CN114181886A - Application of three-dimensional culture system in intestinal epithelial organoids of Bama pigs and cynomolgus monkeys - Google Patents
Application of three-dimensional culture system in intestinal epithelial organoids of Bama pigs and cynomolgus monkeys Download PDFInfo
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Abstract
The invention relates to application of a three-dimensional culture system in intestinal epithelial organoids of Bama pigs and cynomolgus monkeys. The three-dimensional culture system comprises a culture medium or composition for obtaining a three-dimensional organoid and differentiation thereof, wherein the composition for obtaining a three-dimensional organoid comprises: n2, B27, diabody, GlutaMAX, N-acetylcysteine, epidermal growth factor, Noggin, R-spondin1, CHIR-99021, A-83-01, SB202190, nicotinamide, Y27632, Gastrin I, PGE2 and Wnt3 a-CM. The composition can be cultured to form three-dimensional organoids or promote the differentiation of the three-dimensional organoids, so that a medicine screening system with low cost and high efficiency is provided for screening suitable animal models for medicine research.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of a three-dimensional culture system in intestinal epithelial organoids of Bama pigs and cynomolgus monkeys.
Background
The intestinal epithelium is the main structure of the human body performing the digestive absorption function, and is updated rapidly, while the updating is mainly driven by the intestinal epithelium stem cells located in the bottom crater structure, so that the research of the intestinal stem cells is closely related to the health of the human body. In recent years, the appearance of intestinal epithelial stem cell in vitro culture systems (organoids) has greatly promoted the study of intestinal epithelial self-renewal and homeostatic regulation. The in vitro cultured intestinal epithelial organoids can also be integrated into injured intestinal epithelium to help repair injury of the intestinal epithelium, and is hopeful to be applied to treatment of intestinal injury diseases.
Currently, studies on intestinal epithelial cells mostly rely on an in vitro three-dimensional culture system of Matrigel (Matrigel). The three-dimensional culture system can form intestinal organoids with villus-lacuna structures, can be independent of mesenchymal cells and has the capacity of self-renewal and differentiation.
Therefore, there is a need to develop research on organoids close to the inside of the body.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides a composition for obtaining a three-dimensional organoid, an amplification culture medium, a method for obtaining the three-dimensional organoid, a composition for three-dimensional organoid differentiation, a differentiation culture medium, a kit, a use and a method for detecting drug toxicity. Furthermore, the differentiation medium and the composition for three-dimensional organoid differentiation of the invention are beneficial to the formed three-dimensional organoid differentiation, and provide a low-cost and high-efficiency drug screening system for screening a proper animal model for drug research.
It should be noted that the present invention is completed based on the following work of the inventors:
rodents (mainly mice) are widely used for the study of intestinal epithelial cells, but do not mimic well the pathological characteristics of human gastrointestinal disorders. Polyps such as those caused by mutations in the adenomatous polyposis coli cancer suppressor gene (APC) occur predominantly in the human colorectal, whereas in mice they occur predominantly in the small intestine. At present, mouse intestinal organoid culture systems have been established. Based on the research on the genome sequence, the anatomical structure and the drug metabolism of the Bama pigs and the cynomolgus monkeys, the similarity between the Bama pigs and the cynomolgus monkeys and the human is found to be a more ideal animal model for researching the gastrointestinal diseases of the human. However, the intestinal organoids of barnyard pigs and cynomolgus monkeys have been reported rarely, and particularly, the culture method of the colonic epithelial organoids of barnyard pigs and cynomolgus monkeys has greatly limited the development and treatment of drugs for human gastrointestinal diseases.
In view of the above, the inventors have studied a composition for obtaining a three-dimensional organoid, which can form a three-dimensional organoid structure under such conditions by adding a series of growth factors and small molecules, and is particularly suitable for long-term growth of the barnyard pig and cynomolgus monkey organoids. Also, some small molecules (e.g., Wnt3a-CM, PGE2, etc.) are reduced in the composition for three-dimensional organoid differentiation compared to the composition for obtaining three-dimensional organoids, so that Wnt signaling can be reduced, contributing to the formed three-dimensional organoids to be better differentiated, e.g., into colon organoids, to have gene expression levels and cellular composition close to those in vivo. In addition, the invention can more conveniently explore the difference of drug toxicity and drug metabolism in animal models, and provides a low-cost and high-efficiency drug screening system for screening suitable animal models for clinical research.
Thus, in one aspect of the invention, the invention provides a composition for obtaining a three-dimensional organoid. According to an embodiment of the invention, the composition comprises: n2, B27, diabody, GlutaMAX, N-acetylcysteine, epidermal growth factor, Noggin, R-spondin1, CHIR-99021, A-83-01, SB202190, Y27632, nicotinamide, prostaglandin E2 and Wnt3 a-CM.
The N2 and the B27 are serum-free additives, are mostly suitable for most nerve cell culture and can improve the culture effect of cells; the double antibody is a mixed solution of two antibacterial drugs (such as a mixed solution of penicillin and streptomycin) and is used for resisting microbial contamination in a cell culture process; GlutaMAX is a substitute of L-glutamine, has better stability and can improve the health of cells; n-acetylcysteine (N-acetylcysteine) is an antioxidant, and has the functions of interfering the generation of free radicals, eliminating the generated free radicals and regulating the metabolic activity of cells; epidermal Growth Factor (EGF) is a cell Growth Factor and has the function of stimulating cell Growth; noggin is an important molecule for inhibiting BMP signal pathway, and belongs to a group of diffusible proteins; R-Spondin1 is a secretory protein in a Wnt/beta-catenin signal regulatory protein R-Spondin family, and promotes cell proliferation by activating a Wnt signal; CHIR-99021 is a GSK-3 beta inhibitor and can obviously activate a Wnt/beta-catenin signal channel; a-83-01 is inhibitor of TGF-beta (Transforming growth factor beta) type I receptor ALK-5, and can promote cell growth; SB202190 is a p38 MAPK inhibitor; y27632 is a ROCK1 inhibitor, can obviously inhibit the anoikis phenomenon of the organoid, and plays a role in promoting the survival of the primarily cultured organoid; nicotinamide can be used as nutrient component for cell culture to promote long-term survival of organoid; prostaglandin E2(Prostaglandin E2, PGE2 for short) is one of many Prostaglandins (PG), and can be used as cell growth and regulatory factor for promoting organoid proliferation and survival.
