An epithelial organoid culture medium and a culture method

Abstract

The present invention relates to an epithelial organoid culture medium and a culture method, which solve the technical problems of low growth rate and low formation rate of organoids in the prior art. The epithelial organoid culture medium contains basic components and additional components. The basic components include DMEM / F12 basal medium, penicillin-streptomycin solution, Glutamax additive, recombinant spondin, recombinant norrin, Y-27632, epidermal growth factor, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer solution, nicotinamide, A83-01 and CHIR99021. The additional components include Wnt-3A, ITS-X additive and hydrocortisone. The present invention also provides a culture method for epithelial organoids. The present invention can be used for the culture of epithelial organoids.

Description

Technical Field

[0001] The present invention relates to an organoid culture medium and a culture method, and more specifically, to an epithelial organoid culture medium and a culture method. Background Art

[0002] The in vitro three-dimensional cell culture technology was first applied to the in vitro culture of sponge cells in 1907. Since then, the cell sphere model of three-dimensional culture has been widely used in clinical and basic research. In 2009, Dutch scholar Hans Clevers first constructed an organoid model in vitro, which has made great progress in in vitro experiments. An organoid is a three-dimensional cell aggregate formed by the culture of stem cells or progenitor cells in vitro. Compared with the traditional cell culture method, organoids have the ability of self-renewal and self-organization, and maintain the physiological structure and function of their source tissues. As a new in vitro experimental model, organoids combine the advantages of both cell lines and animal models, and can maintain genomic stability during long-term in vitro expansion, and can be used to construct a living biobank for high-throughput screening. In addition, the organoid model can effectively reproduce the personalized characteristics of the host, providing an ideal platform for personalized treatment and drug screening.

[0003] In recent years, organoids have received extensive attention in the research field, but the main research still focuses on the personalized treatment of tumors, and there is less research on the construction of normal tissue organoids. However, the existing methods for constructing organoids in the published literature are still based on the system proposed by Professor Clevers. Currently published literature shows that this system still has the deficiencies of low growth rate and low formation rate of organoids.

[0004] The commonly used methods in the currently published literature are still based on the neural organoid culture system. Although this system can effectively promote the differentiation and self-renewal of stem cells, due to the lack of epithelial cell-specific growth factors, it still has deficiencies in promoting the proliferation of epithelial cells and the development of organoids, which in turn leads to significant differences between the organoid model and in vivo tissues at the gene expression profile and functional levels. This is mainly manifested in the deficiencies of the existing methods for constructing epithelial organoid models, such as low construction success rate, low growth rate of organoids, and large differences in gene expression profiles from in vivo tissues. Summary of the Invention

[0005] The present invention aims to solve the technical problems of low growth rate and low formation rate of organoids in the prior art, and provides an improved culture medium and a culture method, which can improve the growth rate of organoids and the similarity of organoids to in vivo tissues.

[0006] To this end, the present invention provides an epithelial organoid culture medium, which contains basic components and additional components. The basic components include DMEM / F12 basal medium, penicillin-streptomycin solution, Glutamax additive, recombinant spondin, recombinant noggin, Y-27632, epidermal growth factor, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution, N-acetyl-L-cysteine, nicotinamide, A83-01, and CHIR99021, and also contain additional components, which include Wnt-3A growth factor, ITS-X additive, and hydrocortisone.

[0007] Preferably, the content or concentration range of the components is: Wnt-3A growth factor 10 ng / ml to 40 ng / ml; ITS-X additive 0.5× to 1×; hydrocortisone 2.5 to 10 μg / L.

[0008] Preferably, the concentrations of the components are: penicillin-streptomycin solution 100 U / ml, Glutamax additive 1×, recombinant spondin 10 ng / ml, recombinant noggin 10 ng / ml, Y-27632 10 μmol / L, epidermal growth factor 50 ng / ml, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution 10 mmol / L, N-acetyl-L-cysteine 1 mmol / L, nicotinamide 10 mmol / L, A83-01 1 μmol / L, CHIR99021 0.5 μmol / L, Wnt-3A 10 - 40 ng / ml, ITS-X additive 0.5× - 2×, hydrocortisone 2.5 - 10 μg / L.

