Compositions, media and their use in gut organoid culture

Abstract

The present application relates to the field of biotechnology, and particularly relates to a composition, a culture medium and application thereof in intestinal organoid culture. The present application first applies glucosamine hydrochloride to the culture of intestinal organoids, which can effectively promote organ proliferation and differentiation, and significantly increase the number of organ formation. On this basis, the present application constructs a three-dimensional culture system for postpartum neonatal, childhood and adult stages, and realizes the precise construction of mouse intestinal organoids at different development stages by combining dynamic regulation of extracellular matrix and optimization of growth factor combinations, thereby providing an in-vitro model covering multiple time sequences for intestinal development mechanism research and disease modeling.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a composition, a culture medium and the application thereof in intestinal organoid culture. BACKGROUND

[0002] Intestinal development is a complex process regulated by multiple stages and factors, and the cell composition and function are significantly different at different development stages. For example, during the embryonic period, the intestine is mainly composed of stem cell proliferation, cell differentiation begins in the neonatal period, and the mature crypt-villus structure is formed in adulthood. Traditional research methods rely on animal models or two-dimensional cell culture, but animal models are costly and difficult to observe in real time, and two-dimensional culture cannot simulate the three-dimensional structure of the intestine and cell-cell interaction.

[0003] Most of the current intestinal organoids are formed by static culture or single-stage induction, which cannot reproduce the dynamic development process from the postnatal neonatal period to the mature period, limiting their application in developmental biology research. For example, in 2009, Sato et al. (Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262-266) used high-concentration Wnt3a and R-spondin1 to maintain intestinal stem cell proliferation, and obtained embryonic organoids; reducing Wnt signaling and adding BMP4, obtaining partially mature organoids. Specifically, in the culture medium for culturing small intestinal organoids, R-spondin, Noggin, and epidermal growth factor (EGF) are added as Wnt pathway agonists, transforming growth factor-β inhibitors, and epidermal growth factor (EGF), respectively. In addition to these three factors, Wnt-3A is also added in the culture of colon organoids. Although appropriate growth factors and differentiation factors are selected for different parts, this system does not design a differentiated culture system for the development characteristics of the postnatal neonatal period, childhood, and adulthood, and cannot realize the sequential construction of multi-stage organoids, nor can it be integrated into a time sequence model covering the entire development process of the intestine, limiting the study of the dynamic mechanism of intestinal development. Culturing intestinal organoids from mice of different ages is not applicable, especially for postnatal neonatal mice. The intestinal tissue of postnatal and neonatal mice is small, fragile, and difficult to isolate crypts. Therefore, it is crucial to construct a time sequence model containing multi-stage organoids for intestinal development research. SUMMARY

[0004] Therefore, the present application provides a composition, a culture medium and the application thereof in intestinal organoid culture. The present application research found that culturing intestinal crypt stem cells using glucosamine hydrochloride and its composition can significantly improve the formation rate of organoids.

[0005] Use of glucosamine hydrochloride in stem cell culture or organoid culture.

[0006] Further, the stem cells include intestinal stem cells; and the organoids include intestinal organoids.

[0007] Glucosamine hydrochloride is a common dietary supplement, commonly used for joint cartilage protection and repair, mainly suitable for the auxiliary treatment of joint degenerative diseases such as osteoarthritis. The present application first applies glucosamine hydrochloride to the culture of intestinal organoids. It is found through research that glucosamine hydrochloride can effectively promote the proliferation and differentiation of intestinal stem cells, significantly increase the number of intestinal organoids formed, and be suitable for the culture of intestinal organoids. In the present application, the intestinal stem cells include postpartum neonatal intestinal stem cells, childhood intestinal stem cells or adult intestinal stem cells. The intestinal stem cells are small intestinal stem cells or colon stem cells, preferably intestinal crypt stem cells. In specific embodiments of the present application, the intestinal stem cells are crypt stem cells from the duodenum. In the present application, the source of the intestinal stem cells is a mouse or a rat.

[0008] The present application provides a composition comprising a growth factor and glucosamine hydrochloride.

[0009] In some embodiments, the growth factor is selected from at least one of Wnt3a, R-spondin1, EGF, BMP4, FGF2, and HGF.

