Method for rapidly enriching esophageal intrinsic gland organoids in culture and applications thereof

Abstract

The present application belongs to the technical field of cell biology, and particularly relates to a method for rapidly enriching and culturing esophageal intrinsic gland organoids and application. The method of the present application adopts a combination of the methods of stripping an integral esophageal intrinsic gland tissue, enriching the intrinsic gland tissue block and culturing the intrinsic gland organoids. In the enrichment process of the intrinsic gland tissue block, a specific gland tissue enrichment composition is added. In the culture process of the organoids, a specific gland organoid growth composition is added. Finally, a human esophageal intrinsic gland organoid with a purity of intrinsic gland cells greater than or equal to 98% is rapidly obtained.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to a method and application for rapidly enriching and culturing esophageal intrinsic gland organoids. Background Technology

[0002] The esophageal wall is composed of four layers: mucosa, submucosa, muscularis propria, and adventitia. Most of the esophageal glands are located in the loose connective tissue of the submucosa and are mucus-type exocrine glands. These glands open onto the epithelial surface of the mucosa, and their secretions are discharged into the esophageal lumen via ducts, serving a protective and lubricating function.

[0003] Esophageal adenocarcinoma, a cancer with a high mortality rate and an increasing incidence rate, urgently requires experimental models to explore its origin and molecular mechanisms. Current opinion suggests that esophageal adenocarcinoma likely originates from undifferentiated cells in Barrett's esophagus, but the exact origin of Barrett's esophagus remains controversial, possibly stemming from various specific cell populations such as the esophageal glands and cardia. A major obstacle to this research is that the physiological structure of the gastroesophageal cavity in rodents is not entirely similar to that in humans. Standardized experimental animals such as rats and mice have keratinized esophagi and squamous forestomach and lack esophageal glands. For various reasons, research on esophageal adenocarcinoma cannot be conducted as smoothly in animal models as research on other tumors.

[0004] Meanwhile, literature indicates that in various esophageal diseases (such as reflux esophagitis or after Barrett's esophageal ablation), there is significant proliferation of the esophageal glands at the affected site, which may repair damaged epithelial cells in a normal or abnormal manner. This patent provides an in vitro organoid model for evaluating the driving factors in the regeneration of damaged epithelium in esophageal diseases.

[0005] Patent application CN2020106845242, entitled "Method for Constructing an Animal Model of Orthotopic Primary Esophageal Cancer," discloses a technique for culturing mouse esophageal cells into esophageal organoids using a specific culture medium. This patent involves directly digesting mouse esophagus and culturing mouse esophageal cells into organoids. However, mice do not possess intrinsic esophageal gland tissue; therefore, organoids cultured solely from mouse esophageal cells cannot be used to conduct research on diseases related to the intrinsic glands of the human esophagus, particularly esophageal adenocarcinoma, which is often considered a malignant tumor originating from glandular epithelial tissue. Currently, research and treatment of esophageal adenocarcinoma are still in a relatively early stage.

[0006] Furthermore, the esophageal glands in humans are difficult to isolate and accumulate due to their sparse distribution, indistinguishability to the naked eye, and close integration with the submucosa of the esophageal wall. In general, research on esophageal glands in China and abroad is relatively limited, and there are no domestic publications or patent reports on the isolation of esophageal glands. Although there are some reports both domestically and internationally on the molecular mechanisms related to esophageal glands and Barrett's esophagus and esophageal adenocarcinoma at the cellular and RNA levels, there are no reports on the culture of esophageal gland organoids.

[0007] Based on this, the present invention proposes a method for in vitro culture of human esophageal glands to supplement the deficiencies of the prior art. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a method and application for rapidly enriching and culturing esophageal intrinsic gland organoids, the specific technical solution of which is as follows.