[0009] The present invention also provides a method for culturing epithelial organoids, which includes the following steps: (1) Isolation of epithelial tissue layer: The collected epithelial tissue blocks are stored in DMEM / F12 medium containing penicillin-streptomycin double antibody. The epithelial tissue blocks are digested with neutral protease II to isolate the epithelial tissue layer; (2) Obtaining primary epithelial stem cells: The epithelial tissue layer obtained in step (1) is divided into tissue blocks and digested with a mixed digestive solution of type I collagenase and hyaluronidase; (3) Inoculating epithelial stem cells on Matrigel: Use DMEM medium containing 10% fetal bovine serum with a volume twice that of the digestive solution to terminate digestion. Use a pipette to pipette repeatedly. After centrifugation, discard the supernatant. Resuspend with liquid Matrigel and inoculate into a well plate. After inoculation, place the well plate upside down in an incubator; (4) Construction of the culture system: Add epithelial organoid culture medium to the well plate and place the culture plate in an incubator. The organoids inoculated in the well plate are regularly changed the culture medium until they are mature.

[0010] Preferably, in the step (1), the neutral protease II is formulated with serum-free DMEM medium + Rock inhibitor Y-27632 as the solvent; take neutral protease II, dissolve it in the solvent, mix well and filter it through a bacterial filter membrane.

[0011] Preferably, in the step (3), the Matrigel is taken out of the refrigerator in advance and stored in the refrigerator to be converted from a solid state to a liquid state; the pipette tips used to resuspend the cell pellet with Matrigel are placed in the refrigerator for pre-cooling in advance; the centrifuge tube after centrifugation with the supernatant discarded is inserted into ice, and the resuspension operation is carried out on ice to avoid irreversible solidification of Matrigel.

[0012] Preferably, the following steps are further included: (5) Characterization of the growth rate of organoids: Observe the number and diameter of organoids under a light microscope, and then make a qualitative assessment of their growth rate.

[0013] Preferably, the following steps are further included: (6) Characterization of the gene expression profile of organoids: Extract the total RNA of organoids and the total RNA of the preserved epithelial tissue for transcriptome sequencing, and the transcriptome sequencing adopts the illumina second-generation sequencing method.

[0014] Preferably, in the step (6), the organoids in the epithelial tissue and Matrigel are thoroughly ground, and then the total RNA of the cells in the organoids is extracted by the Trizol lysis method.

[0015] The present invention has the following beneficial effects:

[0016] By improving the organoid culture medium and adding components that promote cell proliferation and metabolism, the present invention significantly improves the growth rate of organoids; by adding components that promote organ formation, development, and homeostasis maintenance, the present invention significantly reduces the number of up-regulated and down-regulated differential transcripts between organoids and in vivo tissues; making the functions and pathways of organoids in development, metabolism, and homeostasis maintenance less different from those of in vivo tissues; by adopting the method of grinding with carborundum after quick-freezing in liquid nitrogen + Trizol lysis to extract the total RNA of organoids, compared with the method of using enzyme digestion to isolate single cells + Trizol lysis to extract the total RNA of organoids, higher RNA yield can be obtained, the RNA degradation rate can be reduced, and at the same time, the introduction of protein contamination in RNA can be reduced. Specifically,

[0017] (1) By reducing the non-essential components (B-27 neurotrophic factor, noggin, fibroblast growth factor-2, fibroblast growth factor-10, prostaglandin E2) in the conventional organoid culture medium, the present invention simplifies the medium preparation on the premise of ensuring the unchanged growth rate of organoids and reduces the culture cost;

[0018] (2) By improving the culture medium, the present invention has increased the growth rate (the diameter of the organoids is larger within the same time) and the formation rate (more organoids are formed under the same seeding density) of the organoids;

[0019] (3) By improving the organoid culture medium and adding ITS-X (insulin-transferrin-selenium-ethanolamine), hydrocortisone and Wnt-3A protein, the number of differential transcripts between the improved organoids and the in vivo gingival epithelial tissue is significantly less than that between the conventionally cultured organoids and the in vivo gingival epithelial tissue;

[0020] (4) By improving the organoid culture medium and adding ITS-X (insulin-transferrin-selenium-ethanolamine), hydrocortisone and Wnt-3A protein, the differences between the improved organoids and the in vivo gingival epithelial tissue in the biological processes, cellular components, and molecular functions enriched in GO functions are significantly smaller than those between the conventionally cultured organoids and the in vivo gingival epithelial tissue;

