a rectal tumor single cell suspension in the amounts as indicated in Table 9. The cell suspension is uniformly dripped into the whole flow channel or added into an inoculation groove through an inoculation hole (5×5 microporous lattice organoid chip, 5 flow channels, 5 inoculation macropores for each flow channel), and each flow channel is inoculated with 1mL. After inoculation, the seeds are placed in a incubator for standing for 30 minutes. The flow channel inoculum was aspirated, trypan blue dye was added and the total and viable cell numbers were counted for each well under a microscope. Cell seeding efficiency and cell seeding activity were calculated.
Cell seeding efficiency (%) = average number of single well cells x number of wells (number of wells)/total number of cells to be seeded (number of wells)
Cell plating viability (%) =number of viable cells per single well (number of total cells per single well) ×100%
The test results are detailed in Table 9:
TABLE 9 results of inoculation test of rectal tumor cells (5X 5 chip)
Analysis of test results:
The inoculation liquids of examples 1-5 and comparative example 2 are used for inoculation test of rectal tumor cells, and it can be seen from comparison of results that the cell inoculation efficiency of examples 7-11 is obviously higher than that of example 12, and the cell inoculation activity rate of the liquid reaches more than 90%, so that the formation rate of organoids can be greatly improved in the subsequent organoid culture process, and the use amount of each component of the inoculation liquid is changed in example 12, which indicates that the cell inoculation efficiency and the activity rate are reduced, and it can be stated that the components and the use amount of the inoculation liquid can influence the cell inoculation efficiency and the cell inoculation activity rate more.
Test example 3 organoid culture test
Test methods organoid culture tests were performed by the methods of examples 1-5 and comparative examples 1-3, respectively, and tumor single cells (freshly isolated or organoid derived) were resuspended with inoculum to form single cell suspensions, respectively. The cell suspension was added dropwise to the whole flow channel or to the inoculation tank through the inoculation holes (5X 5 chip plate, 5 flow channels, 5 inoculation macropores per flow channel), each flow channel was inoculated with 1mL. After inoculation, the seeds are placed in a incubator for standing for 30 minutes. Sucking the inoculating liquid from the flow channel and inoculating the inoculating liquid into the organoid culture medium. The organoid formation pore number and organoid diameter were counted the next, fourth, and 6-8 days after inoculation, and the organoid formation rate was calculated. The test results are detailed in Table 10:
Organogenesis rate (%) = number of organoid wells formed (number)/total number of wells (number) ×100%
TABLE 10 results of organoid culture experiments versus Table
The test results show that the organoid is cultured by the method and the kit of the examples 1-5, the organoid formation rate is over 95%, the formation days are shorter, the diameter of the organoid is about 200 mu m when the organoid is cultured for 3 days, the organoid is cultured for 5-7 days, the diameter of the organoid can be about 220 mu m, the morphology size of the organoid is uniform, the components and the dosage of the kit are changed on the basis of the examples, and finally, the organoid formation rate is reduced and the morphology of the organoid is not ideal, so that the formation rate, the size and the uniformity of the organoid can be influenced by the components and the dosage of tissue digestive enzymes, cell inoculation liquid and culture medium.
First, as shown in FIG. 3, example 4 organoids formed at 1 day of culture. As shown in FIGS. 4 and 5, in example 4, only 0.23g of rectal tumor tissue was used, but the tissue was still cultured for 3 days, and the organoids had a diameter of 200 μm (FIG. 4) and a uniform morphology size (FIG. 5), which satisfied the use of drug sensitive detection. Finally, as shown in FIG. 6, example 4 was superior to comparative examples 1 to 3 and other examples in that the diameter of example 4 could reach about 220 μm after 5 days of culture.
The above examples are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (10)
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