[0010] The present application uses postpartum neonatal, childhood or adult intestinal crypt stem cells as culture objects respectively, and specifically combines glucosamine hydrochloride and growth factors according to the growth needs of intestinal organoids at different development stages, significantly improves the morphological and functional accuracy of organoids at each development stage, and realizes the sequential construction of multi-stage organoids. In the present application, the intestinal stem cells are located at the bottom of the intestinal crypt, each intestinal crypt contains 3-4 intestinal stem cells, and the intestinal stem cell refers to the stem cell at the bottom of the intestinal crypt.

[0011] In some embodiments, the composition for postpartum neonatal intestinal stem cell culture comprises Wnt3a, R-spondin1, EGF and glucosamine hydrochloride.

[0012] In some embodiments, the composition for childhood intestinal stem cell culture comprises Wnt3a, R-spondin1, BMP4, FGF2 and glucosamine hydrochloride.

[0013] In some embodiments, the composition for adult intestinal stem cell culture comprises HGF and glucosamine hydrochloride.

[0014] Further, the composition for culturing adult intestinal stem cells further comprises DAPT and / or prostaglandin. The specific type of prostaglandin is not particularly limited in the present application, and any commonly used or known type can be used. In a specific embodiment of the present application, the prostaglandin is prostaglandin E2.

[0015] The present application also provides a culture medium for culturing intestinal organoids, which comprises a basal medium and the composition of the present application.

[0016] In some embodiments, the concentration of glucosamine hydrochloride in the culture medium is 30-250 μg / mL, preferably 50-200 μg / mL, and specifically 50 μg / mL, 100 μg / mL or 200 μg / mL.

[0017] In some embodiments, the culture medium of the present application comprises at least one of the following culture media:

[0018] Culture medium 1 for culturing postnatal neonatal intestinal stem cells, culture medium 2 for culturing pediatric intestinal stem cells, and culture medium 3 for culturing adult intestinal stem cells;

[0019] The culture medium 1 comprises a basal medium, Wnt3a, R-spondin1, EGF and glucosamine hydrochloride;

[0020] The culture medium 2 comprises a basal medium, Wnt3a, R-spondin1, BMP4, FGF2 and glucosamine hydrochloride;

[0021] The culture medium 3 comprises a basal medium, HGF, DAPT, prostaglandin and glucosamine hydrochloride.

[0022] In the present application, the concentration of each culture medium is the working concentration, which refers to the concentration of each culture medium used when culturing cells.

[0023] In some embodiments, the basal medium is DMEM / F12, Advanced DMEM / F12, IMDM, F12 or α-MEM. Among them, Advanced DMEM / F12 refers to a modified culture medium based on DMEM / F12, including commonly modified types in the art.

[0024] In some embodiments, the culture medium 1 comprises a basal medium and the following components at the following concentrations:

[0025] 50-150 ng / mL Wnt3a, 400-600 ng / mL R-spondin1, 30-70 ng / mL EGF, 150-250 μg / mL glucosamine hydrochloride.

[0026] In some embodiments, the medium 1 comprises a basal medium and the following components at the following concentrations:

[0027] 100 ng / mL Wnt3a, 500 ng / mL R-spondin1, 50 ng / mL EGF, 200 μg / mL glucosamine hydrochloride.

[0028] In some embodiments, the medium 2 comprises a basal medium and the following components at the following concentration ranges:

[0029] 30-70 ng / mL Wnt3a, 150-250 ng / mL R-spondin1, 10-30 ng / mL BMP4, 5-15 ng / mL FGF2, and 75-125 μg / mL glucosamine hydrochloride.

[0030] In some embodiments, the medium 2 comprises a basal medium and the following components at the following concentrations:

[0031] 50 ng / mL Wnt3a, 200 ng / mL R-spondin1, 20 ng / mL BMP4, 10 ng / mL FGF2, and 100 μg / mL glucosamine hydrochloride. The basal medium is DMEM / F12.

[0032] In some embodiments, the medium 3 comprises a basal medium and the following components at the following concentration ranges:

[0033] 30-70 ng / mL HGF, 5-15 μM DAPT, 5-15 μM prostaglandin, and 30-70 μg / mL glucosamine hydrochloride.

[0034] In some embodiments, the medium 3 comprises a basal medium and the following components at the following concentrations:

[0035] 50 ng / mL HGF, 10 μM DAPT, 1 μM prostaglandin E2, and 50 μg / mL glucosamine hydrochloride. The basal medium is DMEM / F12.

[0036] The present application also provides a culture reagent for intestinal organoids, which comprises the culture medium and the substrate as described above.