[0009] A method for rapidly enriching and culturing esophageal gland intrinsic organoids includes the following steps: dissection of a whole esophageal gland intrinsic tissue – enrichment of the gland intrinsic tissue block – culture of the gland intrinsic organoids. Step 1: Take esophageal mucosal tissue and peel off as much of the esophageal gland tissue as possible; Step 2: Enrich the esophageal gland tissue stripped in Step 1 by adding an enrichment culture medium, which includes DMEM medium, cytokines, and a gland tissue enrichment composition, wherein the gland tissue enrichment composition includes EGF and ITS, and the cytokines include gastrin and FBS / Noggin. Step 3: The esophageal gland tissue enriched and cultured in Step 2 was seeded onto Matrigel and cultured further in esophageal gland culture medium to form esophageal gland organoids. The esophageal gland organoid culture medium included Advanced DMEM / F12 medium, cytokines, and a gland organoid growth composition. The gland organoid growth composition included recombinant human basic fibroblast growth factor and recombinant human insulin-like growth factor-1. The cytokines included one or more of Y27632, N2 supplement, B27 additive, Noggin, N-acetylcysteine, A83-01, gastrin, nicotinamide, EGF, FGF-10, L-alanyl-L-glutamine, R-Spondin, and Wnt3A.

[0010] Further, in step 1, esophageal mucosal tissue is taken, the submucosa of the esophageal mucosal tissue is stretched to make it thinner and more translucent, and then olive-shaped tissue is selected for peeling.

[0011] Furthermore, in step 2, the ratio of esophageal gland tissue to enrichment culture medium is 4-5 esophageal gland tissues to 1-3 ml of enrichment culture medium.

[0012] Furthermore, the cytokines in step 2 include gastrin and FBS or gastrin and Noggin.

[0013] Furthermore, in step 2, the purity of the intrinsic gland cells in the esophageal intrinsic gland tissue enriched by adding enrichment culture medium is greater than or equal to 57%.

[0014] Further, the glandular organoid growth composition in step 3 comprises 50 ng / ml recombinant human basic fibroblast growth factor and 100 ng / ml recombinant human insulin-like growth factor-1; the cytokines comprise one or more of the following: 10-50 uM Y27632, (1×) N2 supplement, (1×) B27 additive, 100-1000 ng / mL Noggin, 1-2 mM N-acetylcysteine, 200-500 nM A83-01, 1-5 nM gastrin, 10-50 mM nicotinamide, 50-100 ng / mL EGF, 500-1000 ng / mL FGF-10, (1×) L-alanyl-L-glutamine, 1250-5000 ng / mL R-Spondin, and 250-500 ng / mL Wnt3A.

[0015] The esophageal intrinsic gland organoids prepared by the above method have intrinsic gland cells with a purity of ≥98%.

[0016] The application of the aforementioned esophageal intrinsic gland organoids in the preparation of human esophageal adenocarcinoma models.

[0017] The application of the aforementioned esophageal intrinsic gland organoids in the preparation of human esophageal disease models.

[0018] Furthermore, the human esophageal disease models include reflux esophagitis models, drug-induced esophagitis models, corrosive esophagitis models, or Barrett's esophagus models and models after Barrett's esophageal ablation treatment.

[0019] In some specific embodiments, this patent may provide an in vitro esophageal intrinsic gland organoid model for evaluating the driving factors in the regeneration of damaged epithelium in various esophageal diseases (such as reflux esophagitis, drug-induced esophagitis, corrosive esophagitis, Barrett's esophagus and after ablation treatment).

[0020] Beneficial technical effects This invention proposes a method combining whole-piece human esophageal gland intrinsic tissue removal, gland intrinsic tissue enrichment, and gland intrinsic organoid culture. During the enrichment process of the gland intrinsic tissue, a specific glandular tissue enrichment composition (EGF and ITS) is added. During the organoid culture process, a specific glandular organoid growth composition (recombinant human basic fibroblast growth factor and recombinant human insulin-like growth factor-1) is added. Finally, rapid enrichment and culture yields an esophageal gland intrinsic organoid with a gland intrinsic cell purity of ≥98%. This esophageal gland intrinsic organoid can be used for research on human esophageal adenocarcinoma and various intractable human esophageal diseases. Therefore, this invention fully fills the gap in the existing technology in this field and has pioneering and far-reaching clinical application value.

[0021] This invention first employs surgical dissection to extract intact esophageal glandular tissue from human esophageal mucosa. Then, unlike conventional methods that further digest the tissue into single cells, it directly enriches and cultures the glandular tissue blocks using a specific enrichment medium. This approach significantly reduces the types and quantities of added cytokines. Since the in vitro enrichment and culture of esophageal glandular cells is technically challenging, the research team discovered that, unlike in vitro culture of other organoids, more added cytokines do not necessarily lead to better growth. For example, when gastrin, FBS, and Noggin were added simultaneously in the embodiments of this invention, the purity of the cultured glandular cells was actually lower than when only gastrin and FBS or gastrin and Noggin were added. Therefore, this invention uses direct enrichment of tissue blocks, adding only a specific glandular tissue enrichment composition and a very small amount of cytokines, to achieve highly efficient enrichment of glandular cells while simultaneously removing a large number of squamous epithelial cells present in the original glandular tissue.