[0021] (5) By improving the organoid culture medium and adding ITS-X (insulin-transferrin-selenium-ethanolamine), hydrocortisone and Wnt-3A protein, the differences between the improved organoids and the in vivo gingival epithelial tissue in the Reactome pathways are significantly smaller than those between the conventionally cultured organoids and the in vivo gingival epithelial tissue;

[0022] (6) By improving the organoid culture medium and adding Wnt-3A protein, the differences between the improved organoids and the in vivo gingival epithelial tissue in the Wnt-related signaling pathways (including the canonical Wnt signaling pathway and the non-canonical Wnt signaling pathway) are significantly smaller than those between the conventionally cultured organoids and the in vivo gingival epithelial tissue;

[0023] (7) By improving the organoid culture medium and adding insulin, the differences between the improved organoids and the in vivo gingival epithelial tissue in glucose, protein, and lipid metabolism and regulation are significantly smaller than those between the conventionally cultured organoids and the in vivo gingival epithelial tissue;

[0024] (8) By improving the organoid culture medium and adding transferrin, the differences between the improved organoids and the in vivo gingival epithelial tissue in the regulation of cellular iron ion balance and the regulation of DNA / RNA polymerase activity are significantly smaller than those between the conventionally cultured organoids and the in vivo gingival epithelial tissue;

[0025] (9) By improving the organoid culture medium and adding selenium element (sodium selenite), the differences between the improved organoids and the in vivo gingival epithelial tissue in cell cycle regulation, glutathione synthesis metabolism, and the regulation of cellular oxidative stress response are significantly smaller than those between the conventionally cultured organoids and the in vivo gingival epithelial tissue;

[0026] (10) By improving the organoid culture medium and adding ethanolamine, the differences in cell proliferation and stem cell proliferation regulation between the improved organoids and the in-vivo gingival epithelial tissues are significantly smaller than those between the conventionally cultured organoids and the in-vivo gingival epithelial tissues.

[0027] (11) In the present invention, the cells in the organoids are broken by the method of fine grinding after quick freezing in liquid nitrogen, and then the total cellular RNA is extracted with Trizol. Compared with the traditional method of digesting the organoids to obtain single cells and then extracting with Trizol, the O.D.260 / 280 (reflecting the amount of protein contaminants, the higher the value, the less the contamination) of the total RNA obtained by the fine grinding method is increased by 49.46% compared with the enzymatic digestion method, the total amount of RNA (μg) is increased by 1056.14 ± 339.91%, and the RNA integrity (RIN value, the higher the value, the higher the RNA integrity) is increased by 325.00 ± 169.95%. Description of the Drawings

[0028] Figure 1 It shows the differences in the sizes of the organoids cultured with the conventional medium and the improved medium in Example 1 of the present invention under an optical microscope on the 3rd, 5th, and 7th days after inoculation (scale bar under the microscope: 200 μm).

[0029] Figure 2 It shows the differences in the number and size of the organoids cultured with the basal medium, the conventional medium, and the improved medium in Example 1 of the present invention under an optical microscope on the 7th day after inoculation (scale bar under the microscope: 400 μm on the left, 200 μm in the middle, 100 μm on the right).

[0030] Figure 3 It shows the differences in the number and size of the organoids cultured with the basal medium supplemented with 0.5×, 1×, and 2× ITS-X additives respectively in Examples 2, 3, and 4 of the present invention under an optical microscope on the 7th day after culture (scale bar under the microscope: 400 μm on the left, 200 μm in the middle, 100 μm on the right).

[0031] Figure 4 It shows the differences in the number and size of the organoids cultured with the basal medium supplemented with 2.5 μg / L, 5 μg / L, and 10 μg / L hydrocortisone respectively in Examples 5, 6, and 7 of the present invention under an optical microscope on the 7th day after culture (scale bar under the microscope: 400 μm on the left, 200 μm in the middle, 100 μm on the right).