Wnt3a-CM (Wnt3a-conditional medium) is derived from L-Wnt3a cell line (ATCC), the main component is Wnt3a protein, and the specific acquisition method comprises the following steps: culturing the L-Wnt3a cell line by using DMEM + 10% fetal calf serum, and carrying out passage at a ratio of 1:10 after the cell line grows full; adding new DMEM + 10% fetal calf serum after passage, collecting supernatant after the cells grow over the culture dish, and placing the supernatant in a 4-degree refrigerator; adding a new DMEM + 10% fetal calf serum culture medium into the cells, continuously culturing for 3 days, and collecting the supernatant again after the culture medium turns yellow; mixing the two collected supernatants, and filtering through a 0.22 μm filter screen to remove cell debris, namely Wnt3 a-CM.
Through a large number of experiments, the inventor finds that when the composition containing the components is used for culturing the cells of the Bama pigs and the cynomolgus monkeys, the obtained organoid has the capability of long-term growth, and can effectively enrich stem cells and rapidly-proliferating cells, and is particularly suitable for culturing colon epithelial organoids of the Bama pigs and the cynomolgus monkeys.
According to an embodiment of the invention, the composition further comprises gastin I. Wherein Gastrin I is an endogenous gastrointestinal peptide hormone, and promotes the proliferation of gastrointestinal epithelial cells.
The inventor finds that the organoids of the Bama pigs and the cynomolgus monkeys are cultured by adding the composition containing the components through a large number of experiments, so that the survival rate of the organoids can be further improved.
According to an embodiment of the invention, the composition is used for the amplification of the cells of a mare pig and/or a cynomolgus monkey. The inventor finds that the composition has better effect on the cell culture of the Bama pigs and the cynomolgus monkeys through experiments.
According to an embodiment of the invention, the cells are selected from stem cells and/or epidermal cells. The inventor finds out through experiments that the composition has better culture effect on stem cells and epidermal cells of the Bama pigs and the cynomolgus monkeys.
According to an embodiment of the invention, the cells are selected from intestinal stem cells and/or intestinal epithelial cells. The inventor finds out through experiments that the composition has better culture effect on intestinal stem cells and intestinal epidermal cells of the Bama pigs and the cynomolgus monkeys.
According to an embodiment of the invention, the cells are colon epithelial cells. The inventor finds that the composition has better effect on the culture of colon epidermal cells of the mare pigs and the cynomolgus monkeys through experiments.
In another aspect of the invention, an amplification medium is provided. According to an embodiment of the invention, the amplification medium comprises: the above-mentioned composition; and (3) a basic culture medium.
Through a large number of experiments, the inventor finds that when the amplification culture medium is used for culturing the cells (particularly colon epithelial cells) of the Bama pigs and the cynomolgus monkeys, the cultured organoids have the capacity of long-term growth, and stem cells and rapidly proliferating cells can be effectively enriched.
According to an embodiment of the invention, the basal medium is Advanced DMEM/F12. The inventor finds that the culture effect is better when Advanced DMEM/F12 is adopted through a large number of experiments.
According to an embodiment of the invention, the concentration of said GlutaMAX is between 0.5 and 10mM, preferably between 1 and 3 mM. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to an embodiment of the invention, the concentration of N-acetylcysteine is 0.2-5mM, preferably 0.2-2 mM. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to an embodiment of the present invention, the concentration of the epidermal growth factor is 5-100ng/mL, preferably 40-60 ng/mL. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to an embodiment of the present invention, the concentration of Noggin is 5-200ng/mL, preferably 80-120 ng/mL. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to the embodiment of the invention, the concentration of the R-spondin1 is 100-1000ng/mL, preferably 400-600 ng/mL. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to an embodiment of the invention, the concentration of CHIR-99021 is 1-10. mu.M, preferably 4-6. mu.M. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to an embodiment of the invention, the concentration of A-83-01 is 0.5-2. mu.M, preferably 0.5-1. mu.M. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to an embodiment of the invention, the concentration of SB202190 is 1-10. mu.M, preferably 8-10. mu.M. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to an embodiment of the invention, the concentration of Gastrin I is 1-10nM, preferably 1-3 nM. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to an embodiment of the present invention, the concentration of prostaglandin E2 is 1-5. mu.M, preferably 2-3. mu.M. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to an embodiment of the invention, said nicotinamide is present in a concentration of 1-10mM, preferably 8-10 mM. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to an embodiment of the invention, the concentration of said N2 is between 0.5 and 10mM, preferably between 1 and 3 mM. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to an embodiment of the invention, the concentration of B27 is 0.5-10mM, preferably 1-3 mM. The inventor obtains the better adding concentration through a large amount of experiments, so that the cultured organoid has strong growth capacity and high survival rate.
According to the embodiment of the invention, the concentration of the double antibody is 100-1000ng/mL, preferably 400-600 ng/mL. This can inhibit the growth of the bacterial flora in the amplification medium and prevent organoid contamination.
According to an embodiment of the invention, the volume of Wnt3a-CM is 10% to 30%, preferably 20 to 30% of the total volume of the amplification medium. The inventor finds that the survival rate of the cells is dose-dependent on Wnt3a-CM, when 10% -30% of Wnt3a-CM is added, the survival rate of the cultured organoid is higher, and particularly, the survival rate of the organoid can be greatly improved by adding 20% -30% of Wnt3 a-CM.
