Hu sheep rumen epithelial organoid culture medium, in vitro construction method and application of epithelial organoids

Abstract

The present invention relates to a culture medium for Hu sheep rumen epithelial organoids, an in vitro construction method of epithelial organoids and applications. A culture medium for Hu sheep rumen epithelial organoids includes: Advanced DMEM / F-12 basal medium, N-2 supplement, B-27 supplement, GlutaMax, penicillin, streptomycin, HEPES, nicotinamide, N-acetyl-L-cysteine, epidermal growth factor, Wnt3a protein, R-spondin1 protein, Noggin protein, insulin-like growth factor 1, fibroblast growth factor 10, CHIR99021, A8301, SB202190, Y27632. This culture medium can be used to induce the construction of Hu sheep rumen epithelial organoids from primary Hu sheep rumen epithelial cells, and the organoid formation rate can reach more than 15%. The organoids formed by culture have a stratified structure similar to that of tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal tissue culture, and relates to an organoid culture method, specifically to a medium for culturing rumen epithelial organoids of Hu sheep, an in vitro construction method of epithelial organoids and an application thereof. Background Art

[0002] The compound stomach of ruminants includes the rumen, reticulum, omasum, and abomasum. Among them, the rumen of adult ruminants can account for 80% of the total volume of the compound stomach, which is the main energy production site of ruminants and plays an important role in the process of ruminants adapting to complex ecological environments. The rumen epithelium belongs to stratified squamous epithelium and has functions such as barrier, immunity, and energy metabolism. From bottom to top, it is the basal layer, spinous layer, granular layer, and stratum corneum. Keratinocytes migrate upward from the basal layer, continuously divide and differentiate until the stratum corneum of death is formed.

[0003] During the breeding process of ruminants, many problems related to nutritional metabolism are related to the rumen. Directly using ruminants for experiments is costly, affects animal welfare, and has a long experimental cycle. Therefore, it is necessary to explore a method that can reduce experimental costs and reduce the consumption of live animals. Organoids are complex 3D structures induced from adult stem cells or pluripotent stem cells in vitro and have similar structures and functions to tissues in vivo. They have very important application prospects in the fields of developmental biology, disease modeling, drug development, regenerative medicine, etc. Since scientists successfully established mouse intestinal organoids in 2009, organoids including various tissues such as the cortex, adenohypophysis, thyroid gland, optic cup, esophagus, stomach, colon, prostate, liver, and skin have been successfully constructed one after another. In 2017, organoids were named the technology of the year in the field of life sciences by Nature Methods, and the organoids were also included in the National Key Research and Development Program, indicating that organoids have great potential in exploring biology. At present, regarding the in vitro model of rumen epithelium, domestic and foreign literature reports have isolated rumen epithelial cells from rumen epithelial tissues of dairy cows, goats, and sheep and established long-term culture systems, while the 3D organoid culture that can better reflect complex tissue structures compared to 2D culture has not been reported. Summary of the Invention

[0004] The purpose of the present invention is to provide an in vitro construction method of rumen epithelial organoids of Hu sheep. The rumen epithelial organoids of Hu sheep obtained by this method retain the structure similar to rumen epithelial tissues and can be used for in vitro experiments, thereby reducing experimental costs.

[0005] The technical solution adopted by the present invention to solve its technical problems is:

[0006] A medium for culturing rumen epithelial organoids of Hu sheep, the medium comprising: Advanced DMEM / F-12 basal medium, N-2 supplement, B-27 supplement, GlutaMax, penicillin, streptomycin, HEPES, nicotinamide, N-acetyl-L-cysteine, epidermal growth factor (EGF), Wnt3a protein, R-spondin1 protein, Noggin protein, insulin-like growth factor 1 (IGF-1), fibroblast growth factor 10 (FGF10), CHIR99021, A8301, SB202190, Y27632. Due to the addition of these four components, CHIR99021, A8301, SB202190, and Y27632, in this medium, rumen epithelial organoids of Hu sheep can be successfully cultured.

[0007] Organoids can be induced from adult stem cells or pluripotent stem cells. For organoids induced from adult stem cells, the key to organoid culture lies in the isolation of adult stem cells and the construction of an organoid medium. The basal cells of the rumen divide and differentiate to continuously renew the rumen epithelial cells, which have stem cell characteristics. Therefore, it is feasible to isolate the basal cells of the rumen epithelium and induce the formation of organoids through an organoid medium. Various supplements in the organoid medium are the key factors for inducing the division and differentiation of adult stem cells to form organoids. The inventors previously designed dozens of media but were unable to culture rumen epithelial organoids of Hu sheep. Finally, it was found that an organoid medium supplemented with four small molecule inhibitors, CHIR99021, A8301, SB202190, and Y27632, can successfully induce the establishment of rumen epithelial organoids of Hu sheep.

