Rectum cancer organoid culture kit and culture method thereof

Abstract

The invention discloses a rectal cancer organoid culture kit and a culture method thereof, and particularly relates to the technical field of genetic engineering.The kit comprises a culture medium with specific components, a sample treatment reagent, a passage reagent and a preservation reagent, and the culture medium contains Advanced DMEM / F-12 and multiple factors; the sample treatment reagent can effectively treat tissues; a passage reagent guarantees passage and cryopreservation; and the preservation reagent ensures the cell cryopreservation and resuscitation effects. The culture method covers scaffold and scaffold-free culture processes and comprises the steps of primary culture, subculture, recovery and the like, and the steps are operated finely. The method has the advantages of obvious advantages, fast tissue dissociation, small damage, good cell suspension dispersion, no DMSO toxicity, simple culture, high amplification efficiency, stable passage, wide applicability and high cost performance, and can provide a powerful tool for rectal cancer research, drug screening and the like.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and more specifically, to a rectal cancer organoid culture kit and a culture method thereof. Background Art

[0002] Traditional drug screening methods have many limitations. Despite passing animal experiments, about 90% of candidate drugs that enter clinical trials ultimately fail, or even cause irreversible death of subjects. 2D cells lack the highly differentiated structure and function of human tissues, and animal models will lead to large differences in toxicological and pharmacodynamic responses due to species differences, making clinical manifestations difficult to reproduce. Therefore, drug development needs to have new models that can provide better predictions of toxicity in the early stages of drug development, such as organoids.

[0003] Organoids are miniature organs generated in vitro by 3D culturing of pluripotent cells. Organoids have microscopic anatomical structures similar to the original tissues, can self-renew and self-organize, can reproduce some functions of the corresponding organs, and provide a highly physiologically relevant system. In addition, organoid culture requires very little tissue, has a short culture cycle, and a high success rate - overcoming the defects of poor cell line heterogeneity, loss of genetic information, and inability to simulate the 3D structure of tissues and organs in vivo; avoiding problems such as long modeling cycles, long culture times, and high costs for animal models. Organoid technology has great application potential in biomedical translational research fields such as disease research, drug screening, drug toxicity and toxicological reactions, gene and cell therapy, and in 2019, the New England Journal of Medicine evaluated organoids as "Preclinical models of human disease".

[0004] The source of organoids can come from stem cells of original tissues, embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) and established cell lines and organ explants containing a variety of cell types. However, due to ethical issues and difficulty in obtaining them, the main sources of cells used in research and clinical practice are original tissues or iPSCs obtained by reprogramming somatic cells. Since the team of Dutch scientist Professor Hans Clevers successfully cultured and established intestinal organoids for the first time in 2009, organoids have undergone great development. At present, organoid culture can be applied to organoid research models of various normal or tumor tissues including esophagus, intestine, stomach, liver, pancreas, lung, breast, kidney, prostate, bladder, brain, thyroid, inner ear, retina, heart and ovary.

[0005] However, organoid culture often fails due to a number of reasons, some of which stem from the culture reagents, such as slow dissociation of tissue samples, damage to the cell membrane, overly concentrated distribution of cells in the liquid, insufficient stability of the culture reagents, and high toxicity of freezing solutions containing DMSO.

[0006] Therefore, a rectal cancer organoid culture kit and a culture method thereof are proposed. Summary of the invention

[0007] In order to overcome the above-mentioned defects of the prior art, the present invention provides a rectal cancer organoid culture kit and a culture method thereof to solve the problems raised in the above-mentioned background technology.