According to an embodiment of the present invention, the concentration of Y27632 is, preferably, 8-10. mu.M. The inventor finds that the survival rate of the organoid can be improved by adding Y27632 with the concentration of 1-10 mu M through a large number of experiments, and particularly, the survival rate of the colon organoid can be further improved by adding Y27632 with the concentration of 8-10 mu M.
According to an embodiment of the invention, the diabesins are penicillin and streptomycin. Therefore, the antibacterial effect of the penicillin and the streptomycin in the amplification culture medium is better.
In yet another aspect of the present invention, a method for obtaining a three-dimensional organoid is provided. According to an embodiment of the invention, the method comprises: and mixing the cell sample with matrigel, and adding the amplification culture medium for amplification culture to form the three-dimensional organoid.
According to the embodiment of the invention, the cell samples of the Bama pigs and the cynomolgus monkeys are cultured by the method, and the cultured organoids have the capability of long-term growth and high survival rate.
According to the embodiment of the invention, the cell sample is mixed with the matrigel and then solidified for 15-20 minutes in a 4-6% carbon dioxide incubator at 35-38 ℃. Therefore, the culture effect is better when the culture is carried out by adopting the method.
According to an embodiment of the invention, the matrigel has a thickness of 40-60 μm, preferably 50-55 μm. The inventors have found through experiments that three-dimensional structures can be well formed with matrigel having a thickness of 40-60 μm, especially matrigel having a thickness of 50-55 μm.
In yet another aspect of the present invention, a three-dimensional organoid is provided. According to an embodiment of the invention, the three-dimensional organoid is obtained using the method described above.
According to the embodiment of the invention, the three-dimensional organoid has proliferation and differentiation capacities similar to corresponding cells in vivo, can grow for a long time, has high survival rate, can be used for exploring the difference of drug toxicity and drug metabolism in animal models in the later period, screens a proper animal model for clinical research, and provides a drug screening system with low cost and high efficiency.
In yet another aspect of the present invention, the present invention provides a composition for the above three-dimensional organoid differentiation. According to an embodiment of the invention, the composition comprises: n2, B27, double antibody, GlutaMAX, N-acetylcysteine, EGF, Noggin, R-spondin1, Y27632, A-83-01 and Gastrin I.
The inventors have found through extensive experiments that on the basis of the above amplification medium, by adjusting different substances, Wnt3a-CM and PGE2, when not added, could decrease Wnt signaling, promote differentiation of bama pig and cynomolgus monkey organoids, and maintain the organoids for a survival time of at least 1 week, especially colon organoids. And the cultured colon organs have cell composition and gene expression similar to those in vivo, so that the difference of drug toxicity and drug metabolism in animal models can be explored more conveniently, and a drug screening system with low cost and high efficiency is provided for screening suitable animal models for clinical research.
According to an embodiment of the present invention, the composition for the above three-dimensional organoid differentiation further comprises: niacinamide and/or SB 202190. Thereby, organoids can be maintained for a survival time of approximately 2 weeks.
According to an embodiment of the present invention, the composition for the above three-dimensional organoid differentiation further comprises: CHIR-99021. Thereby, organoids can be maintained for a survival time of at least 3 weeks.
According to an embodiment of the invention, the three-dimensional organoid is selected from the group consisting of cells of a mare pig and/or a cynomolgus monkey.
According to an embodiment of the invention, the three-dimensional organoid is selected from stem cells and/or epidermal cells.
According to an embodiment of the invention, the three-dimensional organoid is selected from intestinal stem cells and/or intestinal epithelial cells.
According to an embodiment of the invention, the three-dimensional organoid is a colonic epithelial cell.
In yet another aspect of the present invention, a differentiation medium is provided. According to an embodiment of the invention, the differentiation medium comprises: the above composition for three-dimensional organoid differentiation; and (3) a basic culture medium.
The inventor finds that the three-dimensional organoids (especially colon organoids) of the bama pig and the cynomolgus monkey can be promoted to differentiate by adopting the differentiation medium, and the organoids can survive for at least more than 1 week, even more than 3 weeks.
According to an embodiment of the invention, the basal medium is Advanced DMEM/F12. The inventor finds that the culture effect is better when Advanced DMEM/F12 is adopted through a large number of experiments.
According to an embodiment of the invention, the concentration of said GlutaMAX is between 0.5 and 10mM, preferably between 1 and 3 mM. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to an embodiment of the invention, the concentration of N-acetylcysteine is 0.2-5mM, preferably 0.2-2 mM. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to an embodiment of the present invention, the concentration of the epidermal growth factor is 5-100ng/mL, preferably 40-60 ng/mL. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to an embodiment of the present invention, the concentration of Noggin is 5-200ng/mL, preferably 80-120 ng/mL. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to the embodiment of the invention, the concentration of the R-spondin1 is 100-1000ng/mL, preferably 400-600 ng/mL. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to an embodiment of the invention, the concentration of CHIR-99021 is 1-5. mu.M, preferably 2-3. mu.M. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to an embodiment of the invention, the concentration of A-83-01 is 0.5-2. mu.M, preferably 0.5-1. mu.M. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to an embodiment of the invention, the concentration of SB202190 is 1-10. mu.M, preferably 8-10. mu.M. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to an embodiment of the invention, the concentration of Gastrin I is 1-10nM, preferably 1-3 nM. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to an embodiment of the invention, said nicotinamide is present in a concentration of 1-10mM, preferably 8-10 mM. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to an embodiment of the invention, the concentration of said N2 is between 0.5 and 10mM, preferably between 1 and 3 mM. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to an embodiment of the invention, the concentration of B27 is 0.5-10mM, preferably 1-3 mM. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to the embodiment of the invention, the concentration of the double antibody is 100-1000ng/mL, preferably 400-600 ng/mL. Thereby, the growth of the flora in the differentiation medium can be inhibited, and the organoid can be prevented from being contaminated by the bacteria.
According to an embodiment of the present invention, the concentration of Y27632 is 1-10. mu.M, preferably 8-10. mu.M. The inventor obtains the preferable adding concentration through a large number of experiments, so that the organoid differentiation effect is good, and the organoid survival time is long.