[0008] Preferably, the medium contains the following components: Advanced DMEM / F-12 basal medium, 1×N-2 supplement, 1×B-27 supplement, 1×GlutaMax, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 - 25 mM HEPES, 10 - 12 mM nicotinamide, 1 - 1.5 mM N-acetyl-L-cysteine, 50 - 100 ng / mL epidermal growth factor (EGF), 100 - 200 ng / mL Wnt3a protein, 100 - 200 ng / mL R-spondin1 protein, 100 - 200 ng / mL Noggin protein, 100 - 200 ng / mL insulin-like growth factor 1 (IGF-1), 100 - 200 ng / mL fibroblast growth factor 10 (FGF10), 3 - 6 μΜ CHIR99021, 5 - 10 μΜ A8301, 10 - 20 μΜ SB202190, 10 - 20 μΜ Y27632.

[0009] Preferably, the culture medium contains the following components: Advanced DMEM / F-12 basal medium, 1×N-2 supplement, 1×B-27 supplement, 1×GlutaMax, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 mM HEPES, 10 mM nicotinamide, 1 mM N-acetyl-L-cysteine, 50 ng / mL epidermal growth factor (EGF), 100 ng / mL Wnt3a protein, 100 ng / mL R-spondin1 protein, 100 ng / mL Noggin protein, 100 ng / mL insulin-like growth factor 1 (IGF-1), 100 ng / mL fibroblast growth factor 10 (FGF10), 3 μΜ CHIR99021, 5 μΜ A8301, 10 μΜ SB202190, 10 μΜ Y27632.

[0010] A culture medium for Hu sheep rumen epithelial cells, the culture medium comprising: DMEM, fetal bovine serum, penicillin, streptomycin, insulin, epidermal growth factor (EGF) and Y-27632. Y-27632 is a small molecule inhibitor that maintains stem cell activity and inhibits stem cell apoptosis. The purpose of adding Y-27632 to the epithelial cell culture medium in the present invention is to prevent the death of rumen epithelial cells with stemness during 2D culture.

[0011] Preferably, each 1 L of the epithelial cell culture medium contains: 900 mL of DMEM, 100 mL of fetal bovine serum, 100 U / mL of penicillin, 100 μg / mL of streptomycin, 5 μg / mL of insulin, 10 ng / mL of epidermal growth factor (EGF), 10 μM of Y-27632.

[0012] An in vitro construction method for Hu sheep rumen epithelial organoids, the method comprising the following steps:

[0013] (1) Sampling: Take the rumen of Hu sheep, rinse it with PBS containing 4 antibiotics and strip the muscle layer, and store it in DMEM containing 4 antibiotics; the 4 antibiotics are four antibiotics: penicillin, streptomycin, gentamicin and amphotericin B;

[0014] (2) Isolation of rumen epithelial cells: Divide the epithelial tissue into small pieces, wash it repeatedly with PBS containing 4 antibiotics, add the washed small pieces of epithelial tissue to trypsin-EDTA digestive solution and digest it in a 37°C water bath with shaking. Stop digestion and collect the cells after round epithelial cells are digested;

[0015] (3) Culture of rumen epithelial cells: The isolated cells are suspended in the epithelial cell culture medium and then cultured in a CO2 constant temperature incubator. The rumen epithelial cells can be passaged and cryopreserved;

[0016] Each 1 L of the epithelial cell culture medium contains: 900 mL of DMEM, 100 mL of fetal bovine serum, 100 U / mL of penicillin, 100 μg / mL of streptomycin, 5 μg / mL of insulin, 10 ng / mL of epidermal growth factor (EGF), and 10 μM of Y-27632;

[0017] (4) Construction of rumen organoids: Digest the cells from the culture dish and suspend them in Matrigel, then inoculate them on a 24-well plate, add the organoid culture medium of the present invention, and culture in a CO2 incubator. Change the medium every 2 - 3 days, and after 9 days of culture, obtain Hu sheep rumen epithelial organoids, which can be passaged.

[0018] Preferably, the PBS containing 4 antibiotics in step (1) refers to a 10 mM PBS solution containing 1000 U / mL of penicillin, 1 mg / mL of streptomycin, 500 μg / mL of gentamicin, and 25 μg / mL of amphotericin B; the DMEM containing 4 antibiotics in step (1) refers to a DMEM culture medium containing 1000 U / mL of penicillin, 1 mg / mL of streptomycin, 500 μg / mL of gentamicin, and 25 μg / mL of amphotericin B.