[0037] In some embodiments, the substrate comprises: a substrate 1 for culturing postpartum neonatal intestinal stem cells, a substrate 2 for culturing childhood intestinal stem cells, and a substrate 3 for culturing adult intestinal stem cells.

[0038] The matrix 1 comprises Matrigel and collagen type I; preferably, the volume ratio of the Matrigel and collagen type I is 3:1.

[0039] The matrix 2 comprises Matrigel and basal medium; preferably, the volume ratio of the Matrigel and basal medium is 1:1.

[0040] The matrix 3 comprises Matrigel and fibronectin; preferably, the volume ratio of the Matrigel and fibronectin is 2:1.

[0041] The application further provides application of the composition, the culture medium or the culture reagent in culturing intestinal stem cells. In the application, the intestinal stem cells include postpartum neonatal intestinal stem cells, childhood intestinal stem cells and adult intestinal stem cells. Further, the intestinal stem cells are small intestinal stem cells or colon stem cells. Still further, the intestinal stem cells are derived from mice or rats.

[0042] The application further provides application of the composition, the culture medium or the culture reagent in constructing intestinal organoids. In the application, the intestinal organoids are mouse intestinal organoids or rat intestinal organoids.

[0043] The application further provides a culture method of intestinal stem cells, which comprises selecting a corresponding culture medium in the culture medium 1-3 according to the development period of the intestinal stem cells.

[0044] The application further provides a construction method of intestinal organoids, which comprises: selecting an EDTA treatment time according to the development period of mice, isolating intestinal crypts, selecting a matrix in the matrix 1-3 and mixing the intestinal crypts with a corresponding 1-3 culture medium, and culturing.

[0045] In the application, the step of treating the intestinal stem cells with EDTA is further included before mixing the matrix and the intestinal stem cells. In some specific embodiments, the treatment time of the postpartum neonatal intestinal stem cells is 10-15 min. In some specific embodiments, the treatment time of the childhood intestinal stem cells is 15-25 min. In some specific embodiments, the treatment time of the adult intestinal stem cells is 25-35 min.

[0046] In the present application, the crypt + matrix liquid mixture is dropped into the center of a 24-well culture plate using a pipette, and is solidified at 37 DEG C to form a liquid structure containing crypts inside, similar to the principle of amber formation. The volume of a droplet is 50-60 microliters, and the number of crypts in a droplet should not be too high, otherwise it may affect the growth state of the crypts and the imaging effect. In the present application, one droplet contains 50-100 crypts, and the seeding density of the intestinal crypts is 50-100 crypts per 60 microliters of matrix. Specifically, it can be 50 crypts per 60 microliters of matrix, 60 crypts per 60 microliters of matrix, 70 crypts per 60 microliters of matrix, 80 crypts per 60 microliters of matrix, 90 crypts per 60 microliters of matrix, or 100 crypts per 60 microliters of matrix. In the present application, the culture time is 5-7 days, specifically 5 days, 6 days or 7 days. The background is clear and does not affect growth.

[0047] In the present application, after mixing the intestinal crypts and the matrix, the system after mixing is placed at 37 DEG C to promote the matrix to solidify into a gel. Research has found that the optimal temperature for the solidification of the matrix 1-3 is 37 DEG C. If the temperature is lower than this, the solidification speed will be significantly reduced, and the gel structure may be loose and unstable. If the temperature is too high (such as more than 40 DEG C), the biological active ingredients in the matrix gel may be damaged, affecting its function.

[0048] The present application also provides an intestinal organoid obtained by the above construction method.

[0049] The present application also provides the use of the above intestinal organoid or the intestinal organoid obtained by the above construction method in any one of the following:

[0050] (1) constructing an intestinal disease model;

[0051] (2) constructing a drug screening or drug evaluation model;

[0052] (3) food safety evaluation.

[0053] The present application upgrades from "single growth factor regulation" to "matrix component-metabolic precursor-growth factor" synergistic regulation, and solves the problem of incomplete organoid development period in the prior art by changing the intestinal stem cell separation conditions, matrix components, and growth factors according to the growth needs of intestinal organoids at different development stages. This improvement not only significantly improves the morphological and functional accuracy of organoids at each development stage, but also constructs the first intestinal development timing model based on glycosaminoglycan metabolism, providing a more in-vivo physiological state in-vitro tool for developmental biology research and disease modeling, which has significant technical breakthroughs and application value.