According to an embodiment of the invention, the diabesins are penicillin and streptomycin. Therefore, the antibacterial effect of the penicillin and the streptomycin in the differentiation medium is better.
In yet another aspect of the present invention, a kit is provided. According to an embodiment of the present invention, the composition for obtaining a three-dimensional organoid as described above, the expansion medium as described above, the composition for three-dimensional organoid differentiation as described above, and/or the differentiation medium as described above are contained. The inventor finds that the kit can be used for culturing three-dimensional organoids or promoting the differentiation of the three-dimensional organoids through a large number of experiments, and has the advantages of convenience in use and the like.
In yet another aspect of the present invention, the present invention provides a use of the above-mentioned composition for obtaining a three-dimensional organoid, the above-mentioned amplification medium, the above-mentioned composition for three-dimensional organoid differentiation, the above-mentioned differentiation medium or the above-mentioned kit for drug screening or drug analysis. According to the embodiment of the invention, the three-dimensional organoid composition, the amplification medium or the kit for obtaining the three-dimensional organoid composition can be used for obtaining the three-dimensional organoid by culture, and can be used for drug screening or drug analysis at the later stage; the composition, the differentiation medium or the kit for the three-dimensional organoid differentiation can be used for promoting the differentiation of the three-dimensional organoid and can be used for drug screening or drug analysis.
In yet another aspect of the present invention, a method for detecting drug toxicity is provided. According to an embodiment of the invention, the method comprises: culturing the candidate drug and the cell sample cultured in the amplification culture medium; carrying out differentiation culture on the cultured cells in the differentiation culture medium to obtain differentiated cells; and taking the detection result of the cell viability before and after the differentiation culture as an indication of whether the drug toxicity exists. According to the embodiment of the invention, the method can be used for detecting the drug toxicity, and has the advantages of simple operation, high accuracy of detection results and the like.
According to an embodiment of the present invention, the cultivation time is 2 to 10 days. Therefore, the result accuracy of the drug toxicity detection is high.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a long-term growth of colon organoids of Bama pigs and cynomolgus monkeys in example 1 according to the present invention;
FIG. 2 is a graph comparing the effects of Wnt3a-CM on the presence or absence of cultured three-dimensional organoids in amplification medium according to example 1 of the present invention;
FIG. 3 is a graph showing the differentiation of a colon organoid of Bama pigs in different differentiation media according to example 2 of the present invention;
FIG. 4 is a graph showing the differentiation ability of colon organoids of Bama pigs in example 3 according to the present invention;
FIG. 5 is a graph showing the differentiation ability of colon organoids of cynomolgus monkeys according to example 3 of the present invention;
FIG. 6 shows the results of the test of the toxicity of drugs in colon organoids of Bama pigs, cynomolgus monkeys and humans according to example 4 of the present invention.
Detailed Description
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
In the following examples, matrigel was purchased from BD Bioscience under the reference 356231.
Double antibody was purchased from Invitrogen, cat #: 15140-148, original concentration: 100X, working concentration: 1X.
N2 was purchased from Invitrogen, cat #: 17502-048, original concentration: 100X, working concentration: 1X.
B27 was purchased from Invitrogen, cat #: 17504-044, original concentration: 50X, working concentration: 1X.
The basal medium used was Advanced DMEM/F12.
The three-dimensional culture system used (hereinafter also referred to as amplification medium) comprised Advanced DMEM/F12, N2, B27, diabodies (penicillin and erythromycin), 2mM GlutaMAX and 1mM N-acetylcysteine, 50ng/mL EGF, 100ng/mL Noggin, 500ng/mL R-spondin1, 5. mu.M CHIR-99021, 0.5. mu. M A-83-01, 10. mu.M SB202190, 1nM Gastrin I, 10. mu. M Y27632, 2.5. mu.M PGE2, 10mM Nicotinamide (hereinafter also referred to as Nicotinamide) and 30% Wnt3a conditioned medium.
The differentiation-1 medium used (hereinafter also referred to as differentiation medium) contained Advanced DMEM/F12, N2, B27, diabody, 2mM GlutaMAX and 1mM N-acetylcysteine, 50ng/mL EGF, 100ng/mL Noggin, 500ng/mL R-spondin1, 2.5. mu.M CHIR-99021, 0.5. mu. M A-83-01, 10. mu.M SB 190, 1nM Gastrin I, 10. mu. M Y27632 and 10mM Nicotinamide.
The differentiation-2 medium used contained Advanced DMEM/F12, N2, B27, diabodies, 2mM GlutaMAX and 1mM N-acetylcysteine, 50ng/mL EGF, 100ng/mL Noggin, 500ng/mL R-spondin1, 0.5. mu. M A-83-01, 10. mu.M SB202190, 1 GanM strin I, 10. mu. M Y27632 and 10mM Nicotinamide.
The differentiation-3 medium used contained Advanced DMEM/F12, N2, B27, diabodies, 2mM GlutaMAX and 1mM N-acetylcysteine, 50ng/mL EGF, 100ng/mL Noggin, 500ng/mL R-spondin1, 0.5. mu. M A-83-01, 1nM Gastrin I and 10. mu. M Y27632.
Example 1: colon organoid culture of Bama pigs and cynomolgus monkeys based on 3D culture system
The isolated bacon pigs and cynomolgus monkeys coloboma prepared in this example were cultured according to the 3D culture system and amplification medium established in the present invention and optimized for coloboma Isolation as reported in the literature (Jung, p., et al (2011) Isolation and in vitro expansion of human colonic cells, nature Medicine) as follows:
(1) the colons were isolated from adult Bama pigs and cynomolgus monkeys, slit and washed three times with pre-cooled PBS buffer at pH 7.4 to remove chyme. The surrounding adipose and vascular tissue was then removed using a scalpel, and the remaining portion was cut into 10cm long pieces of intestine and incubated with 10mM EDTA in PBS buffer on ice for 40 minutes. The PBS buffer containing EDTA was replaced with fresh pre-cooled PBS buffer and then vigorously scraped using a scalpel to obtain a large number of colobomas. The resulting dimple suspension was transferred to a 1.5ml EP tube and left to stand for 1 minute to take the supernatant to remove the bulk tissue. Then, the scattered single cells were removed by centrifugation at 600rpm for 3 minutes. The pellet after centrifugation is a purer colostomy, which can be used for in vitro culture.