[0019] Preferably, in step (2), the termination of digestion is to add DMEM containing 20% fetal bovine serum in an equal volume to the digestive solution.

[0020] Preferably, in step (3), passage is carried out at a ratio of 1:3 every 4 - 5 days. After resuspending the cells with the cryopreservation solution, they can be stored in liquid nitrogen for a long time; the composition of the cryopreservation solution is: 90% fetal bovine serum and 10% dimethyl sulfoxide, mixed evenly and prepared immediately before use under light protection conditions.

[0021] This method further includes (5) organoid frozen section and immunofluorescence: Digest the Matrigel with cell recovery solution to collect the organoids, and after fixation, dehydration, and embedding, section and perform immunofluorescence staining. Preferably, the fixing solution is a 4% paraformaldehyde solution, the dehydrating solution is 15% and 30% sucrose solutions, and the embedding agent is OCT embedding agent.

[0022] Preferably, in step (4), the volume ratio of cells to Matrigel during inoculation is 1:4.

[0023] Use of the Hu sheep rumen epithelial organoid culture medium of the present invention in culturing Hu sheep rumen epithelial organoids.

[0024] The beneficial effects of the present invention are:

[0025] 1. By using the rumen epithelial cell isolation and culture method of the present invention, primary rumen epithelial cells can be isolated from rumen tissues, and rumen epithelial cells within 8 passages can all successfully form organoids;

[0026] 2. The in vitro construction method of rumen epithelial organoids provided by the present invention can induce the construction of rumen epithelial organoids from primary rumen epithelial cells of Hu sheep. The organoid formation rate can reach more than 15%, and the cultured organoids have a stratified structure similar to that of tissues. The structure of rumen epithelial organoids is closer to that of rumen epithelial tissues and is more suitable for in vitro experiments compared with rumen cells. It is a good in vitro research model. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a bright-field photograph of rumen epithelial cells cultured in a culture dish (scale bar: 100 μm);

[0028] Figure 2 It is the immunofluorescence result of rumen epithelial cells (scale bar: 100 μm);

[0029] Figure 3 It is the morphological change of organoids during a 9-day culture period (scale bar: 100 μm);

[0030] Figure 4 It is the change of organoid diameter over time. The diameter of organoids can reach 250 μm after 9 days of culture (scale bar: 100 μm);

[0031] Figure 5 It is the immunofluorescence result of frozen sections of rumen epithelial tissue and rumen epithelial organoids. KRT14 is used to label basal layer cells, IVL is used to label granular layer and stratum corneum cells, ZO-1 is used to label tight junction proteins between epithelial cells, and Ki67 is used to label proliferating cells. Among them, Figure A is rumen epithelial tissue, and Figure B is rumen epithelial organoids (scale bar: 100 μm);

[0032] Figure 6 It is the formation rate of organoids cultured from rumen epithelial cells of different passages;

[0033] Figure 7 It is the passage result of organoids. After continuous passage, the organoids maintain similar morphological characteristics (scale bar: 100 μm);

[0034] Figure 8 It is the culture condition of organoids after removing any component from the culture medium. Figure A is the organoid formation rate, and Figure B is the organoid diameter. Different superscript letters indicate significant differences (P < 0.05). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] The present invention will be further described below in conjunction with specific examples. The following examples are only for explaining the present invention and do not constitute a limitation to the present invention. The test samples and test procedures used in the following examples include the following content (if the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels).

[0038] In the following examples, PBS and DMEM medium are of Biosharp brand; penicillin-streptomycin mixture, HEPES, and trypsin-EDTA digestion solution are purchased from Beijing Solarbio Science & Technology Co., Ltd.; gentamicin-amphotericin B mixture is purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; fetal bovine serum is purchased from Zhejiang Geneo Biopharmaceutical Technology Co., Ltd.; Advanced DMEM / F-12 basal medium, N-2 supplement, B-27 supplement, GlutaMax, and TrypLE are purchased from Invitrogen (Shanghai) Trading Co., Ltd.; nicotinamide and N-acetyl-L-cysteine are purchased from Sigma-Aldrich; epidermal growth factor (EGF), Wnt3a protein, Noggin protein, R-spondin1 protein, insulin-like growth factor 1 (IGF-1), fibroblast growth factor 10 (FGF-10), insulin, CHIR99021, A8301, SB202190, and Y27632 are purchased from MCE; Matrigel, 100 mm culture dishes, 24-well plates, 96-well plates, 25 cm 2 culture flasks, Cell Recovery Solution, and 2 mL cryotubes are purchased from Corning; 4% paraformaldehyde solution and DAPI are purchased from Shanghai Beyotime Biotechnology Co., Ltd.; OCT is of SAKURA brand; KRT14 primary antibody, ZO-1 primary antibody, Ki67 primary antibody, IVL primary antibody, and Cy3 secondary antibody are purchased from Abclonal Technology Co., Ltd.; Triton X-100 is purchased from Aladdin Biochemical Technology Co., Ltd.