[0054] The present application is directed to postpartum neonatal period, childhood, adulthood, and designs different cell separation conditions (such as EDTA treatment time), culture medium formula (growth factor combination) and matrix composition, introduces hydrochloric acid glucosamine, and forms a ternary regulation system of "matrix composition + precursor + growth factor" through different concentrations (50-200 μg / mL). Experiments show that the matrix composition, growth factor and precursor (such as hydrochloric acid glucosamine) in the culture medium provided by the present application have obvious synergistic effect on the culture of intestinal organoids, and are indispensable.

[0055] The culture medium and culture method provided by the present application have at least one of the following advantages:

[0056] Stage-specific function enhancement:

[0057] Postpartum neonatal period: hydrochloric acid glucosamine and type I collagen synergistically improve the adhesion and proliferation efficiency of stem cells;

[0058] Childhood: inducing intestinal stem cells to differentiate into secretory cells (such as goblet cells and chromaffin cells) through glycosaminoglycans;

[0059] Adulthood: enhancing the mechanical properties of the matrix to maintain the structure and function of the mature organoid.

[0060] Time sequence model integration: integrating different development period organoids in the order of postpartum neonatal period→childhood→adulthood to form a time sequence model covering the whole process of intestinal development.

[0061] Specific culture system: three-dimensional culture system of organoids at each stage, including matrix composition ratio, culture medium formula and culture operation process.

[0062] Stage-specificity: optimizing the proliferation environment for postpartum neonatal period to improve the formation rate of organoids, precisely inducing differentiation in childhood to enrich cell types, and promoting functional maturation in adulthood to make the organoids have a structure closer to the in-vivo crypt-villus structure.

[0063] Time sequence integrity: through stage-by-stage culture and integration, a time sequence model covering the postpartum neonatal period to adulthood is constructed, which can continuously study the dynamic changes of cell proliferation, differentiation and functional maturation during intestinal development.

[0064] High application value: provides multi-stage organoids for modeling intestinal development related diseases (such as children's intestinal development disorder and adult intestinal degenerative diseases), and can also be used for drug efficacy evaluation at different development periods, improving research reliability.

[0065] The application constructs a three-dimensional culture system induced by phased timing, and dynamically optimizes the EDTA processing time (10-15 min of precise dissociation of fragile crypts in the postnatal neonatal period), matrix composition (such as mixing Matrigel and collagen type I 3:1 in the postnatal neonatal period to simulate a soft matrix microenvironment), and growth factor combination (high-concentration Wnt3a and R-spondin1 in the postnatal neonatal period to promote stem cell proliferation), and innovatively introduces hydrochloric acid glucosamine (50 μg / mL in the postnatal neonatal period) to enhance glycosaminoglycan synthesis, so that the organoid formation rate in the postnatal neonatal period is increased from 30% (the highest organ formation rate reported by the traditional method) to 85%, and the crypt-villus structure integrity rate is more than 90%, effectively solving the problems of easy loss and structure damage of neonatal organoids caused by the complexity of traditional methods and poor matrix matching, and becoming the first intestinal organoid culture system covering the whole development stage of mice. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 The technical flowchart of the application is shown in the figure;

[0067] Figure 2 The mouse intestinal development timing constructed by the method of the application is shown in the figure, wherein A~D are respectively representative images of intestinal organoids in the postnatal neonatal period, childhood, and adulthood of the traditional method, and E~H are respectively representative images of intestinal organoids in the postnatal neonatal period, childhood, and adulthood of the method of the application;

[0068] Figure 3 The number of mouse intestinal organoids constructed by the method of the application is shown in the figure; wherein A~D are respectively the results of the 10th day after birth, the 12th day after birth, childhood, and adulthood;

[0069] Figure 4 The percentage of the number of mouse intestinal organoids constructed by the method of the application is shown in the figure; wherein A~D are respectively the results of the 10th day after birth, the 12th day after birth, childhood, and adulthood. DETAILED DESCRIPTION

[0070] The present application provides compositions, culture media and their use in intestinal organoid culture. Those skilled in the art can improve the process parameters as appropriate based on the content herein. In particular, it is pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0071] Related term explanation

[0072] Organoid: A miniature organ structure formed by stem cells in vitro three-dimensional culture, with similar cell types and functions to in vivo organs, supporting cell growth and differentiation in three-dimensional space through a combination of extracellular matrix (such as Matrigel) and nutritional factors.

[0073] Budding (organoid): The number of bud-like structures protruding outward from the main structure of a single organoid in a three-dimensional culture system. In the development process of intestinal organoids, "budding" is an important feature of organoid to form functional substructures (such as crypts, villus-like protrusions), reflecting the ability of cell proliferation, differentiation and tissue structure construction.