[0032] Figure 5The difference in the number and size of organoids under an optical microscope after the basal culture medium in Examples 8, 9, and 10 of the present invention was additionally supplemented with 10 ng / mL, 20 ng / mL, and 40 ng / mL of Wnt-3A additives, respectively, was cultured to day 7 (microscope scale: 400 μm on the left, 200 μm in the middle, and 100 μm on the right);

[0033] Figure 6a is a bar graph showing the difference in OD260 / 280 values ​​of total RNA of organoids extracted by conventional digestion and grinding methods respectively in Example 12 of the present invention measured by a fluorescence spectrophotometer;

[0034] Figure 6b A bar graph showing the difference in total RNA mass obtained by converting the RNA concentration measured by Nanodrop into total RNA extracted from organoids using conventional digestion and grinding methods in Example 12 of the present invention;

[0035] Figure 6c The difference bar graph of the RIN value of the total RNA of the organoids extracted by the conventional digestion method and the grinding method in Example 12 of the present invention was measured by Agilent 2100 Bioanalyzer;

[0036] Figure 7 This is a histogram of differential transcripts obtained by comparing conventional cultured organoids and improved cultured organoids with in vivo mucosal epithelial tissues in Example 13 of the present invention; the vertical axis represents the number of differential transcripts, the horizontal axis shows the sample grouping, dark colors represent differentially up-regulated transcripts, and light colors represent differentially down-regulated transcripts;

[0037] Figure 8a , Figure 8b and Figure 8c The biological processes obtained by GO functional enrichment analysis of the differential transcripts obtained by comparing the conventional cultured organoids and the improved cultured organoids with the in vivo mucosal epithelial tissues in Example 13 of the present invention are respectively Figure 8a ), cellular components ( Figure 8b )、Molecular Function( Figure 8c ) Heat map of the main differential entries in the three aspects of function; the vertical axis represents the differential function entries, and the horizontal axis represents the sample grouping; the darker the color, the more differential transcripts are annotated under the entry;

[0038] Figure 9 This is a heat map of the main differential entries obtained by Reactome pathway enrichment analysis of the differential transcripts obtained by comparing the conventional cultured organoids and the improved cultured organoids with the in vivo mucosal epithelial tissues in Example 13 of the present invention; the vertical axis represents the differential function entries, and the horizontal axis represents the sample grouping; the darker the color, the more differential transcripts annotated under the entry;

[0039] Figure 10 It is a heat map of differential transcripts enriched in Wnt-related signaling pathways obtained by comparing the conventional cultured organoids and the improved cultured organoids with the in vivo mucosal epithelial tissues in Example 13 of the present invention; the vertical axis represents the name of the relevant pathway, and the horizontal axis represents the sample grouping; the darker the color, the more differential transcripts annotated under the entry;

[0040] Figure 11a , Figure 11b and Figure 11c The differential transcripts obtained by comparing the conventional cultured organoids and the improved cultured organoids with the in vivo mucosal epithelial tissues in Example 13 of the present invention are enriched in glucose ( Figure 11a ),protein( Figure 11b ) and lipids ( Figure 11c )Heat map of metabolism and regulation related functions; the vertical axis represents the name of the related pathway, and the horizontal axis represents the sample grouping; the darker the color, the more differential transcripts annotated under the item;

[0041] Figure 12 It is a heat map of differential transcripts enriched in functions related to cellular iron ion balance regulation and DNA / RNA polymerase activity regulation obtained by comparing the conventional cultured organoids and the improved cultured organoids with the in vivo mucosal epithelial tissues in Example 13 of the present invention; the vertical axis represents the name of the relevant pathway, and the horizontal axis represents the sample grouping; the darker the color, the more differential transcripts are annotated under the item;

[0042] Figure 13a , Figure 13b and Figure 13c The differential transcripts obtained by comparing the conventional cultured organoids and the improved cultured organoids in Example 13 of the present invention with the mucosal epithelial tissue in vivo are enriched in cell cycle regulation ( Figure 13a ), glutathione anabolism ( Figure 13b ) and regulation of cellular oxidative stress response ( Figure 13c ) Heat map of related functions; the vertical axis represents the name of the related pathway, and the horizontal axis represents the sample grouping; the darker the color, the more differential transcripts annotated under the entry;

[0043] Figure 14 This is a heat map of differential transcripts enriched in cell proliferation and stem cell proliferation regulation-related functions obtained by comparing the standard cultured organoids and the improved cultured organoids with the in vivo mucosal epithelial tissues in Example 13 of the present invention. The vertical axis represents the name of the relevant pathway, and the horizontal axis represents the sample grouping; the darker the color, the more differential transcripts are annotated under the item. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with embodiments.