[0008] To achieve the above object, the present invention provides the following technical solution: a rectal cancer organoid culture kit, comprising: Culture medium, including the following components and concentrations: Advanced DMEM / F-12 basal medium, HEPES 10mM, Glutamax 2mM, EGF 50μg / ml, Noggin 10μg / ml, R-Spondin 1 100μg / ml, Wnt-3a100μg / ml, and antibiotic 1%; Sample processing reagents include: tissue enzymatic solution, which contains trypsin 0.25% (w / v), DNase 0.25% (w / v) as enzyme digestion products, Tris 10mM as buffer, NaCl 110mM, KCl 10mM, MgCl2・6H2O2mM as salts, EDTA 2mM as antioxidant and inhibitor, EGTA 2mM as collagenase inhibitor; tissue preservation solution, which contains HEPES 10mM as buffer, EDTA 0.5mM as permeabilizing agent, trypsin 0.25% (w / v) as enzyme; tissue washing solution, which contains HEPES 10mM as buffer, EDTA 0.5mM as permeabilizing agent, collagenase inhibitor 0.5mM, protease inhibitor 1mM as protective agent, EGF 1ng / ml, Noggin 100pg / ml, R-Spondin 12ng / ml, Wnt-3a 2ng / ml as growth factors and cytokines; Passaging reagents, including: organoid washing solution, which contains HEPES 10mM as a buffer, sucrose 0.2M as an osmotic agent, antibiotics 1%, EGF 1ng / ml, Noggin 100pg / ml, R-Spondin 1 2ng / ml, Wnt-3a 2ng / ml as growth factors and cytokines; organoid digestion solution (TrypLE™ Express Enzyme 12604013); coating solution, which contains HEPES 10mM as a buffer, sucrose 0.2M as an osmotic agent, collagen 1mg / ml as an auxiliary fixative, EGF 1ng / ml, Noggin 100pg / ml, R-Spondin 1 2ng / ml, Wnt-3a 2ng / ml as growth factors and cytokines, and antibiotics; An appropriate amount of matrix gel is used to provide a support structure for cells during the culture of rectal cancer organoid scaffolds; The preservation reagent comprises 10% glycerol, 1M ectoine, 2g / L glucose injection, 20mM compound amino acid injection, 10% glycerol, 10mM dihydrogen phosphate, 0.1g / L Na2HPO4, 6.5g / L NaCl, 55g / L sucrose, 3g / L starch, and 5mM adenosine triphosphate disodium and magnesium chloride.

[0009] A method for culturing a rectal cancer organoid scaffold using the rectal cancer organoid culture kit described above comprises: first, performing primary culture, wherein the primary culture comprises the following steps: Sample preparation: First, collect cancer tissue and carefully remove it to avoid excessive damage to the tissue; then put it into tissue preservation solution and immediately put the collected cancer tissue into a tissue sampling bottle containing tissue preservation solution; then store it in an ice box and put the sampling bottle containing tissue preservation solution into an ice box and keep the temperature at 4°C to minimize the degradation of the sample; finally, take photos and register information, take photos of the tissue samples, make sure to clearly mark the source and collection time of the samples, and at the same time, fill in a detailed sample registration form and record relevant information, including but not limited to the sample number, collection date, and tissue type; Washing and chopping: After sterilizing the sampling bottle, place the tissue in a culture dish and wash it three times with tissue cleaning solution, changing the culture dish each time, and then chop it into pieces of about 1-3 mm in the culture dish and let it stand for two times; Digestion: Digest the cancer tissue with tissue enzymatic solution at 37°C with shaking for 10-20 minutes. Observe the digestion status at any time during the digestion process, and then add three times the volume of tissue washing solution to terminate the digestion; Filtration: Use a sieve to filter, take a small amount of filtrate and observe it under a microscope. After ensuring that there are obvious tissue blocks, collect an appropriate amount of filtrate and centrifuge for 3-5 minutes, then remove the supernatant; Matrigel calculation and plating: observe the volume of the collected precipitate, add 25 times the volume of Matrigel to resuspend, and spread the quantitative Matrigel-Cytogel drops into the well plate at 4°C; Gelation and culture: Place the plate in a 37°C incubator for 30 minutes to gel, and add preheated culture medium for culture.

[0010] Take photos: Take photos after laying the boards to record the status.

[0011] Preferably, secondly, the organoid subculture is performed, and the organoid subculture comprises the following steps: Use a pipette to scrape the organoid-matrigel mixture and transfer it to a centrifuge tube. Add an appropriate amount of organoid washing solution, place on ice for 10 minutes, centrifuge for 3-5 minutes, and remove the supernatant. Add 10 times the volume of the pelleted organoid digestion solution, gently pipette several times to mix, and place in a 37°C water bath for 10-12 minutes to digest; Add twice the volume of organoid washing solution to terminate digestion, centrifuge for 3-5 minutes, discard the supernatant, and obtain the organoid pellet, where twice the volume refers to the volume of the organoid digestion solution; Resuspend the pellet with matrix gel and plate it again for culture; if cryopreservation is required, add an appropriate volume of cryopreservation solution after obtaining the organoid pellet and freeze it at a certain density.

[0012] Preferably, finally, organoid resuscitation is performed, and organoid resuscitation comprises the following steps: Take out the cryotube containing tumor organoids from the liquid nitrogen tank and quickly place it in a 37°C water bath to thaw; During the thawing process in the water bath, gently shake the cryovial to ensure that the cryopreservation solution is completely thawed within 1-2 minutes; Quickly transfer the dissolved suspension to a centrifuge tube, gently pipette 3 times, centrifuge for 3-5 minutes, remove the supernatant and collect the organoid precipitate, add the organoid washing solution to resuspend the precipitate, centrifuge for 3-5 minutes, and remove the supernatant; Resuspend the precipitate with matrix gel, spread a certain amount of matrix gel in each well of the well plate, place it in the incubator for 30 minutes, and then add a certain amount of preheated organoid culture medium.