[0074] Temporal development: Refers to the continuous and orderly development process of an organism from development maturity through postnatal neonatal, childhood and adulthood stages.

[0075] Dynamic regulation: In the culture process, the separation conditions and culture conditions (such as growth factors, matrix components, etc.) are adjusted according to the needs of different development stages.

[0076] Glucosamine hydrochloride: Amino derivative of glucose, as a precursor of glycosaminoglycans (such as hyaluronic acid, chondroitin sulfate), involved in extracellular matrix synthesis, regulating cell adhesion, signal transduction and matrix mechanical properties, used in the present application to optimize the microenvironment of organoid culture at each stage.

[0077] In this paper, "including", "containing" and "having" are described, including a closed technical solution consisting of the listed features, and also including an open technical solution containing the listed features.

[0078] In this paper, the term "and / or" includes any and all combinations of one or more related listed items.

[0079] As used herein, it is intended that values disclosed herein include all values customarily used within the range that is being described. It is also intended that each successive range build on the preceding ranges and are thus disclosed. For example, it is intended that claims which state a range include any and all sub-ranges falling within the range. It is also intended that amounts provided herein include any and all impurities that occur to the compounds during the usual manufacturing practices of such compounds. Further, the disclosure of ranges includes endpoints.

[0080] As used herein, units associated with a range of data, if only the right end point is provided with a unit, it means that the units of the left end point and the right end point are the same. For example, 50-150 ng / mL means that the units of the left end point "50" and the right end point "150" are both ng / mL.

[0081] As used herein, P10 refers to the 10th day after birth of an animal, P12, P40, P60 refer to the 12th day, the 40th day, the 60th day after birth, respectively.

[0082] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market.

[0083] The present application is further described below in conjunction with examples:

[0084] Example 1

[0085] 1.1 Isolation and enrichment of intestinal stem cells at different developmental stages

[0086] (1) Sample processing

[0087] The duodenum of mice at postnatal neonatal stage (P10, P12), childhood stage (P40), and adult stage (P60) was taken, the inside of the intestinal tube was washed with cold DPBS to remove food residues, connective tissue was removed, the intestinal tube was opened longitudinally to expose the intestinal cavity, and was shaken and washed until no visible fragments were left.

[0088] The intestinal segments were cut into pieces (1-2 mm) and 20 mM EDTA was added. The EDTA treatment time was adjusted according to the developmental stage: postnatal neonatal stage: 10-15 minutes, childhood stage: 15-25 minutes, and adult stage: 25-35 minutes.

[0089] (2) Crypt enrichment

[0090] The intestinal segment pieces were blown and filtered through a 70 μm filter to collect crypt tissue. Centrifugation (4°C, 400-500g, 5min) was performed, and DMEM / F-12 medium containing 10% fetal bovine serum (FBS) was added to terminate the EDTA effect. Microscopic counting was performed to ensure that about 100 crypts were inoculated per well.

[0091] 1.2 Construction of the three-dimensional culture system in stages

[0092] (1) Postnatal neonatal period (P10, P12) culture

[0093] Matrix: Matrigel and collagen type I were mixed at a volume ratio of 3:1 in a 1.5 ml-EP centrifuge tube and placed in an ice bath.

[0094] Mixed inoculation: The isolated postnatal neonatal intestinal crypts were counted under a microscope and mixed with the prepared matrix (Matrigel and collagen type I mixed at a volume ratio of 3:1). Ensure 50-100 crypts / 60ul matrix. Add the crypt-containing matrix to the center of the culture plate, place it in a 37℃-CO2 incubator for 10-15 min, solidify to form a 3D droplet-shaped dome structure, and add the corresponding culture medium along the side wall of the culture plate for 5-7 days.

[0095] Culture medium: DMEM / F12 with Wnt3a (100 ng / mL), R-spondin1 (500 ng / mL), EGF (50 ng / mL), and glucosamine hydrochloride (200μg / mL).

[0096] Culture: After inoculation, solidify the matrix glue in a 37℃ incubator, add complete medium along the side wall, change the medium every 2 days, and culture for 5-7 days.

[0097] (2) Childhood (P40) culture:

[0098] Matrix: Matrigel and basal medium were mixed at a volume ratio of 1:1 in a 1.5 ml-EP centrifuge tube and placed in an ice bath.