[0045] The culture medium used in the present invention is a modified culture medium, and the culture medium used in the control group is a conventional culture medium. Their common components include DMEM / F12 basal medium, penicillin-streptomycin solution, Glutamax additive, recombinant R-Spondin, recombinant Noggin, Y-27632, epidermal growth factor (EGF), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer solution, nicotinamide, A83-01, CHIR99021. The additional components contained in the conventional culture medium include B-27 additive, fibroblast growth factor-2 (FGF-2), fibroblast growth factor-10 (FGF-10), follistatin, prostaglandin E2 (PGE2). The additional components contained in the modified culture medium include Wnt-3A, ITS-X additive and hydrocortisone.

[0046] The components and concentrations of the conventional culture medium and the modified culture medium are shown in Table 1 and Table 2 respectively.

[0047] Table 1. Composition and Concentration Table of Conventional Culture Medium

[0048]

[0049]

[0050] Table 2. Composition and Concentration Table of Modified Culture Medium

[0051]

[0052] Example 1

[0053] (1) Acquisition and preservation of epithelial tissue: Human gingival epithelium is derived from discarded gingiva in clinical periodontal surgery. After rinsing the surface blood with normal saline, it is stored at 4°C in DMEM / F12 medium containing 10 μmol / L Y-27632 and 5% penicillin-streptomycin double antibody. The epithelial tissue is ensured to start the next step of separation within 2 hours;

[0054] (2) Isolation and acquisition of epithelial cells: The epithelial layer and the subepithelial tissue are separated by digestion with type II neutral protease at 4°C for 8 hours. The isolated epithelial tissue is cut into tissue blocks of 1x1 mm 2 in size, and added to a mixed digestive solution composed of 2 mg / mL type I collagenase + 1 mg / mL hyaluronidase for digestion for 1 hour. After reaching the digestion time, 2 volumes of DMEM medium containing 10% fetal bovine serum are added to terminate the digestion. After filtration through a 100 μm cell strainer, it is centrifuged at 1000 rpm for 5 minutes (4°C), the supernatant is discarded, and the precipitate is resuspended with d-PBS and centrifuged again at 1000 rpm for 5 minutes (4°C);

[0055] (3) Inoculation and culture of oral mucosal epithelial organoids: Discard the supernatant after centrifugation. Resuspend the precipitate with Matrigel matrix gel rewarmed at 4°C and inoculate 50 μL per well into a 24-well cell culture plate. Invert the cell culture plate and place it in the cell culture incubator for 30 - 40 minutes (37°C). Add 500 μL of organoid medium per well and culture it in an incubator at 37°C, with saturated humidity and a CO₂ volume fraction of 5%. Replace the fresh medium every 48 hours during the culture period.

[0056] The components and concentrations of the basic organoid medium used in this example are: DMEM / F12 medium, penicillin-streptomycin solution 100 U / ml, Glutamax additive 1×, recombinant spondin 10 ng / ml, recombinant noggin 10 ng / ml, Y-27632 10 μmol / L, epidermal growth factor 50 ng / ml, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer solution 10 mmol / L, N-acetyl-L-cysteine 1 mmol / L, nicotinamide 10 mmol / L, A83-01 1 μmol / L, CHIR99021 0.5 μmol / L.

[0057] The components and concentrations of the modified organoid medium used in this example are: DMEM / F12 medium, penicillin-streptomycin solution 100 U / ml, Glutamax additive 1×, recombinant spondin 10 ng / ml, recombinant noggin 10 ng / ml, Y-27632 10 μmol / L, epidermal growth factor 50 ng / ml, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer solution 10 mmol / L, N-acetyl-L-cysteine 1 mmol / L, nicotinamide 10 mmol / L, A83-01 1 μmol / L, CHIR99021 0.5 μmol / L, Wnt-3A 20 ng / ml, ITS-X additive 1×, and hydrocortisone 5 μg / L.

[0058] The components and concentrations of the control organoid medium used in this example are: DMEM / F12 medium, penicillin-streptomycin solution 100 U / ml, Glutamax additive 1×, recombinant dickkopf-1 10 ng / ml, recombinant noggin 10 ng / ml, Y-27632 10 μmol / L, epidermal growth factor 50 ng / ml, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution 10 mmol / L, N-acetyl-L-cysteine 1 mmol / L, nicotinamide 10 mmol / L, A83-01 1 μmol / L, CHIR99021 0.5 μmol / L, B-27 additive 1×, fibroblast growth factor-2 (FGF-2) 5 ng / ml, fibroblast growth factor-10 (FGF-10) 10 ng / ml, follistatin 1 μmol / L, prostaglandin E2 (PGE2) 1 μmol / L.