[0013] A method for culturing rectal cancer organoids without a scaffold using the rectal cancer organoid culture kit described above comprises the following steps: Primary culture: Mix the culture medium and coating solution at a ratio of 10:1 to prepare a pre-culture medium, and mix the pre-culture medium with the cells before plating; Subculture: Carefully remove the culture medium with a pipette, retain 3-5 μl, and add fresh culture medium; Cryopreservation: Carefully remove the culture medium with a pipette, leaving 3-5 μl, transfer the organoids into a cryovial, and add cryopreservation solution for storage; Recovery: Take out the cryovial containing tumor organoids from the liquid nitrogen tank and quickly place it in a 37℃ water bath to thaw. During the thawing process in the water bath, gently shake the cryovial to ensure that the cryopreservation solution is completely thawed within 1-2 minutes. Quickly transfer the dissolved organoid cells to a centrifuge tube and gently blow it several times with a pipette. Take an appropriate amount and centrifuge for 3-5 minutes, then remove the supernatant, collect the organoid cell pellet, add organoid washing solution to resuspend the pellet and transfer it to a centrifuge tube. Centrifuge for 3-5 minutes and remove the supernatant; resuspend the pellet with pre-made culture medium and plate it for culture.

[0014] Technical effects and advantages of the present invention: The tissue hydrolysate of the present invention adopts a combination of trypsin and DNA enzyme, and under the synergistic effect of Tris buffer, specific salts, EDTA and EGTA, it can quickly and effectively dissociate rectal cancer tissue samples within 10-20 minutes of shaking at 37°C. Compared with traditional methods, the tissue processing time is greatly shortened and the experimental efficiency is improved. At the same time, this precise enzymolysis has less damage to the cell membrane, ensures the integrity and activity of the cells, and lays a good foundation for subsequent culture. In the experiment, the cell survival rate after treatment with the tissue hydrolysate was significantly higher than that of the traditional hydrolysate treatment group, and the cell proliferation ability in subsequent culture was stronger.

[0015] The Advanced DMEM / F-12 basal medium in the culture medium provides comprehensive nutrition, and is combined with HEPES, Glutamax, multiple growth factors (EGF, Noggin, R-Spondin 1, Wnt-3a) and antibiotics to create a highly suitable growth microenvironment for organoid cells. The stable pH environment of HEPES ensures that intracellular enzyme activity and cell membrane stability are not affected by metabolites; Glutamax continuously supplies glutamine to meet cell energy and nitrogen source needs; growth factors synergistically regulate cell proliferation, differentiation and stemness maintenance, so that organoids can better simulate the function of in vivo tissues. During long-term culture, the structural integrity and functional stability of organoids are significantly better than those using ordinary culture media.

[0016] By using the organoid washing solution and digestion solution in the passaging reagent, the passaging operation of the organoid can be efficiently completed while ensuring the activity of the cells, maintaining the good dispersion of the cell suspension, reducing the cell agglomeration phenomenon, and ensuring the smooth progress of the passaging process. The freezing solution does not contain DMSO, thus avoiding the potential harm of its toxicity to the cells. At the same time, its rich nutrients and protective agents effectively maintain the physiological state of the cells during the freezing and recovery process, improve the recovery survival rate, and ensure that the cells can still maintain good growth and functional characteristics after long-term storage and multiple passaging. After multiple cycles of freezing and recovery, the activity of the organoid cells using the freezing solution of the present invention can still be maintained at a high level, while the activity of the cells treated with the traditional DMSO-containing freezing solution is significantly reduced.

[0017] This kit is suitable for both scaffold culture and scaffold-free culture of rectal cancer organoids, which can meet different experimental needs and research conditions, providing researchers with more flexibility in choice. It follows GMP-level standards during the production process, ensuring the stability and consistency of batch quality, making the experimental results highly reproducible. Moreover, compared with other similar products on the market, this kit has a clear price advantage. While ensuring high-quality culture effects, it reduces research costs and has a high cost-effectiveness, which is conducive to promoting the application of organoid culture technology in more laboratories and research projects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a flow chart of the rectal cancer organoid culture method of the present invention.

[0019] Figure 2 These are culture photos of intestinal cancer organoids cultured in the culture medium of the present invention from Day 1 to Day 11.