[0099] Mixed inoculation: The isolated childhood intestinal crypts were counted under a microscope and mixed with the prepared matrix (Matrigel and basal medium mixed at a volume ratio of 3:1). Ensure 50-100 crypts / 60ul matrix. Add the crypt-containing matrix to the center of the culture plate, place it in a 37℃-CO2 incubator for 10-15 min, solidify to form a 3D droplet-shaped dome structure, and add the corresponding culture medium along the side wall of the culture plate for 5-7 days.

[0100] Culture medium: Add Wnt3a (50 ng / mL), R-spondin1 (200 ng / mL), BMP4 (20 ng / mL), FGF2 (10 ng / mL), and glucosamine hydrochloride (100μg / mL).

[0101] Culture: solidify Matrigel after inoculation in 37℃ incubator, add complete medium along the side wall, change liquid every 2 days, culture for 5-7 days.

[0102] (3) Adult (P60) culture:

[0103] Matrix: Matrigel and fibronectin are mixed at a volume ratio of 2:1 in a 1.5ml-EP centrifuge tube and placed in an ice bath.

[0104] Mixed inoculation: the separated adult intestinal crypts are counted under a microscope and mixed with the prepared matrix (Matrigel and fibronectin mixed at a volume ratio of 2:1), ensuring 50-100 crypts / 60ul matrix. The matrix containing the crypts is added to the center of the culture plate and placed in a 37℃-CO2 incubator for 10-15min to solidify and form a 3D droplet-shaped dome structure. The corresponding medium is added along the side wall of the culture plate and cultured for 5-7 days.

[0105] Culture medium: add HGF (50 ng / mL), DAPT (10 μM), prostaglandin E2 (1 μM), and glucosamine hydrochloride (50 μg / mL).

[0106] Culture: solidify Matrigel after inoculation in 37℃ incubator, add complete medium along the side wall, change liquid every 2 days, culture for 5-7 days.

[0107] 1.3 Analysis and identification of organoids

[0108] Morphology and growth characterization analysis: observe the morphology of organoids under a microscope and compare the morphological characteristics of organoids at different developmental stages.

[0109] Formation rate analysis: statistically analyze the number of three-dimensional spatial structure organoids formed at different developmental stages.

[0110] Sprouting number analysis: statistically analyze the number of sprouts per organoid at different developmental stages, and the proportion of organoids with different sprout numbers.

[0111] 1.4 Technical process Figure 1 .

[0112] Example 2

[0113] This example designs a control experiment to compare the culture effect when a key factor is missing or changed, and to clarify the obvious synergistic effect between the components.

[0114] (I) Experimental design

[0115] Each group used 3 C57BL / 6 mice of the same age, after the duodenal crypt was isolated, inoculated into the culture medium of the corresponding stage (with / without glucosamine hydrochloride), 6 biological replicates were set in each group, and the number of organoids formed was counted after 7 days of culture (defined as three-dimensional structures with a diameter > 100 μm).

[0116] (II) Experimental method

[0117] 1. The culture method of the application - experimental group: For postpartum newborn, childhood and adult mice, culture according to the method of Example 1 while adding glucosamine hydrochloride (the concentration is optimized according to different stages, such as 50 μg / mL for adult mice).

[0118] 2. Traditional method - control group: According to the culture medium and method adopted by Sato et al. in "Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche" in 2009, the general method is as follows, and the specific method and parameters are referred to the method summary in the last paragraph of the article:

[0119] (1) Isolation of mouse intestinal crypts

[0120] Take the proximal 20 cm small intestine of the mouse after death, remove the membrane, blood vessels and fat, and place it in cold PBS.

[0121] Colon samples need to be taken 3-6 cm, a small amount of tissue below the cecum and above the rectum is removed to avoid toxin contamination.

[0122] Rinse the intestinal segment with cold PBS, cut the intestinal tube longitudinally, and wash the intestinal cavity 3 times.

[0123] Cut the intestinal tube into 2 mm segments, rinse with PBS for 15-20 times until the supernatant is clear (colon needs to be rinsed 15 times). Incubate with a mild cell dissociation reagent at room temperature for 15 minutes (colon for 20 minutes), collect the cell suspension by filtration, and obtain the crypt precipitate after centrifugation.

[0124] (2) Isolation of crypt organoids

[0125] Crypt quality assessment: Under an inverted microscope, select crypts with regular morphology (rectangular or circular, smooth edges), count and calculate the concentration. Mix the crypts with Matrigel® at a ratio of 1:1, inoculate into a preheated 24-well plate, form a dome structure, and solidify at 37°C for 10 minutes.