(2) Each 200 separated colostomy cells were mixed with Matrigel matrix (BD Biosciences), applied to a cell culture plate, coagulated in a 5% carbon dioxide cell culture chamber at 37 ℃ for 15-20 minutes, and then cultured with an amplification medium. The amplification medium contains basal medium Advanced DMEM/F12, serum substitutes N2 and B27, double antibody, 2mM GlutaMAX and 1mM N-acetyl cysteine, and a series of growth factors and small molecules as required: 50ng/mL EGF, 100ng/mL Noggin, 500ng/mL R-spondin1, 5. mu.M CHIR-99021, 0.5. mu.M-83-01, 10. mu.M SB202190, 1nM Gastrin I, 10. mu. M Y27632, 2.5. mu.M PGE2, 10mM Nicotinamide and 30% Wnt3a conditioned medium. The inventors found that colon organoids of Bama pigs and cynomolgus monkeys can grow continuously and are continuously passaged under the culture conditions for at least more than 3 months. As shown in fig. 1A (growth state of bama pig colon organoid, taken by orthomicroscope) and 1C (growth state of cynomolgus monkey colon organoid, taken by orthomicroscope), the colon of bama pig and cynomolgus monkey was changed from columnar epithelium to spherical organoid and maintained spherical morphology with continuous passage. FIG. 1B shows that, under successive passages, Colon organoids of Bama pigs grow in size, indicating a rapid increase in size. Fig. 1D shows that colon organoids of cynomolgus monkeys increased in size before passage P6, and then remained unchanged in size.
In addition, the inventor adopts the method to culture the colon of the Bama pigs by two amplification culture mediums respectively, the two amplification culture mediums only differ in the presence or absence of the added 30% Wnt3a conditioned medium, and the rest conditions are the same. The inventors found that adding 30% Wnt3a conditioned medium to the amplification medium significantly improved the survival efficiency of colon organoids, as shown in fig. 2 (the effect of 30% Wnt conditioned medium on the growth of pig colon organoids, photographed by an upright microscope). Thus, during subsequent culture, 30% Wnt3a conditioned medium was also added to the amplification medium.
Example 2: 3D culture system-based exploration of colon organoid differentiation conditions
The isolated Bama pig colon fossa prepared in this example was cultured according to the 3D culture system and amplification medium established in the present invention. In example 1 the inventors have been able to successfully culture and maintain the growth of colon organoids for a long period of time, but the current colon organoids are mainly composed of proliferating cells and stem cells, and therefore, in order to increase the proportion of differentiated cells, the inventors have optimized the culture medium by withdrawing the different components, as follows:
(1) the colon was isolated from adult Bama pigs by the method of example 1. It was slit and washed three times with pre-cooled PBS buffer pH 7.4 to remove chyme. The surrounding adipose and vascular tissue was then removed using a scalpel, and the remaining portion was cut into 10cm long pieces of intestine and incubated with 10mM EDTA in PBS buffer on ice for 40 minutes. The PBS buffer containing EDTA was replaced with fresh pre-cooled PBS buffer and then vigorously scraped using a scalpel to obtain a large number of colobomas. The resulting dimple suspension was transferred to a 1.5ml EP tube and left to stand for 1 minute to take the supernatant to remove the bulk tissue. Then, the scattered single cells were removed by centrifugation at 600rpm for 3 minutes. The pellet after centrifugation is a purer colostomy, which can be used for in vitro culture.
(3) Each 200 separated colostomy cells were mixed with Matrigel matrix (BD Biosciences), applied to a cell culture plate, coagulated in a 5% carbon dioxide cell culture chamber at 37 ℃ for 15-20 minutes, and then cultured with an amplification medium. The amplification medium contains basal medium Advanced DMEM/F12, serum substitutes N2 and B27, double antibody, 2mM GlutaMAX and 1mM N-acetyl cysteine, and a series of growth factors and small molecules as required: 50ng/mL EGF, 100ng/mL Noggin, 500ng/mL R-spondin1, 5. mu.M CHIR-99021, 0.5. mu. M A-83-01, 10. mu.M SB202190, 1nM Gastrin I, 10. mu. M Y27632, 2.5. mu.M PGE2, 10mM Nicotinamide and 30% Wnt3a conditioned medium. After several passages of colon organoids were cultured, the inventors cultured cells with four media of expansion, differentiation-1, differentiation-2 and differentiation-3, respectively, and observed the cell state for a long period of time. FIG. 3 (effect of different differentiation conditions on long-term culture of porcine colon organoids, photographed by an upright microscope) shows that colon organoids are still predominantly spherical within 18 days under the treatment of the expanded medium, whereas the proportion of budding or solid organoids increases significantly after 5 days of treatment with the three differentiation media, suggesting the appearance of differentiated cells. Colon organoids can only be maintained for about 9 days under differentiation-3 medium treatment, with later apparent death. Colon-like organ function was maintained for approximately 2 weeks under differentiation-2 medium treatment. While colon organoids can be maintained for at least 18 days under differentiation-1 medium treatment. It has been observed that the growth state is not significantly inhibited for 34 days. Therefore, in the subsequent studies, the differentiation-1 medium was used as an ideal differentiation medium.