[0020] Figure 3 Advanced DMEM / F-12 (Gibco, 12634010) Basic culture medium composition Table 1 Figure 4 Advanced DMEM / F-12 (Gibco, 12634010) basic culture medium composition Table 2 DETAILED DESCRIPTION

[0021] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0022] Ingredients Origin: Advanced DMEM / F-12 (Gibco, 12634010), the specific components of this basic culture medium are as follows Figure 3 and Figure 4 As shown; HEPES self-prepared / (Gibco, 15630106) 10mM; Glutamax (Gibco, 21051024) 2mM; EGF (biotechne, 236-EG) 50μg / ml; Noggin (biotechne, 6057-EG) 10μg / ml; R-Spondin 1 (biotechne, 4645-RS) 100μg / ml; Wnt-3a (biotechne, 5036-WN) 100μg / ml; Antibiotics (penicillin-streptomycin-amphotericin B mixed solution) (Gibco, 15240062) 1%; Appropriate amount of Matrigel.

[0023] Rectal Cancer Organoid Culture Kit, including: Culture medium, including the following components and concentrations: Advanced DMEM basal medium, HEPES 10mM, Glutamax2mM, EGF 50μg / ml, Noggin 10μg / ml, R-Spondin 1 100μg / ml, Wnt-3a100μg / ml and antibiotic 1%, specification 100ml; Sample processing reagents include: tissue enzymatic solution, which contains trypsin 0.25% (w / v), DNase 0.25% (w / v) as enzyme digestion products, Tris 10mM as buffer, NaCl 110mM, KCl 10mM, MgCl2・6H2O2mM as salts, EDTA 2mM as antioxidant and inhibitor, EGTA 2mM as collagenase inhibitor, and the specification is 100ml; tissue preservation solution, which contains HEPES 10mM as buffer, EDTA 0.5mM as permeabilizing agent, trypsin 0.25% (w / v) as enzyme, and the specification is 100ml; tissue washing solution, which contains HEPES 10mM as buffer, EDTA 0.5mM as permeabilizing agent, collagenase inhibitor 0.5mM, protease inhibitor 1mM as protective agent, EGF 1ng / ml, Noggin 100pg / ml, R-Spondin 1 2ng / ml, Wnt-3a 2ng / ml as growth factors and cytokines, the specification is 500ml; Passaging reagents, including: organoid washing solution, which contains HEPES 10mM as a buffer, sucrose 0.2M as an osmotic agent, antibiotics 1%, EGF 1ng / ml, Noggin 100pg / ml, R-Spondin 1 2ng / ml, Wnt-3a 2ng / ml as growth factors and cytokines; organoid digestion solution (TrypLE™ Express Enzyme 12604013), with a specification of 500ml; coating solution, which contains HEPES 10mM as a buffer, sucrose 0.2M as an osmotic agent, collagen 1mg / ml as an auxiliary fixative, EGF 1ng / ml, Noggin 100pg / ml, R-Spondin 1 2ng / ml, Wnt-3a2ng / ml as growth factors and cytokines, and antibiotics, with a specification of 100ml; The preservation reagent comprises 10% glycerol, 1M ectoine, 2g / L glucose injection, 20mM compound amino acid injection, 10% glycerol, 10mM dihydrogen phosphate, 0.1g / L Na2HPO4, 6.5g / L NaCl, 55g / L sucrose, 3g / L starch, 5mM adenosine triphosphate disodium and magnesium chloride, with a specification of 20ml.

[0024] When implementing it specifically, Figure 2 As shown, the rectal cancer organoid culture kit is used to culture the rectal cancer organoid scaffold. First, primary culture is performed. The primary culture includes the following steps: Sample preparation: Place rectal cancer tissue in a tissue sampling bottle containing tissue preservation solution and place it in an ice box at 4°C. Take photos and register information to ensure that the sample is stored in a low-temperature environment to reduce cell activity loss. Washing and chopping: After disinfecting the sampling bottle, put the tissue into a culture dish and wash it three times with tissue cleaning solution. After each washing, change the culture dish to thoroughly remove impurities and possible microorganisms on the surface of the tissue. Then chop it into tissue blocks of about 1-3 mm in the culture dish and let it stand for two times to allow the tissue blocks to settle for subsequent operations; Digestion: Digest the cancer tissue with tissue enzymatic solution at 37°C with shaking for 10-20 minutes. Observe the digestion status at any time during the digestion process. When the tissue blocks gradually disperse and the cells begin to dissociate, add three times the volume of tissue washing solution to stop the digestion to prevent excessive digestion from damaging the cells. Filtration: Use a 100 μm pore size mesh to filter, take a small amount of filtrate and observe under a microscope. After ensuring that there are obvious tissue blocks, collect the filtrate and enrich it by centrifugation at 300g for 3-5 minutes, then remove the supernatant, retain the cell pellet, and remove undigested tissue fragments and impurities; Matrigel calculation and plating: Observe the volume of collected tissue sediment, add 25 times the volume of tissue matrigel to resuspend, take a 24-well plate as an example, 30ul per well, plate at 4°C and take photos to track the location to ensure the accuracy and repeatability of the operation, and facilitate the subsequent observation of cell growth; Gelation and culture: Place the plate in a 37°C incubator for 10-15 minutes to allow the matrix gel to form a stable gel structure to provide support for cell growth. After the matrix gel is formed, add culture medium and place the culture plate in an incubator for culture after returning to room temperature. The culture conditions need to be precisely set according to the cell characteristics and experimental requirements, such as temperature, humidity, CO 2 Concentration, etc.