[0126] Culture and observation: add complete culture medium, cultivate at 37°C, 5% CO2. Intestinal organoids form spherical structures in 3 hours, start to bud in 2-4 days, and form multilobular structures in 5-7 days; colon organoids grow more slowly, form cystic structures in 2 days, and bud in 7-10 days.

[0127] (3) The culture medium composition used in the culture process refers to the method summary in the last paragraph of the article.

[0128] (Three) Experimental results

[0129] 1. Morphological photographs of intestinal organoids formed by postpartum newborn, childhood and adult mice at 7 days of culture

[0130] Organoid morphology and structure: The organoids in the method group of the application exhibit a crypt-villus structure that is closer to the in vivo intestinal tissue at each developmental stage, with clear crypts and regular villus arrangement. For example, the crypt depth of childhood organoids can reach 50-70 pm on average, and the villus length is 100-150 pm. The crypt-villus structure of the organoids in the traditional method group is not fully developed, with a crypt depth of only 20-30 pm, short and sparse villi, and some organoids even exhibit structural disorder and cannot form a typical intestinal epithelial structure.

[0131] Figure 2 To show the morphology of intestinal organoids at different developmental stages at 7 days of culture. Among them, A-D are the traditional organoid construction method, and the intestinal organoids formed at postpartum 10, 12 days (A-B), childhood (E), and adulthood (D). E-H are the organoids constructed by the method of the application at the corresponding periods, and their morphology at 7 days of culture (Day7). By comparison, it can be directly observed that the traditional method is not suitable for the construction of postpartum newborn intestinal organoids, and it is difficult to form a 3D organoid spatial structure (A-B), while the postpartum newborn, childhood, and adult intestinal organoids constructed by the method of the application have complete 3D spatial structure (E-H), showing the morphological evolution from postpartum development to adulthood, and clearly showing the growth differences and morphological characteristics of organoids at each stage.

[0132] 2. Statistical analysis of the number of intestinal organoids formed by postpartum newborn, childhood and adult mice

[0133] Compared with the traditional method, the number of organoids formed in each stage was significantly increased (P<0.01); the formation rate in the postnatal neonatal period was 200% higher than that of the traditional method, verifying the synergistic effect of glucosamine hydrochloride and matrix components (Matrigel / Type I collagen) in promoting the survival of fragile neonatal crypts. The traditional method group almost cannot form organoids with three-dimensional structure in the postnatal neonatal period, suggesting that the intestinal crypts in the postnatal neonatal period are fragile, and glucosamine hydrochloride (50 μg / mL) can enhance crypt-matrix adhesion by promoting glycosaminoglycan synthesis, significantly increasing the number of organoids, and gradually increasing with the culture period (1-7 days in a culture cycle), which confirms the key regulatory role of the component in the formation of organoids in the postnatal neonatal period (corresponding to the initial formation of intestinal villi in vivo).

[0134] Figure 3 For the analysis results of the number of intestinal organoids formed at different development stages under the same inoculation density. A-D correspond to the organoids on the 10th day after birth, the 12th day after birth, the childhood period, and the adult period, respectively. The number of organoids was analyzed and compared, and it was found that the number of organoids formed by the method of the application in the postnatal neonatal period, childhood period and adult period was significantly higher than that of the traditional method. ns: no significant difference compared with the control group; * represents p<0.05 compared with the control group; ** represents p<0.01 compared with the control group; *** represents p<0.001 compared with the control group.

[0135] 3. Statistical analysis of the percentage of the number of sprouts of mouse intestinal organoids in the postnatal neonatal period, childhood period and adult period

[0136] Postnatal neonatal period specific breakthrough: the proportion of 3-5 sprouted organoids by the method of the application is significantly higher than that of the traditional method, which confirms that glucosamine hydrochloride (50 μg / mL) and type I collagen matrix synergistically promote the sprouting of neonatal organoids (corresponding to the peak of intestinal epithelial proliferation in vivo).

[0137] Development stage dynamic matching: the proportion of adult organoids with more than 5 sprouts is 55.0%, which is consistent with the branching characteristics of mature intestinal crypt-villus in vivo, while the number of sprouts in each stage by the traditional method has no significant difference, indicating that the staged regulation (such as the addition of glucosamine hydrochloride) of the application effectively induces the functional maturation of organoids;

[0138] Necessity of glucosamine hydrochloride: almost no organoids with more than 1 sprout can be formed in the postnatal neonatal period by the traditional method, suggesting that the lack of this component will lead to insufficient proliferation and differentiation of organoids, which confirms its key role as a glycosaminoglycan precursor in matrix adhesion and organoid growth at this growth stage.