[0022] Finally, esophageal gland tissue enriched to a certain standard was cultured into organoids using a specific esophageal gland culture medium. The advantage of this method is that it significantly increases the number of glandular cells and significantly reduces impurity cells in the enriched tissue, resulting in a significantly improved success rate and quality of esophageal gland organoids. Compared to the control, the organoids cultured using the method of this invention exhibit superior cell count and size. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0024] Figure 1This is a flowchart of the method for enriching and culturing esophageal intrinsic gland organoids according to the present invention; Figure 2 A schematic diagram illustrating the surgical procedure for removing the intrinsic glands of the human esophagus. Figure 3 An image of the esophageal glands in the human esophageal mucosa; Figure 4 These are esophageal intrinsic gland organoids cultured using the method of this invention; Figure 5 Light microscopic comparison of the purity of intrinsic gland cells in surgically removed and unremoved esophageal glands; Figure 6 A bar chart showing the purity of intrinsic gland cells in surgically removed and unremoved esophageal glands; Figure 7 Light micrographs showing the purity of intrinsic glandular cells enriched in different culture media; Figure 8 Bar chart showing the purity of intrinsic glandular cells after enrichment in different culture media; Figure 9 Light micrographs of intrinsic glandular organs cultured in different organoid cultures; Figure 10 A statistical chart showing the size of esophageal glandular organs after culturing in different organoid cultures; Figure 11 Light micrographs showing the purity of esophageal glandular cells; Figure 12 Bar chart showing the purity of esophageal intrinsic gland cells; Figure 13 DAPI staining image of esophageal intrinsic glandular organoids; Figure 14 Immunofluorescence staining image of CK7 on esophageal intrinsic glandular organoids; Figure 15 Immunofluorescence staining image of muc1 in esophageal intrinsic glandular organoids. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0027] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0028] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0029] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0030] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered as having specifically disclosed all possible subranges and independent numerical values ​​within those ranges. For example, range 1... The description of 6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0031] The "purity" mentioned in this invention refers to the proportion of esophageal gland cells in the entire esophageal gland tissue or esophageal gland organoids.

[0032] The "glandular tissue enrichment composition" described in this invention refers to a composition specifically used for the enrichment and growth of cell numbers in the intrinsic glandular tissue of the esophagus.

[0033] The “glandular organoid growth composition” described herein refers to a composition specifically designed for in vitro culture that increases both the number and volume of esophageal intrinsic glandular organs.

[0034] Example 1 Surgical dissection of the entire esophageal gland tissue under a stereomicroscope 1) Obtain human esophageal mucosa tissue of 1×1cm size and sufficient thickness from clinical practice.

[0035] 2) Immerse the esophageal mucosal tissue in pre-cooled PBS and wash the sample as thoroughly as possible. At this point, the tissue consists of a tough mucosal layer and a loose submucosa. The mucosal layer is mainly composed of squamous epithelium, while the esophageal glands are located in the submucosa. Use forceps to fix the tissue sample so that the submucosa is exposed upwards.

[0036] 3) Use surgical scissors or a scalpel to peel away the loose submucosa.

[0037] 4) Under a stereomicroscope, use tweezers to stretch the submucosa to make it thinner and more transparent.

[0038] 5) Select high-density, opaque olive-shaped tissue (esophageal glands), see [reference needed]. Figure 3 .

[0039] 6) Use tweezers to hold the olive-shaped tissue selected in step 5) and use a scalpel to peel it off in one piece, removing as much excess connective tissue as possible.

[0040] This embodiment illustrates a schematic diagram of a surgical procedure for removing intact esophageal gland tissue. Figure 2 .

[0041] Example 2 Rapidly enrich esophageal glandular tissue cells (or simply glandular tissue cells) in vitro and perform the first round of impurity cell removal. 1) Take a number of esophageal gland tissues that were peeled off in Example 1 and wash them repeatedly with pre-cooled PBS containing 1% penicillin and antibiotics.