[0059] Example 2

[0060] The components and concentrations of the modified organoid medium used in this example are: DMEM / F12 medium, penicillin-streptomycin solution 100 U / ml, Glutamax additive 1×, recombinant dickkopf-1 10 ng / ml, recombinant noggin 10 ng / ml, Y-27632 10 μmol / L, epidermal growth factor 50 ng / ml, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution 10 mmol / L, N-acetyl-L-cysteine 1 mmol / L, nicotinamide 10 mmol / L, A83-01 1 μmol / L, CHIR99021 0.5 μmol / L, ITS-X additive 0.5×.

[0061] Except for the components and concentrations of the above modified medium, the control medium is the same as that in Example 1 for other aspects.

[0062] Example 3

[0063] The components and concentrations of the modified organoid medium used in this example are: DMEM / F12 medium, penicillin-streptomycin solution 100 U / ml, Glutamax additive 1×, recombinant dickkopf-1 10 ng / ml, recombinant noggin 10 ng / ml, Y-27632 10 μmol / L, epidermal growth factor 50 ng / ml, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution 10 mmol / L, N-acetyl-L-cysteine 1 mmol / L, nicotinamide 10 mmol / L, A83-01 1 μmol / L, CHIR99021 0.5 μmol / L, ITS-X additive 1×.

[0064] Except for the components and concentrations of the above-mentioned improved culture medium, the control culture medium and others are the same as those in Example 1.

[0065] Example 4

[0066] The components and concentrations of the improved organoid culture medium used in this example are: DMEM / F12 medium, penicillin-streptomycin solution 100 U / ml, Glutamax additive 1×, recombinant hedgehog protein 10 ng / ml, recombinant dickkopf-1 protein 10 ng / ml, Y-27632 10 μmol / L, epidermal growth factor 50 ng / ml, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution 10 mmol / L, N-acetyl-L-cysteine 1 mmol / L, nicotinamide 10 mmol / L, A83-01 1 μmol / L, CHIR99021 0.5 μmol / L, ITS-X additive 2×.

[0067] Except for the components and concentrations of the above-mentioned improved culture medium, the control culture medium and others are the same as those in Example 1.

[0068] Example 5

[0069] The components and concentrations of the improved organoid culture medium used in this example are: DMEM / F12 medium, penicillin-streptomycin solution 100 U / ml, Glutamax additive 1×, recombinant hedgehog protein 10 ng / ml, recombinant dickkopf-1 protein 10 ng / ml, Y-27632 10 μmol / L, epidermal growth factor 50 ng / ml, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution 10 mmol / L, N-acetyl-L-cysteine 1 mmol / L, nicotinamide 10 mmol / L, A83-01 1 μmol / L, CHIR99021 0.5 μmol / L, hydrocortisone 2.5 μg / L.

[0070] Except for the components and concentrations of the above-mentioned improved culture medium, the control culture medium and others are the same as those in Example 1.

[0071] Example 6

[0072] The components and concentrations of the improved organoid culture medium used in this example are as follows: DMEM / F12 medium, penicillin-streptomycin solution at 100 U / ml, Glutamax additive at 1×, recombinant spondin at 10 ng / ml, recombinant norrin at 10 ng / ml, Y-27632 at 10 μmol / L, epidermal growth factor at 50 ng / ml, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution at 10 mmol / L, N-acetyl-L-cysteine at 1 mmol / L, nicotinamide at 10 mmol / L, A83-01 at 1 μmol / L, CHIR99021 at 0.5 μmol / L, hydrocortisone at 5 μg / L.

[0073] Except for the components and concentrations of the above-mentioned improved culture medium, the control culture medium and others are the same as in Example 1.

[0074] Example 7

[0075] The components and concentrations of the improved organoid culture medium used in this example are as follows: DMEM / F12 medium, penicillin-streptomycin solution at 100 U / ml, Glutamax additive at 1×, recombinant spondin at 10 ng / ml, recombinant norrin at 10 ng / ml, Y-27632 at 10 μmol / L, epidermal growth factor at 50 ng / ml, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution at 10 mmol / L, N-acetyl-L-cysteine at 1 mmol / L, nicotinamide at 10 mmol / L, A83-01 at 1 μmol / L, CHIR99021 at 0.5 μmol / L, hydrocortisone at 10 μg / L.