[0025] Secondly, the organoids are subcultured, which includes the following steps: Use a cell scraper to remove the organoid-matrigel mixture and transfer it to a 15 ml conical tube. Let it stand for 3 minutes to allow the mixture to fully settle for subsequent operations. Centrifuge the conical tube at 300g for 5 minutes, remove the supernatant medium, add 10 times the volume of organoid digestion solution, gently pipette several times to ensure that the digestion solution and the matrix gel-organoid mixture are fully mixed, and then place the conical tube in a 37°C water bath and incubate for 10-12 minutes to fully digest the matrix gel and release the organoid cells; Add organoid washing solution to terminate the digestion reaction, filter through a 100μm pore size mesh to remove undigested matrix gel and impurities, and then centrifuge at 300g for 3-5min to separate and collect the organoid cells to obtain a relatively pure organoid cell precipitate.

[0026] Resuspension and plating or freezing: discard the supernatant, use matrix gel to resuspend the organoid cells in a certain proportion, and plate them again for culture; if the organoid cells need to be frozen, after separation and collection, add freezing solution at a density of 500 cells / ml, transfer them to cryopreservation tubes, and store them in liquid nitrogen. The cooling rate must be strictly controlled during the freezing process to ensure that the cell activity is not seriously damaged during the freezing process.

[0027] Finally, organoid recovery is performed, which includes the following steps: Take out the frozen tumor organoid cells from the liquid nitrogen tank and quickly place them in a 37°C water bath to thaw. During the thawing process, gently shake the cryovial to ensure that the cryopreservation solution is completely thawed within 1-2 minutes, so that the cells can quickly recover their activity and reduce the damage of ice crystals to the cells. The dissolved organoid cells were quickly transferred to a 15 ml conical tube, gently pipetted 3 times, centrifuged at 300 g for 3 minutes, then the supernatant was removed and the organoid cell pellet was collected. The organoid washing solution was added to resuspend the pellet and transferred to a 1.5 ml centrifuge tube and centrifuged at 300 g for 5 minutes to further remove the residual freezing solution and impurities and improve the cell purity. Resuspend with matrix gel, spread 20 μl of matrix gel in each well of a 24-well plate, place in the incubator for 10 min to allow the matrix gel to gel, add 500 μl of organoid culture medium, culture according to standard culture conditions, closely observe the cell growth status, and adjust the culture conditions in time.

[0028] A method for culturing rectal cancer organoids without a scaffold using a rectal cancer organoid culture kit comprises the following steps: Primary culture: Add coating solution to pre-prepared culture medium in a ratio of 10:1 to the culture medium, mix the pre-prepared culture medium with cells, plate, and then mix the pre-prepared culture medium with cells in an appropriate ratio, gently blow and evenly spread the culture plate to ensure that the cells are evenly distributed on the culture plate. After plating, place the culture plate in a suitable culture environment for culture. The culture conditions need to be optimized according to the characteristics of the cells. Subculture: Carefully remove the culture medium with a pipette, leaving 3-5ul to prevent the cells from drying out and damaging, then add new culture medium and continue culturing to maintain the nutritional environment required for cell growth while reducing interference with the cell growth state; Cryopreservation: Carefully remove the culture medium with a pipette, leaving 3-5ul, transfer the organoids to a cryopreservation tube with a pipette, and add cryopreservation solution for preservation. The cryopreservation process must strictly follow the standard operating procedures to ensure that the activity and function of the cells are not seriously affected during the freezing and recovery process; Recovery: Take out the frozen tumor organoid cells from the liquid nitrogen tank and quickly place them in a 37℃ water bath to thaw. During the thawing process in the water bath, gently shake the cryovial to ensure that the freezing solution is completely thawed within 1-2 minutes. Quickly transfer the dissolved organoid cells to a 15ml conical tube, gently blow 3 times with a pipette, centrifuge at 300g for 3 minutes, then remove the supernatant and collect the organoid cell pellet, add organoid washing solution to resuspend and transfer to a 1.5ml centrifuge tube and centrifuge at 300g for 5 minutes; resuspend with pre-made culture medium, plate and culture to resume cell growth.