[0042] 2) Cut the above-mentioned esophageal gland tissue into fragments of about 1mm × 1mm.

[0043] 3) Add esophageal gland enrichment medium. The ratio of esophageal gland tissue blocks to enrichment medium is 1-3 ml of enrichment medium for every 4-5 esophageal gland tissue blocks. The enrichment medium includes DMEM medium, and the gland tissue enrichment composition is: 50 ng / mL EGF (epidermal growth factor) and (1×) ITS (insulin-transferrin-selenium supplement), and cytokines: 100 ng / ml Noggin (endogenous inhibitor of BMP bone morphogenetic protein) and 1 nM gastrin. Mix thoroughly by pipetting and aspiration, and place in a culture flask for culture.

[0044] 4) Incubate at 37℃ and 5% CO2 for one week.

[0045] 5) Observe the cell condition daily and change the culture medium according to the color change of the culture medium.

[0046] Esophageal gland tissue blocks, detached from human esophageal mucosa, mainly contain esophageal glandular cells, immune cells, and squamous epithelial cells. The enrichment culture medium provided in this embodiment can significantly remove squamous epithelial cells present in the original tissue while enriching esophageal glandular cells, achieving simultaneous enrichment and removal of the first round of impurity cells. After successful enrichment, the purity of esophageal glandular cells can reach 57% or more of the total cells in the esophageal glandular tissue.

[0047] Example 3 Rapidly culture a high-purity esophageal intrinsic gland organoid. 1) Discard the culture medium from Example 2 and wash the enriched tissue once with PBS containing 1% penicillin and antibiotics.

[0048] 2) Add 10ml of Trypsin to digest the tissue, aspirate and inhale thoroughly, and digest at 37°C for 1.5 hours, inverting and resuspending the tissue block every ten minutes during the process.

[0049] 3) Add 10ml of DMEM + 10% FBS to the culture flask to stop digestion.

[0050] 4) Filter the tissue suspension from 3) through a 70μm filter.

[0051] 5) Centrifuge at 1500 rpm for 10 minutes, discard the supernatant and collect the cells.

[0052] 6) Add 5 ml of red blood cell lysis buffer to resuspend the cells on ice and let stand for 3 mins.

[0053] 7) Centrifuge at 1500 rpm for 5 minutes, discard the supernatant and collect the cells.

[0054] 8) Depending on the cell number, add Matrigel to resuspend the cells on ice and seed them in 96-well plates at 5 μl / well.

[0055] 9) Place in a 37℃ incubator and let it solidify for 10 minutes.

[0056] 10) Add 60 μl / well of esophageal intrinsic gland organoid culture medium (which can be simply referred to as organoid culture medium) and incubate at 37°C and 5% CO2. The esophageal adenocarcinoma culture medium consists of Advanced DMEM / F12 medium, cytokines, and a glandular organoid growth composition, specifically including Advanced DMEM / F12, 10-50 μM Y27632 (ROCK inhibitor), N2 supplement (1×), B27 supplement (1×), 100-1000 ng / mL Noggin (endogenous inhibitor of BMP bone morphogenetic protein), 1-2 mM N-acetylcysteine, 200-500 nM A83-01 (ALK5 inhibitor), 1-5 nM Gastrin, 10-50 mM Nicotinamide, 50-100 ng / mL EGF (epidermal growth factor), and 500-1000 ng / mL... FGF-10 (fibroblast growth factor 10), Glutamax (1×) (L-alanyl-L-glutamine), 1250-5000 ng / mL R-Spondin (activator of the classical Wnt signaling pathway), 250-500 ng / mL Wnt3A (recombinant Wnt3A protein), 50 ng / mL human recombinant FGF-2 (recombinant human basic fibroblast growth factor), 100 ng / mL human recombinant IGF-1 (recombinant human insulin-like growth factor-1).

[0057] 11) Observe the growth of esophageal gland organoids daily, change the culture medium according to the color change of the culture medium, and passage the organoids according to their growth.

[0058] 12) After one week of culture, photograph and record the growth of the esophageal intrinsic glandular organoids. Figure 4 .

[0059] Comparative Example 1 Comparison of cell percentages between tissues with and without esophageal gland tissue after separation and whole-piece tissue culture. 1) Obtain two 1×1cm human esophageal mucosal tissues with sufficient thickness from the clinic.