[0076] Except for the components and concentrations of the above-mentioned improved culture medium, the control culture medium and others are the same as in Example 1.

[0077] Example 8

[0078] The components and concentrations of the improved organoid culture medium used in this example are as follows: DMEM / F12 medium, penicillin-streptomycin solution at 100 U / ml, Glutamax additive at 1×, recombinant spondin at 10 ng / ml, recombinant norrin at 10 ng / ml, Y-27632 at 10 μmol / L, epidermal growth factor at 50 ng / ml, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution at 10 mmol / L, N-acetyl-L-cysteine at 1 mmol / L, nicotinamide at 10 mmol / L, A83-01 at 1 μmol / L, CHIR99021 at 0.5 μmol / L, Wnt-3A at 10 ng / ml.

[0079] Except for the components and concentrations of the above-mentioned improved culture medium, the control culture medium and others are the same as in Example 1.

[0080] Example 9

[0081] The components and concentrations of the improved organoid culture medium used in this example are as follows: DMEM / F12 medium, penicillin-streptomycin solution 100 U / ml, Glutamax additive 1×, recombinant spondin 10 ng / ml, recombinant noggin 10 ng / ml, Y-27632 10 μmol / L, epidermal growth factor 50 ng / ml, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer solution 10 mmol / L, N-acetyl-L-cysteine 1 mmol / L, nicotinamide 10 mmol / L, A83-01 1 μmol / L, CHIR99021 0.5 μmol / L, Wnt-3A 20 ng / ml.

[0082] Except for the components and concentrations of the above-mentioned improved culture medium, the control culture medium and others are the same as in Example 1.

[0083] Example 10

[0084] The components and concentrations of the improved organoid culture medium used in this example are as follows: DMEM / F12 medium, penicillin-streptomycin solution 100 U / ml, Glutamax additive 1×, recombinant spondin 10 ng / ml, recombinant noggin 10 ng / ml, Y-27632 10 μmol / L, epidermal growth factor 50 ng / ml, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer solution 10 mmol / L, N-acetyl-L-cysteine 1 mmol / L, nicotinamide 10 mmol / L, A83-01 1 μmol / L, CHIR99021 0.5 μmol / L, Wnt-3A 40 ng / ml.

[0085] Except for the components and concentrations of the above-mentioned improved culture medium, the control culture medium and others are the same as in Example 1.

[0086] Example 11

[0087] Characterization of the growth rate of oral mucosa organoids: On the 3rd, 5th, and 7th days after organoid inoculation, the number and diameter of organoids were observed under a light microscope. Nine fields of view at the same magnification were taken from both the experimental group and the control group for observation, and their growth rates were evaluated.

[0088] Example 12

[0089] Total RNA extraction from oral mucosa organoids: On the 12th day of organoid culture, discard the culture medium, rinse 3 times with sterile PBS, aspirate all the liquid in the well plate, quickly freeze the well plate together with the solid matrix gel adsorbed on it in liquid nitrogen for 10 minutes, separate the well plate from the solid matrix gel, place the matrix gel in a cryopreservation tube and store it at -80 °C. For total RNA extraction, use carborundum for fine grinding. After grinding the solid matrix gel, add 10 times the volume of Trizol to extract total RNA.

[0090] (1) Use fluorescence spectrophotometry to measure the O.D.260 / 280 value of the RNA sample;

[0091] (2) Use Nanodrop to measure the RNA concentration (RNA content);

[0092] (3) Use an Agilent 2100 bioanalyzer to measure the RIN value of the RNA.

[0093] Example 13

[0094] High-throughput identification of the phenotype of oral mucosa organoids: For oral mucosa epithelial tissue, routinely culture oral mucosa organoids, and perform transcriptomic sequencing on the improved cultured oral mucosa organoids. Compare the differential transcripts between mucosal epithelial tissue - routinely cultured mucosal organoids and mucosal epithelial tissue - improved cultured mucosal organoids respectively, and perform functional and pathway enrichment analysis on the differential transcripts.