Example 3: 3D culture system-based colon organoid differentiation capacity detection of Bama pigs and cynomolgus monkeys
According to the 3D culture system and the differentiation culture medium established by the invention, the isolated Bama pigs and the cynomolgus monkey colocrine fossils prepared in the embodiment are cultured and the differentiation capability is detected. The inventor uses a differentiation medium to carry out Q-PCR detection and staining observation on three-dimensionally cultured colon organoids, and the specific method is as follows:
(1) the colon was isolated from adult Bama pigs and cynomolgus monkeys by the method of example 1. It was slit and washed three times with pre-cooled PBS buffer pH 7.4 to remove chyme. The surrounding adipose and vascular tissue was then removed using a scalpel, and the remaining portion was cut into 10cm long pieces of intestine and incubated with 10mM EDTA in PBS buffer on ice for 40 minutes. The PBS buffer containing EDTA was replaced with fresh pre-cooled PBS buffer and then vigorously scraped using a scalpel to obtain a large number of colobomas. The resulting dimple suspension was transferred to a 1.5ml EP tube and left to stand for 1 minute to take the supernatant to remove the bulk tissue. Then, the scattered single cells were removed by centrifugation at 600rpm for 3 minutes. The pellet after centrifugation is a purer colostomy, which can be used for in vitro culture.
(3) Each 200 separated colostomy cells were mixed with Matrigel matrix (BD Biosciences), applied to a cell culture plate, coagulated in a 5% carbon dioxide cell culture chamber at 37 ℃ for 15-20 minutes, and then cultured with an amplification medium. The amplification medium contains basal medium Advanced DMEM/F12, serum substitutes N2 and B27, double antibody, 2mM GlutaMAX and 1mM N-acetyl cysteine, and a series of growth factors and small molecules as required: 50ng/mL EGF, 100ng/mL Noggin, 500ng/mL R-spondin1, 5. mu.M CHIR-99021, 0.5. mu. M A-83-01, 10. mu.M SB202190, 1nM Gastrin I, 10. mu. M Y27632, 2.5. mu.M PGE2, 10mM Nicotinamide and 30% Wnt3a conditioned medium. After several passages of colon organoids culture, the inventors induced colon organoids differentiation in bama pigs and cynomolgus monkeys using differentiation medium, and after 7 days of treatment, the inventors first observed morphological changes in the bama pig colon organoids. As shown in fig. 4A, the number of budding organoids increased significantly under differentiation medium treatment. Meanwhile, total mRNA is extracted, the expression change of each cell type marker gene is detected by using real-time fluorescent quantitative PCR, and GAPDH is used as an internal reference. As shown in fig. 4B, compared to the amplification medium, the expression of the intestinal epithelial cell marker genes Vil1 and Fabp1 was increased in the differentiation medium, and the expression of the goblet cell marker genes Muc2 and Tff3 was also significantly increased, accompanied by the increase in the expression of the enteroendocrine cell marker genes Chga and Chgb.
To further verify the presence of differentiated cells, the inventors stained intestinal epithelial cells, goblet cells and enteroendocrine cells, which were fixed with 4% paraformaldehyde for 15 minutes at room temperature. After three resuspension in PBS buffer, the cells were permeabilized with 0.5% Triton for one hour at room temperature, followed by blocking with 3% BSA and 0.1% Triton for two hours at room temperature, and then incubated overnight at 4 degrees after addition of Fabp1 (Abcam), Muc2 antibody (Santa-Cruz), ChgA antibody (Abcam) and E-cadherin antibody (BD). The next day, three rinses with PBS were performed, followed by addition of 488-coupled secondary mouse antibody, 561-coupled secondary rabbit antibody, and DAPI for incubation for one hour in the dark at room temperature, followed by rinsing with PBS and mounting, followed by photographing observation using a laser confocal microscope (FV 3000, Olympus). As shown in fig. 4C and 4D, Fabp1 positive cells and ChgA positive cells were both significantly elevated under differentiation medium treatment. To further verify goblet cell differentiation, the inventors first paraffin-embedded and sectioned Bama pig colon organoids and tissues, respectively, in expansion and differentiation medium using Alsinoblue staining, followed by deparaffinization and Alsinoblue staining (kit from BASO). As shown in fig. 4E, positive cells were significantly increased in differentiation medium, with a positive cell proportion similar to that of colon tissue in vivo. At the same time, this was also demonstrated by fluorescent staining with Muc2 (fig. 4F). The experiments show that the differentiation medium can well promote the differentiation of the colon organoid of the Bama pigs to enterocyte, goblet cell and enteroendocrine cell.
(4) The inventors also applied the differentiation medium to colon organoids of cynomolgus monkeys, as determined in the same manner as described above for the barnyard pig colon organoids. After 7 days of treatment, the inventors first observed morphological changes in cynomolgus monkey colon organoids. As shown in fig. 5A, growth of colon organoids was significantly inhibited and their size was significantly reduced with concomitant appearance of solid organoids. Meanwhile, the inventor extracts total mRNA, detects the expression change of each cell type marker gene by using real-time fluorescent quantitative PCR, and uses GAPDH as an internal reference. As shown in fig. 5B, compared to the amplification medium, the expression of the intestinal epithelial cell marker genes Vil1 and Fabp1 was significantly increased and the expression of the goblet cell marker genes Muc2 and Tff3 was also significantly increased under the differentiation medium. However, the expression of the intestinal secretory cell marker gene Chgb is only slightly increased, and Chga expression is too low to detect a significant change. Significant decreases in stem cells and proliferation-related genes occurred. To further verify the presence of differentiated cells, the inventors performed staining of intestinal epithelial cells, goblet cells and enteroendocrine cells. The inventors fixed it with 4% paraformaldehyde at room temperature for 15 minutes. After three resuspension rinses in PBS buffer, 0.5% Triton was used for one hour at room temperature for permeabilization. After subsequent blocking for two hours at room temperature using 3% BSA and 0.1% Triton, Fabp1(Abcam Co.), Muc2 antibody (Santa-Cruz Co.), ChgA antibody (Abcam Co.) and E-cadherin antibody (BD Co.) were added and incubated overnight at 4 degrees. The next day, three rinses with PBS were performed, followed by addition of 488-coupled secondary murine antibody, 561-coupled secondary rabbit antibody, and DAPI for incubation for one hour at room temperature in the dark, followed by rinsing with PBS and mounting. Then, the image was observed by a confocal laser microscope (FV 3000, Olympus). As shown in fig. 5C and 5D, Fabp 1-positive cells and Muc 2-positive cells were both significantly elevated under differentiation medium treatment. At this time, no positive cells for ChgA were detected. This also indicates that in cynomolgus monkey colon organoids, differentiation media mainly promote the differentiation of intestinal epithelial cells and goblet cells, while promoting enteroendocrine cells is very limited. The establishment of the colon organoid differentiation system makes the colon organoid differentiation system more approximate to the in vivo cell composition and structure, and provides an important basis for subsequent drug screening and steady state regulation and control research.