[0060] 2) Immerse the esophageal mucosal tissue in pre-cooled PBS and wash the sample as thoroughly as possible with tweezers; at this point, the tissue includes a tough mucosal layer and a loose submucosa, with the mucosal layer mainly composed of squamous epithelium.

[0061] 3) The esophageal glands were dissected from one piece of tissue according to the method in Example 1 and labeled as tissue A; the other piece of tissue was not dissected and labeled as tissue B (i.e., the complete esophageal mucosa).

[0062] 4) Cut tissue A and tissue B into small pieces, add 10 ml of Trypsin for digestion, aspirate thoroughly, and digest at 37°C for 1.5 hours, inverting and resuspending the tissue pieces every ten minutes during digestion; add 10 ml of DMEM + 10% FBS to the culture flask and stop digestion; filter the tissue suspension through a 70 μm filter; centrifuge at 1500 rpm for 10 minutes, discard the supernatant and collect the cells.

[0063] 5) After obtaining the cells, add an appropriate amount of culture medium (10ml DMEM + 10% FBS) to resuspend them. The specific dilution factor should be adjusted according to the cell density so that the cells can be monolayered in the culture plate for easy observation under a light microscope.

[0064] 6) Take pictures of at least 3 fields of view under a 10x objective lens and embed a scale.

[0065] 7) Cell counting is performed based on the following morphological characteristics: ① Immune cells are mostly 5-20 μm in size, and are round in shape with uniform cytoplasm distribution; ② Glandular cells are generally larger than immune cells, and are round in shape with uneven cytoplasm distribution, often containing vacuoles of varying sizes; ③ Squamous epithelial cells are mostly flat and irregular in shape.

[0066] 8) The percentage of esophageal glandular cells is shown in Table 1 and... Figure 5 , Figure 6 It can be seen that the proportion of glandular cells in the surgically dissected esophageal glandular tissue (tissue A) is greater than that in the whole esophageal mucosa tissue (tissue B), while the number of squamous epithelial cells is less and the size of the squamous epithelial cells is significantly smaller.

[0067] Table 1. Percentage of intrinsic glandular cells in surgically dissected / non-surgically dissected tissues Comparative Example 2 By comparing different formulations of esophageal gland enrichment media, esophageal gland tissue was enriched and cultured, and the proportion of esophageal gland cells after enrichment was calculated. The specific procedures were as follows: 1) Based on the method in Example 2, cultured for one week using different intrinsic gland enrichment media.

[0068] 2) Discard the culture medium and wash the tissue once with PBS containing 1% penicillin and antibiotics; add 10 ml of Trypsin to digest, aspirate thoroughly, and digest at 37°C for 1 hour, inverting and resuspending the tissue block every 10 minutes during digestion; add 10 ml of DMEM + 10% FBS to the culture flask and stop digestion; filter the tissue suspension through a 70 μm filter; centrifuge at 1500 rpm for 10 minutes, discard the supernatant and collect the cells.

[0069] 3) After obtaining the cells, add an appropriate amount of culture medium to resuspend them. The specific dilution factor should be adjusted according to the cell density so that the cells can be laid in a monolayer in the culture plate for easy observation under a light microscope.

[0070] 4) Take pictures of at least 3 fields of view under a 10x objective lens and embed a scale.

[0071] 5) Cell counting is performed based on the following morphological characteristics: ① Immune cells are mostly 5-20 μm in size, round in shape, and have uniform cytoplasm distribution; ② Glandular cells are generally larger than immune cells, round in shape, with uneven cytoplasm distribution, and often contain vacuoles of varying sizes; ③ Squamous epithelial cells are mostly flat and irregular in shape.

[0072] The results are shown in Tables 2 and 3, and Figure 7 , Figure 8 The comparison shows that the glandular tissue enrichment composition consisting of EGF and ITS plays the most important role in the enrichment process of intrinsic glandular tissue. Without either of these components (Control A and Control D), the proportion of intrinsic glandular cells in the enriched intrinsic glandular tissue cannot reach 50%. Furthermore, the addition of the cytokine Gastrino plays a secondary role; without its addition (Control C), the proportion of intrinsic glandular cells reaches approximately 55%, but this is below the target (≥57%). Finally, when both FBS and Noggin are added (Control E), the proportion of intrinsic glandular cells is lower than that when only one of them is added (Example 2 and Control B). Therefore, as the most preferred method, the present invention adds Noggin and Gastrino to the enrichment medium.