[0095] Only the above-mentioned are specific embodiments of the present invention, and the scope of implementation of the present invention cannot be limited thereby. Therefore, the replacement of equivalent components or equivalent changes and modifications made according to the scope of protection of the present invention patent shall still fall within the scope covered by the claims of the present invention.

Claims

1. A method for culturing oral mucosa epithelial organoids, characterized in that, It includes the following steps: (1) Isolation of the epithelial layer tissue: The collected gingival epithelial tissue blocks are stored in DMEM / F12 medium containing penicillin-streptomycin double antibody. The epithelial tissue blocks are digested with neutral protease II to isolate the epithelial layer tissue; (2) Obtaining of primary epithelial stem cells: The epithelial layer tissue obtained in step (1) is divided into tissue blocks; Digestion is carried out using a mixed digestive solution of type I collagenase and hyaluronidase; (3) Seeding of epithelial stem cells on Matrigel: Use DMEM medium containing fetal bovine serum to terminate digestion, repeatedly pipette with a pipette gun, after centrifugation, discard the supernatant, resuspend with liquid Matrigel, and seed in a well plate. After seeding, invert the well plate in an incubator; (4) Construction of the culture system: Add oral mucosal epithelial organoid medium to the well plate, and place the culture plate in an incubator; The organoids seeded in the well plate are regularly changed with medium and cultured until mature. The oral mucosal epithelial organoid medium consists of a basic component and an additional component. The basic component is DMEM / F12 basal medium, penicillin-streptomycin solution, Glutamax additive, recombinant dickkopf-1, recombinant noggin, Y-27632, epidermal growth factor, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution, N-acetyl-L-cysteine, nicotinamide, A83-01 and CHIR99021, and the additional component is Wnt-3A growth factor, ITS-X additive and hydrocortisone.

2. The method for culturing oral mucosa epithelial organoids according to claim 1, characterized in that, The concentration range of the additional component is: Wnt-3A growth factor 10 ng / ml to 40 ng / ml; ITS-X additive 0.5× to 1×; Hydrocortisone 2.5 to 10 μg / L.

3. The method for culturing oral mucosa epithelial organoids according to claim 1, characterized in that, The concentrations of the basic component and the additional component are: penicillin-streptomycin solution 100 U / ml, Glutamax additive 1×, recombinant dickkopf-1 10 ng / ml, recombinant noggin 10 ng / ml, Y-27632 10 μmol / L, epidermal growth factor 50 ng / ml, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution 10 mmol / L, N-acetyl-L-cysteine 1 mmol / L, nicotinamide 10 mmol / L, A83-01 1 μmol / L, CHIR99021 0.5 μmol / L, Wnt-3A 10 - 40 ng / ml, ITS-X additive 0.5× - 2×, hydrocortisone 2.5 - 10 μg / L.

4. The method for culturing oral mucosa epithelial organoids according to claim 1, characterized in that, In step (1), the neutral protease II is prepared using serum-free DMEM medium + Rock inhibitor Y-27632 as the solvent; Take neutral protease II, dissolve it in the solvent, mix well and filter through a bacterial filter membrane.

5. The method for culturing oral mucosa epithelial organoids according to claim 1, characterized in that, In step (3), the Matrigel is taken out of the refrigerator in advance and stored in the refrigerator to transform from a solid state to a liquid state; The pipette tips used to resuspend the cell pellet by aspirating Matrigel are pre-cooled in the refrigerator in advance; The centrifuge tube after centrifugation and discarding the supernatant is inserted into ice, and the resuspension operation is carried out on ice to avoid irreversible solidification of Matrigel.

6. The method for culturing oral mucosa epithelial organoids according to claim 1, characterized in that, It also includes the following steps: (5) Characterization of the growth rate of organoids: Observe the number and diameter of organoids under a light microscope, and then conduct a qualitative assessment of their growth rate.

7. The method for culturing oral mucosa epithelial organoids according to claim 1, characterized in that, It also includes the following steps: (6) Characterization of the gene expression profile of organoids: Extract the total RNA of organoids and the total RNA of the preserved epithelial tissue for transcriptome sequencing, and the transcriptome sequencing uses the Illumina second-generation sequencing method.

8. The method for culturing oral mucosa epithelial organoids according to claim 7, characterized in that, In step (6), the epithelial tissue and the organoids in Matrigel are thoroughly ground, and then the Trizol lysis method is used to extract the total RNA of the cells in the organoids.

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