Example 4: colonic organoid drug toxicity sensitivity testing based on 3D culture system
In accordance with the 3D culture system and the differentiation medium established in the present invention, isolated Bama pigs and cynomolgus monkeys prepared in this example were cultured and differentiated for colonic fossa. To better perform drug development, screening animal models close to humans, the inventors developed a colon organoid drug toxicity sensitivity test based on a 3D culture system. The selected medicaments are all clinically common medicaments for resisting colorectal cancer. The specific method is as follows:
(1) the colon was isolated from adult Bama pigs, cynomolgus monkeys and humans by the method of example 1. It was slit and washed three times with pre-cooled PBS buffer pH 7.4 to remove chyme. The surrounding adipose and vascular tissue was then removed using a scalpel, and the remaining portion was cut into 10cm long pieces of intestine and incubated with 10mM EDTA in PBS buffer on ice for 40 minutes. The PBS buffer containing EDTA was replaced with fresh pre-cooled PBS buffer and then vigorously scraped using a scalpel to obtain a large number of colobomas. The resulting dimple suspension was transferred to a 1.5ml EP tube and left to stand for 1 minute to take the supernatant to remove the bulk tissue. Then, the scattered single cells were removed by centrifugation at 600rpm for 3 minutes. The pellet after centrifugation is a purer colostomy, which can be used for in vitro culture.
(2) Each 200 separated colostomy cells were mixed with Matrigel matrix (BD Biosciences), applied to a cell culture plate, coagulated in a 5% carbon dioxide cell culture chamber at 37 ℃ for 15-20 minutes, and then cultured with an amplification medium. The amplification medium contains basal medium Advanced DMEM/F12, serum substitutes N2 and B27, double antibody, 2mM GlutaMAX and 1mM N-acetyl cysteine, and a series of growth factors and small molecules as required: 50ng/mL EGF, 100ng/mL Noggin, 500ng/mL R-spondin1, 5. mu.M CHIR-99021, 0.5. mu. M A-83-01, 10. mu.M SB202190, 1nM Gastrin I, 10. mu. M Y27632, 2.5. mu.M PGE2, 10mM Nicotinamide and 30% Wnt3a conditioned medium. After the colon organoids were cultured for several generations, they were passaged to 96-well plates and differentiation medium was added to induce colon organoids differentiation in Bama pigs and cynomolgus monkeys. For human colon organoids, the inventors used expanded medium without Wnt3 a-conditioned medium to induce differentiation.
(3) While adding the differentiation medium, drugs with different concentration gradients, such as clinically common anti-colorectal cancer drugs Irinotecan (Irinotecan) and Regorafenib (Regorafenib), are added. The drug concentration was set based on previous reports. After 4 days, the inventors determined drug toxicity by recording colon organoid morphological changes (observed by upright microscopy) and cell viability assays. The Cell Viability Assay method is described in Cell-Titer Glo 3D Cell Viability Assay of Promega corporation. Add 25. mu.l Cell titer reagent into the cells, incubate them for 30 minutes at room temperature, centrifuge them for 1 minute at 500g, transfer the supernatant to a new 96-well plate, and detect the activity with a microplate reader.
As shown in fig. 6A and 6B, the colon organoids of bama pigs were very sensitive to irinotecan and nearly 70% of the cells had died under 1 μ M irinotecan treatment. While colon organoids of cynomolgus monkeys were relatively resistant to irinotecan, even with 50 μ M irinotecan treatment, 40% of the cells survived. Human colon organoid toxicity responses range between the two, similar to the Bama pig colon organoids.
As shown in fig. 6C and 6D, low concentrations of regorafenib were similarly toxic to the three organoids, while high concentrations of regorafenib induced significant cell death in the bama pig colon organoids, while cynomolgus monkey colon organoids remained antagonistic throughout. Human colon organoid toxicity responses are intermediate between those of Bama pigs and cynomolgus monkeys, and are more similar to those of Bama pigs. The colon of the Bama pig can be used as a better animal model to simulate the toxicity reaction of the human colon, and the establishment of the three-dimensional culture system provides an important basis for subsequent drug toxicity detection and drug screening.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (13)
1. A composition for obtaining a three-dimensional organoid, comprising:
n2, B27, diabody, GlutaMAX, N-acetylcysteine, epidermal growth factor, Noggin, R-spondin1, CHIR-99021, A-83-01, SB202190, Y27632, nicotinamide, prostaglandin E2 and Wnt3 a-CM.
2. The composition as claimed in claim 1, wherein said composition further comprises: gastrin I;
optionally, the composition is used to amplify a cell of a Bama pig and/or a cynomolgus monkey;
optionally, the cells are selected from stem cells and/or epidermal cells;
optionally, the cells are selected from intestinal stem cells and/or intestinal epithelial cells;
optionally, the cell is a colonic epithelial cell.
3. An amplification medium comprising:
the composition of any one of claims 1 to 2;
and (3) a basic culture medium.