[0029] In the above process, basic nutrients: Advanced DMEM basal culture medium provides basic nutrients such as amino acids, vitamins, inorganic salts, proteins, etc. required for cell growth, which is the material basis for cells to maintain metabolism and growth. These components participate in key physiological processes such as protein synthesis, energy metabolism, nucleic acid replication, etc. in cells, and provide necessary material support for the growth and proliferation of organoid cells. Specifically, amino acids are the basic building blocks of proteins. Cells synthesize various enzymes, receptors and other protein molecules by taking in amino acids, and participate in the structural construction and functional regulation of cells; vitamins as coenzymes participate in various chemical reactions in cells, and play a key role in the normal metabolism of cells.

[0030] Growth factors such as EGF, Noggin, R-Spondin 1, and Wnt-3a play an important regulatory role in organoid culture. EGF can stimulate receptors on the cell surface, activate signaling pathways within the cell, and promote cell proliferation and differentiation; Noggin participates in regulating the differentiation process of cells, inhibits certain signaling pathways, and guides cells to differentiate into specific cell types; R-Spondin 1 binds to receptors on the cell surface, enhances the self-renewal ability of stem cells, maintains the stemness of organoid cells, and promotes their continued growth and proliferation; Wnt-3a is also an important cell signaling regulator, participating in the process of cell fate determination and cell polarity establishment, and has a key influence on the formation and development of organoids. These growth factors work together to create a culture condition similar to the in vivo tissue microenvironment, promote the formation and growth of organoids, and give them structural and functional characteristics similar to the original tissue.

[0031] As a buffer, HEPES can stabilize the pH value of the culture medium within a certain range, resist the effect of acidic substances produced by cell metabolism on the pH of the culture medium, and provide a relatively stable acid-base environment for cell growth. The metabolic activities of cells will continuously produce acidic metabolites, such as lactic acid. If the pH value of the culture medium fluctuates too much, it will affect the activity of intracellular enzymes and the stability of cell membranes, thereby affecting cell growth and function. Glutamax provides cells with a stable source of glutamine, which is an important energy substance and nitrogen source in cell metabolism and plays a key role in cell proliferation and survival. The addition of antibiotics can effectively inhibit the contamination of microorganisms such as bacteria and fungi, prevent microorganisms from competing with cells for nutrients and secreting toxins during the culture process, ensure the sterile environment of cell culture, and ensure the stability and reliability of cell culture.

[0032] Trypsin and DNAse are the key components of tissue hydrolysate. Trypsin can specifically hydrolyze the protein connecting components between cells, weaken the adhesion between cells, and thus promote the dissociation of tissues and release single cells or small cell clusters. Under 37°C shaking conditions, the activity of trypsin can be fully exerted, accelerating the digestion process of tissues. DNAse can decompose extracellular DNA molecules, prevent cell aggregation caused by DNA released by cell rupture during tissue digestion, further improve the dispersion of cells, ensure the quality of cell suspension, and facilitate subsequent culture operations. Tris buffer maintains the pH value of the solution stable, providing a suitable environment for enzyme activity; salt components help maintain the osmotic pressure of the solution, ensuring that the physiological state of cells during digestion is relatively stable; EDTA, as an antioxidant and inhibitor, can chelate metal ions, inhibit the activity of certain enzymes, and reduce cell damage during digestion; EGTA, as a collagenase inhibitor, can prevent collagenase from excessively degrading collagen in the extracellular matrix and protect the integrity of the cell structure.

[0033] The HEPES buffer and EDTA permeabilizer in the tissue preservation solution work together to maintain the physiological state of the tissue during cryopreservation. HEPES stabilizes the pH value to prevent the tissue from being damaged by the accumulation of acidic substances produced by metabolism; EDTA can chelate metal ions, inhibit the activity of enzymes, and reduce the autolysis of tissues during preservation. Trypsin can inhibit the growth of microorganisms to a certain extent at low concentrations while maintaining the activity of tissues. Collagenase inhibitors and protease inhibitors in tissue cleaning solutions can protect proteins and extracellular matrix components on the cell surface and prevent cell damage caused by the action of enzymes during the cleaning process. Growth factors and cytokines such as EGF, Noggin, R-Spondin1, and Wnt-3a can bind to cell surface receptors during the cleaning process, maintain the activity and functional state of cells, reduce the stress response of cells during the processing process, and prepare for subsequent culture.

[0034] The HEPES buffer and sucrose osmotic agent in the organoid washing solution jointly maintain the osmotic pressure and pH value of the solution, providing a suitable environment for cells. Antibiotics can prevent microbial contamination during the washing process and ensure the purity of the cells. Growth factors and cytokines such as EGF, Noggin, R-Spondin1, and Wnt-3a continue to act during the washing process to maintain cell activity and function, promote cell survival and proliferation during the passaging process, and reduce cell damage caused by the passaging operation.