[0073] Table 2. Different enrichment culture medium formulations and the percentage of esophageal gland cells Table 3. Calculation of each cell type in different enrichment media. Comparative Example 3 Comparison of esophageal intrinsic gland organoids cultured using different organoid culture media 1) Tissue blocks cultured using the enrichment medium in Comparative Example 2 were further digested into cells, and intrinsic glandular organs were cultured for one week using the following culture media based on the technology in Example 3.

[0074] Table 4. Culture medium for esophageal intrinsic gland organoids 2) Take pictures of at least 3 fields of view under a 4x objective lens and embed a scale.

[0075] 5) Organoids larger than 40 μm were selected for statistical analysis. The number and average size of intrinsic gland organoids are shown in Table 5. Figure 9 , Figure 10 It is evident that the organoids cultured from tissues enriched in Example 2 exhibited the best activity. Figure 9 (More cells are visible in the field of view); further, data shows that the esophageal gland intrinsic organoids cultured in the present invention have a larger diameter (the diameter of the entire 3D cell mass, not the diameter of a single cell) compared to control medium I. It is evident that adding the glandular organoid growth composition consisting of human recombinant FGF-2 and human recombinant IGF-1 to the esophageal gland intrinsic organoid culture medium can significantly promote the growth of human esophageal gland intrinsic organoids, especially promoting the size growth of the organoids. Furthermore, increasing the amount of R-Spondin and Wnt3A also promotes organoid growth.

[0076] Table 5. Comparison of organoids after culture in different organoid culture media Note: Table 5 shows organoid data measured within the same culture time, including the number of cells in the organoids and the average cell size (organoid size divided by the number of cells). Example 4 This example illustrates the surgical removal of the intrinsic gland, enrichment of the intrinsic gland, and the proportion of intrinsic gland cells in organoids in Examples 1-3. The purity of intrinsic gland cells was initially determined under a light microscope.

[0077] 1) Add 10 ml of Trypsin to the tissue blocks obtained in Examples 1 and 2 for digestion, thoroughly aspirate and incubate at 37°C for 1.5 hours, resuspending the tissue blocks by inversion every ten minutes during digestion; add 10 ml of DMEM + 10% FBS to the culture flask and stop digestion; filter the tissue suspension through a 70 μm filter; centrifuge at 1500 rpm for 10 minutes, discard the supernatant and collect the cells.

[0078] 2) After culturing the organoids for one week in Example 3, discard the culture medium, add PBS for washing; add TryplE, and crush the matrix gel with the tip of a pipette, incubate at 37 degrees for 15 mins; repeatedly pipette the organoids, filter the suspension through a 70 μm filter; centrifuge at 1500 rpm for 10 minutes, discard the supernatant and collect the cells.

[0079] 3) After obtaining the cells, add an appropriate amount of culture medium to resuspend them. The specific dilution factor should be adjusted according to the cell density so that the cells can be laid in a monolayer in the culture plate for easy observation under a light microscope.

[0080] 4) Take pictures of at least 3 fields of view under a 10x objective lens and embed a scale.

[0081] 5) Cell counting is performed based on the following morphological characteristics: ① Immune cells are mostly 5-20 μm in size, round in shape, and have uniform cytoplasm distribution; ② Glandular cells are generally larger than immune cells, round in shape, with uneven cytoplasm distribution, and often contain vacuoles of varying sizes; ③ Squamous epithelial cells are mostly irregular in shape.

[0082] 6) Comparison of the purity of intrinsic glandular cells is shown in Table 6. Figure 11 , Figure 12 It is evident that the combination of surgical dissection, tissue enrichment, and organoid culture techniques can produce high-purity intrinsic gland organoids (intrinsic gland cells accounting for ≥98%).

[0083] Table 6. Percentage of esophageal glandular cells under different methods Example 5 Identification of esophageal intrinsic glandular organs by immunofluorescence staining of muc1, CK7, and DAPI. 1) Aspirate the culture medium from the organoids in the 96-well plate, add 4% PFA, and fix at room temperature for 30 mins.

[0084] 2) Remove the supernatant and wash with PBS 3 times, 5 minutes each time.

[0085] 3) Add 0.5% Triton X-100 to PBS and punch holes for 30 minutes.

[0086] 4) Remove the supernatant and wash with PBS 3 times, 5 minutes each time.