[0139] Figure 4Statistical analysis results of the percentage of the total number of intestinal organoids for different development stages (the more the number of organoid sprouts, the stronger the differentiation ability). A-D correspond to the percentage of different sprout numbers of organoids at the 10th day after birth, the 12th day after birth, childhood, and adulthood, respectively. Analysis and comparison found that the percentage of the number of organoids with more sprouts formed by the method of the application in the postnatal newborn period, childhood, and adulthood was significantly higher than that by the traditional method. ns: no significant difference compared with the control group; * represents p<0.05 compared with the control group; ** represents p<0.01 compared with the control group; *** represents p<0.001 compared with the control group.

[0140] Black: percentage of the total number of organoids with 0-2 sprouts; green: percentage of the total number of organoids with 3-5 sprouts; purple: percentage of the total number of organoids with >5 sprouts. Among them, old: traditional method; new: method of the application.

[0141] The above is only the preferred embodiment of the application, and it should be noted that for those skilled in the art, without departing from the principles of the application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the protection scope of the application.

Claims

1. Use of glucosamine hydrochloride in intestinal stem cell culture or intestinal organoid culture; the use is non-disease treatment purpose.

2. A composition for intestinal stem cell culture or intestinal organoid culture, characterized by, including a growth factor and glucosamine hydrochloride; the growth factor is at least one selected from Wnt3a, R-spondin1, EGF, BMP4, FGF2, HGF.

3. The composition of claim 2, wherein, DAPT and / or prostaglandin are further included.

4. A medium for culturing enteroids, characterized in that, including a basal medium and the composition of any one of claims 2-3; the concentration of glucosamine hydrochloride in the medium is 30-250 μg / mL.

5. The medium of claim 4, characterized in that, the medium includes at least one of the following media 1-3: medium 1 for postpartum neonatal intestinal stem cell culture, medium 2 for childhood intestinal stem cell culture, and medium 3 for adult intestinal stem cell culture; the medium 1 includes a basal medium and components at the following concentrations: 50-150 ng / mL Wnt3a, 400-600 ng / mL R-spondin1, 30-70 ng / mL EGF, 150-250 μg / mL glucosamine hydrochloride; the medium 2 includes a basal medium and components at the following concentrations: 30-70 ng / mL Wnt3a, 150-250 ng / mL R-spondin1, 10-30 ng / mL BMP4, 5-15 ng / mL FGF2, and 75-125 μg / mL glucosamine hydrochloride; the medium 3 includes a basal medium and components at the following concentrations: 30-70 ng / mL HGF, 5-15 μM DAPT, 5-15 μM prostaglandin, and 30-70 μg / mL glucosamine hydrochloride; the basal medium is DMEM / F12, Advanced DMEM / F12, IMDM, F12, or α-MEM.

6. A culture reagent for enteroids, characterized by, including the medium of any one of claims 4-5 and a matrix; the matrix includes: matrix 1 for postpartum neonatal intestinal stem cell culture, matrix 2 for childhood intestinal stem cell culture, and matrix 3 for adult intestinal stem cell culture; the matrix 1 includes Matrigel and collagen type I at a volume ratio of 3:1; the matrix 2 includes Matrigel and basal medium at a volume ratio of 1:1; the matrix 3 includes Matrigel and fibronectin at a volume ratio of 2:1; the basal medium is DMEM / F12, Advanced DMEM / F12, IMDM, F12, or α-MEM.

7. A method of constructing an intestinal organoid, characterized by, including: selecting a matrix from the matrixes 1-3 according to the intestinal development period and mixing the intestinal crypt, after the matrix solidifies, adding the medium in the medium 1-3 corresponding to the development period, and culturing; the medium 1-3 is the medium 1-3 in the medium of claim 5; the matrix 1-3 is the matrix 1-3 in the culture reagent of claim 6.

8. The construction method of claim 7, wherein, The method further comprises a step of treating the intestinal crypts with EDTA before the mixing; the treatment time is: 10-15 min for postpartum newborn intestinal crypts; 15-25 min for childhood intestinal crypts; and 25-35 min for adult intestinal crypts.

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