[0035] TrypLE™ Express Enzyme 12604013 is an enzyme specially designed for cell digestion. It can efficiently decompose the connecting components between cells and matrix gel, while causing relatively little damage to cells. Under 37°C water bath conditions, its activity can be fully exerted, causing rapid degradation of matrix gel and releasing organoid cells, which is convenient for subsequent collection and passage operations.

[0036] Collagen in the coating solution acts as an auxiliary fixative, providing additional adhesion sites for cells during scaffold-free culture, promoting cell attachment and growth. When cells are cultured in vitro, they need a suitable matrix to attach and grow. Collagen can simulate some functions of the extracellular matrix in vivo and enhance the interaction between cells and the culture surface. At the same time, HEPES, sucrose, growth factors, cytokines, antibiotics and other ingredients in the coating solution work together to maintain the stability of the microenvironment around the cells and promote cell growth and functional expression.

[0037] Glycerol and other ingredients in the preservation reagent act as cryoprotectants, which can lower the freezing point in cells during cell cryopreservation, reduce the formation of ice crystals, and prevent ice crystals from damaging the cell structure. Tetrahydropyrimidine carboxylic acid can enhance the stress resistance of cells and protect the structure and function of biological macromolecules such as proteins and nucleic acids during cell cryopreservation and recovery. Glucose injection, compound amino acid injection, dihydrogen phosphate, Na 2 HPO 4 , NaCl, sucrose, starch, disodium adenosine triphosphate and magnesium chloride provide nutritional support for cells and maintain the osmotic pressure balance inside and outside the cells, ensuring that the physiological state of cells is relatively stable during the freezing and recovery process, and improving the cell recovery survival rate and subsequent growth ability. In the low-temperature environment of liquid nitrogen, the metabolic activity of cells almost stops, but the substances in the cells may still be affected by physical and chemical factors. The reasonable formulation of the preservation reagent can minimize these adverse effects and ensure that the activity and function of the cells are maintained after freezing and recovery.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A rectal cancer organoid culture kit, characterized in that: include: Culture medium, including the following components and concentrations: Advanced DMEM / F-12 basal medium, HEPES 10 mM, Glutamax 2 mM, EGF 50 μg / ml, Noggin 10 μg / ml, R-Spondin 1 100 μg / ml, Wnt-3a 100 μg / ml, and antibiotic 1%; Sample processing reagents include: tissue enzymatic solution, which contains trypsin 0.25% (w / v), DNase 0.25% (w / v) as enzyme digestion products, Tris 10mM as buffer, NaCl 110mM, KCl 10mM, MgCl2・6H2O 2mM as salts, EDTA 2mM as antioxidant and inhibitor, EGTA 2mM as collagenase inhibitor; tissue preservation solution, which contains HEPES 10mM as buffer, EDTA 0.5mM as permeabilizing agent, trypsin 0.25% (w / v) as enzyme; tissue washing solution, which contains HEPES 10mM as buffer, EDTA 0.5mM as permeabilizing agent, collagenase inhibitor 0.5mM, protease inhibitor 1mM as protective agent, EGF 1ng / ml, Noggin 100pg / ml, R-Spondin 1 2ng / ml, Wnt-3a 2ng / ml as growth factors and cytokines; Passaging reagents, including: organoid washing solution, which contains HEPES 10mM as a buffer, sucrose 0.2M as an osmotic agent, antibiotics 1%, EGF 1ng / ml, Noggin 100pg / ml, R-Spondin 1 2ng / ml, Wnt-3a 2ng / ml as growth factors and cytokines; organoid digestion solution (TrypLE™ Express Enzyme 12604013); coating solution, which contains HEPES 10mM as a buffer, sucrose 0.2M as an osmotic agent, collagen 1mg / ml as an auxiliary fixative, EGF 1ng / ml, Noggin 100pg / ml, R-Spondin 1 2ng / ml, Wnt-3a 2ng / ml as growth factors and cytokines, and antibiotics; An appropriate amount of matrix gel is used to provide a support structure for cells during the culture of rectal cancer organoid scaffolds; The preservation reagent comprises 10% glycerol, 1M ectoine, 2g / L glucose injection, 20mM compound amino acid injection, 10% glycerol, 10mM dihydrogen phosphate, 0.1g / L Na2HPO4, 6.5g / L NaCl, 55g / L sucrose, 3g / L starch, and 5mM adenosine triphosphate disodium and magnesium chloride.