[0087] 5) Add 1% bovine serum albumin (BSA) to PBS and block at room temperature for 1 hour.

[0088] 6) Remove the supernatant, add primary antibody muc1 and CK7 prepared with 1% bovine serum albumin (BSA) in PBS, and incubate overnight at 4 degrees Celsius.

[0089] 7) Remove the supernatant and wash with PBS 3 times, 5 minutes each time.

[0090] 8) Add secondary antibody prepared with PBS and incubate at room temperature in the dark for 2 hours.

[0091] 9) Remove the supernatant and wash with PBS 3 times, 5 minutes each time.

[0092] 10) Add DAPI prepared with PBS and incubate at room temperature for 5 minutes.

[0093] 11) Remove the supernatant and wash with PBS 3 times, 5 mins each time.

[0094] 12) Use the tip of a pipette to draw the organoids from the 96-well plate into a single concave slide or a confocal dish.

[0095] 13) Apply the mounting medium, gently cover with a glass slide, and seal with nail polish. The DAPI staining image of the esophageal intrinsic glandular organoids in this embodiment is shown below. Figure 13 Immunofluorescence staining image of CK7 in esophageal intrinsic glandular organoids is shown below. Figure 14 Immunofluorescence staining image of muc1 in esophageal intrinsic glandular organoids is shown below. Figure 15 .

[0096] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A method for rapidly enriching and culturing esophageal intrinsic gland organoids, characterized in that, The process includes dissecting a whole piece of esophageal glandular tissue, enriching the glandular tissue mass, and culturing glandular organoids. The specific steps are as follows: Step 1: Take esophageal mucosal tissue and peel off the entire piece of esophageal gland tissue, which includes esophageal gland cells, immune cells and squamous epithelial cells; Step 2: After the esophageal gland tissue stripped in Step 1 is broken up, it is added to an enrichment culture medium for enrichment culture. The enrichment culture medium consists of DMEM medium, cytokines and a gland tissue enrichment composition. The gland tissue enrichment composition is 50 ng / ml EGF and 1×ITS, and the cytokines are 1 nM gastrin and 100 ng / ml Noggin. Step 3: The esophageal gland tissue enriched and cultured in Step 2 is first digested and separated, then added to Matrigel. After solidification, it is cultured in esophageal gland organoid culture medium. The esophageal gland organoid culture medium consists of Advanced DMEM / F12 medium, additives, and gland organoid growth composition. The gland organoid growth composition is 50 ng / ml recombinant human basic fibroblast growth factor and 100 ng / ml recombinant human insulin-like growth factor-1. The additives are 10-50 μM Y27632, 1× N2 supplement, 1× B27 additive, 100-1000 ng / mL Noggin, 1-2 mM N-acetylcysteine, 200-500 nM A83-01, 1-5 nM gastrin, 10-50 mM nicotinamide, 50-100 ng / mL EGF, 500-1000 ng / mL FGF-10, 1× L-alanyl-L-glutamine, 1250-5000 ng / mL; R-Spondin, 250-500 ng / mL; Wnt3A.

2. The method as described in claim 1, characterized in that, In step 1, human esophageal mucosal tissue is taken, and the submucosa of the human esophageal mucosal tissue is stretched to make it thinner and more translucent. Then, olive-shaped tissue is selected for peeling.

3. The method as described in claim 1, characterized in that, In step 2, the ratio of esophageal gland tissue to enrichment culture medium is 1-3 ml of enrichment culture medium for every 4-5 fragmented esophageal gland tissues.

4. The method as described in claim 1, characterized in that, In step 2, the purity of the intrinsic gland cells in the esophageal intrinsic gland tissue enriched by adding enrichment culture medium is greater than or equal to 57%.

5. The esophageal intrinsic gland organoid prepared by the method according to any one of claims 1-4, characterized in that, The purity of the intrinsic gland cells in the esophageal intrinsic gland organoids is greater than or equal to 98%.

6. The application of the esophageal intrinsic gland organoids as described in claim 5 in the preparation of a human esophageal adenocarcinoma model.

7. The application of the esophageal intrinsic gland organoids as described in claim 5 in the preparation of human esophageal disease models.

8. The application as described in claim 7, characterized in that, The human esophageal disease models include reflux esophagitis models, drug-induced esophagitis models, corrosive esophagitis models, or Barrett's esophagus models.

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