4. The amplification medium of claim 3, wherein the basal medium is Advanced DMEM/F12;
optionally, the concentration of said GlutaMAX is 0.5-10mM, preferably 1-3 mM;
optionally, the concentration of N-acetylcysteine is 0.2-5mM, preferably 0.2-2 mM;
optionally, the concentration of the epidermal growth factor is 5-100ng/mL, preferably 40-60 ng/mL;
optionally, the concentration of Noggin is 5-200ng/mL, preferably 80-120 ng/mL;
optionally, the concentration of the R-spondin1 is 100-1000ng/mL, preferably 400-600 ng/mL;
optionally, the concentration of CHIR-99021 is 1-10. mu.M, preferably 4-6. mu.M;
optionally, the concentration of A-83-01 is 0.5-2. mu.M, preferably 0.5-1. mu.M;
optionally, the concentration of SB202190 is 1-10. mu.M, preferably 8-10. mu.M;
optionally, the concentration of Gastrin I is 1-10nM, preferably 1-3 nM;
optionally, the concentration of prostaglandin E2 is 1-5 μ M, preferably 2-3 μ M;
optionally, the concentration of nicotinamide is 1-10mM, preferably 8-10 mM;
optionally, the concentration of said N2 is 0.5-10mM, preferably 1-3 mM;
optionally, the concentration of B27 is 0.5-10mM, preferably 1-3 mM;
optionally, the concentration of the double antibody is 100-1000ng/mL, preferably 400-600 ng/mL;
optionally, the Wnt3a-CM volume is 10% -30%, preferably 20-30% of the total volume of the amplification medium;
optionally, the concentration of Y27632 is 1-10. mu.M, preferably 8-10. mu.M;
optionally, the diabesin is penicillin and streptomycin.
5. A method of obtaining a three-dimensional organoid, comprising:
mixing a cell sample with matrigel, and then adding the amplification culture medium of any one of claims 3-4 for amplification culture to form a three-dimensional organoid;
optionally, after mixing the cell sample with the matrigel, coagulating the cell sample in a 4-6% carbon dioxide incubator at 35-38 ℃ for 15-20 minutes;
optionally, the matrigel has a thickness of 40-60 μm, preferably 50-55 μm.
6. A three-dimensional organoid, wherein said three-dimensional organoid is obtained using the method of claim 5.
7. A composition for three-dimensional organoid differentiation according to claim 6, comprising:
n2, B27, diabody, GlutaMAX, N-acetylcysteine, epidermal growth factor, Noggin, R-spondin1, Y27632, A-83-01, Gastrin I.
8. The composition as claimed in claim 7, wherein said composition further comprises: nicotinamide and/or SB 202190;
optionally, the composition further comprises CHIR-99021;
optionally, the three-dimensional organoid is selected from a cell of a mare pig and/or a cynomolgus monkey;
optionally, the three-dimensional organoid is selected from stem cells and/or epidermal cells;
optionally, the three-dimensional organoid is selected from intestinal stem cells and/or intestinal epithelial cells;
optionally, the three-dimensional organoid is a colonic epithelial cell.
9. A differentiation medium, comprising:
the composition of any one of claims 7 to 8;
and (3) a basic culture medium.
10. The differentiation medium according to claim 9, wherein the basal medium is Advanced DMEM/F12;
optionally, the concentration of said GlutaMAX is 0.5-10mM, preferably 1-3 mM;
optionally, the concentration of N-acetylcysteine is 0.2-5mM, preferably 0.2-2 mM;
optionally, the concentration of the epidermal growth factor is 5-100ng/mL, preferably 40-60 ng/mL;
optionally, the concentration of Noggin is 5-200ng/mL, preferably 80-120 ng/mL;
optionally, the concentration of the R-spondin1 is 100-1000ng/mL, preferably 400-600 ng/mL;
optionally, the concentration of CHIR-99021 is 1-5. mu.M, preferably 2-3. mu.M;
optionally, the concentration of A-83-01 is 0.5-2. mu.M, preferably 0.5-1. mu.M;
optionally, the concentration of SB202190 is 1-10. mu.M, preferably 8-10. mu.M;
optionally, the concentration of Gastrin I is 1-10nM, preferably 1-3 nM;
optionally, the concentration of nicotinamide is 1-10mM, preferably 8-10 mM;
optionally, the concentration of said N2 is 0.5-10mM, preferably 1-3 mM;
optionally, the concentration of B27 is 0.5-10mM, preferably 1-3 mM;
optionally, the concentration of the double antibody is 100-1000ng/mL, preferably 400-600 ng/mL;
optionally, the concentration of Y27632 is 1-10. mu.M, preferably 8-10. mu.M;
optionally, the diabesin is penicillin and streptomycin.
11. A kit comprising the composition of claims 1 to 2, the amplification medium of claims 3 to 4, the composition of claims 7 to 8, and/or the differentiation medium of claims 9 to 10.
12. Use of the composition of claims 1-2, the amplification medium of claims 3-4, the composition of claims 7-8, the differentiation medium of claims 9-10, or the kit of claim 11 in drug screening or drug analysis.
13. A method for detecting drug toxicity, comprising:
culturing a candidate drug with a cell sample cultured in the amplification medium according to any one of claims 3 to 4;
carrying out differentiation culture on the cultured cells obtained in the previous step in a differentiation culture medium according to any one of claims 9-10 to obtain differentiated cells;
taking the detection result of the cell viability before and after the differentiation culture as an indication of whether the drug toxicity exists;
optionally, the culturing time is 2-10 days.
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| CN114908037A (en) * | 2022-05-17 | 2022-08-16 | 郑州大学第五附属医院 | Culture medium of human intestinal metaplasia gas-liquid interface model, construction method and application |
| CN116751749A (en) * | 2023-08-15 | 2023-09-15 | 南昌大学 | Body fluid tumor organoid culture method and drug sensitivity detection method |
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| CN116751749A (en) * | 2023-08-15 | 2023-09-15 | 南昌大学 | Body fluid tumor organoid culture method and drug sensitivity detection method |
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