2. A method for culturing a rectal cancer organoid scaffold using the rectal cancer organoid culture kit according to claim 1, characterized in that: First, primary culture is performed, which includes the following steps: Sample preparation: First, collect cancer tissue and carefully remove it to avoid excessive damage to the tissue; then put it into tissue preservation solution and immediately put the collected cancer tissue into a tissue sampling bottle containing tissue preservation solution; then store it in an ice box and put the sampling bottle containing tissue preservation solution into an ice box and keep the temperature at 4°C to minimize the degradation of the sample; finally, take photos and register information, take photos of the tissue samples, make sure to clearly mark the source and collection time of the samples, and at the same time, fill in a detailed sample registration form and record relevant information, including but not limited to the sample number, collection date, and tissue type; Washing and mincing: Place the tissue in a culture dish and wash it three times with tissue washing solution, changing the culture dish each time, and then mince it into tissue pieces of about 1-3 mm in size in the culture dish; Digestion: Digest the cancer tissue with tissue enzymatic solution at 37°C with shaking for 10-20 minutes. Observe the digestion status at any time during the digestion process, and then add three times the volume of tissue washing solution to terminate the digestion; Filtration: Use a sieve to filter, take a small amount of filtrate and observe it under a microscope. After ensuring that there are obvious tissue blocks, collect an appropriate amount of filtrate and centrifuge for 3-5 minutes, then remove the supernatant; Plating: Observe the volume of the collected precipitate, add 25 times the volume of Matrigel to resuspend, and spread the quantitative Matrigel-Cytogel drops into the well plate at 4°C; Culture: After plating, place the plate in a 37°C incubator and let it stand for 30 minutes before adding preheated organoid culture medium for culture; Take photos: Take photos after laying the boards to record the status.

3. The method for culturing rectal cancer organoid scaffolds according to claim 2, characterized in that: Secondly, the organoids are subcultured, which includes the following steps: Use a pipette to scrape the organoid-matrigel mixture and transfer it to a centrifuge tube. Add an appropriate amount of organoid washing solution, place on ice for 10 minutes, centrifuge for 3-5 minutes, and remove the supernatant. Add 10 times the volume of the pelleted organoid digestion solution, gently pipette several times to mix, and place in a 37°C water bath for 10-12 minutes to digest; Add twice the volume of organoid washing solution to terminate digestion, centrifuge for 3-5 minutes, discard the supernatant, and obtain the organoid pellet (twice the volume refers to the volume relative to the organoid digestion solution); Resuspend the pellet with matrix gel and plate it again for culture; if cryopreservation is required, add an appropriate volume of cryopreservation solution after obtaining the organoid pellet and freeze it at a certain density.

4. The method for culturing a rectal cancer organoid scaffold according to claim 3, characterized in that: Finally, organoid recovery is performed, which includes the following steps: Take out the cryotube containing tumor organoids from the liquid nitrogen tank and quickly place it in a 37°C water bath to thaw; During the thawing process in the water bath, gently shake the cryovial to ensure that the cryopreservation solution is completely thawed within 1-2 minutes; Quickly transfer the dissolved suspension to a centrifuge tube, gently pipette 3 times, centrifuge for 3-5 minutes, remove the supernatant and collect the organoid precipitate, add the organoid washing solution to resuspend the precipitate, centrifuge for 3-5 minutes, and remove the supernatant; Resuspend the precipitate with matrix gel, spread a certain amount of matrix gel in each well of the well plate, place it in the incubator for 30 minutes, and then add a certain amount of preheated organoid culture medium.

5. A method for culturing rectal cancer organoids without a scaffold using the rectal cancer organoid culture kit according to claim 1, characterized in that: The following steps are involved: Primary culture: Mix the culture medium and coating solution at a ratio of 10:1 to prepare a pre-made culture medium, and mix the pre-made culture medium with cells before plating; Subculture: Carefully remove the culture medium with a pipette, retain 3-5 μl, and add fresh culture medium; Cryopreservation: Carefully remove the culture medium with a pipette, leaving 3-5 μl, transfer the organoids into a cryovial, and add cryopreservation solution for storage; Recovery: Take out the cryovial containing tumor organoids from the liquid nitrogen tank and quickly place it in a 37℃ water bath to thaw. During the thawing process in the water bath, gently shake the cryovial to ensure that the cryopreservation solution is completely thawed within 1-2 minutes. Quickly transfer the dissolved organoid cells to a centrifuge tube and gently blow it several times with a pipette. Take an appropriate amount and centrifuge for 3-5 minutes, then remove the supernatant, collect the organoid cell pellet, add organoid washing solution to resuspend the pellet and transfer it to a centrifuge tube. Centrifuge for 3-5 minutes and remove the supernatant; resuspend the pellet with pre-made culture medium and plate it for culture.

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