[0039] Example 1

[0040] Isolation and culture of Hu sheep rumen epithelial cells, the specific steps are as follows:

[0041] 1.1 Sampling

[0042] Prepare scissors, forceps, gloves, trays, PBS containing 4 antibiotics and DMEM (stored on ice) in advance.

[0043] PBS containing antibiotics: 10 mM PBS solution containing 1000 U / mL penicillin, 1 mg / mL streptomycin, 500 μg / mL gentamicin and 25 μg / mL amphotericin B;

[0044] DMEM containing antibiotics: DMEM medium containing 1000 U / mL penicillin, 1 mg / mL streptomycin, 500 μg / mL gentamicin and 25 μg / mL amphotericin B.

[0045] After taking the rumen of Hu sheep, rinse the rumen contents thoroughly. Use forceps to bluntly separate the muscular layer and serosa layer of the rumen tissue. Place the serosa layer in PBS containing antibiotics and rinse it more than 5 times, then soak it in DMEM containing antibiotics for preservation and bring it back.

[0046] 1.2 Isolation of rumen epithelial tissue

[0047] Take out the sample on the operating table. After repeatedly washing the serosa layer in PBS containing antibiotics more than 5 times, cut it into small pieces of 5 mm × 5 mm with scissors and wash it by oscillation in PBS containing antibiotics 3 times.

[0048] 1.3 Isolation of rumen epithelial cells

[0049] Transfer the washed small pieces of rumen epithelium to a culture flask, add 5 times the volume of trypsin-EDTA digestive solution, and digest it by oscillation in a 37 °C water bath at a rotation speed of 120 - 140 revolutions. Observe under a microscope every 20 minutes. When round cells are digested, start collecting the digestive solution, collect it once every 10 minutes, terminate the reaction with DMEM medium containing 20% fetal bovine serum equal in volume to the digestive solution, and end the digestion when the rumen epithelial tissue turns white.

[0050] DMEM medium containing 20% fetal bovine serum: Add fetal bovine serum to the DMEM medium so that the final concentration of fetal bovine serum in the DMEM medium is 20%.

[0051] 1.4 Rumen epithelial cell culture

[0052] Preparation of epithelial cell medium: 1 L of epithelial cell medium contains: 900 mL of DMEM medium, 100 mL of fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 5 μg / mL insulin, 10 ng / mL epidermal growth factor (EGF), 10 μM Y-27632.

[0053] The collected digestive fluids were passed through 70-μm and 40-μm cell sieves respectively, and then centrifuged at 300 g for 5 minutes. The precipitate was resuspended in epithelial cell medium and inoculated on a culture dish. After culturing in a CO2 incubator for 1 hour, the supernatant was aspirated and inoculated on a new culture dish (epithelial cell medium) to remove fibroblasts. The rumen epithelial cells after several days of culture gradually merged and formed a pavement-like morphology, as Figure 1 shown. The culture medium was changed every 2 days during the culture process. (In this invention, Hu sheep rumen epithelial cells were cultured on 100-mm culture dishes, and 10 mL of epithelial cell medium was added to each culture dish; when performing immunofluorescence staining, rumen epithelial cells were cultured in 96-well plates, and 100 μL of epithelial cell medium was added to each well)

[0054] When the fusion degree of rumen epithelial cells reached about 80%, they could be used for subsequent construction of rumen organoids.

[0055] Example 2

[0056] Method for subculturing, cryopreserving and resuscitating Hu sheep rumen epithelial cells, the specific steps are as follows:

[0057] 2.1 Subculturing

[0058] When the cell fusion degree reached about 80%, subculturing could be carried out. Take out the culture dish from the incubator and remove the epithelial cell medium, add PBS and gently shake to wash the culture dish, then add TrypLE and incubate in a CO2 incubator for 10-15 minutes. After the rumen epithelial cells became round, gently pipette and transfer the digestive fluid to a centrifuge tube, centrifuge at 300 g for 5 minutes, remove the supernatant, resuspend the precipitated cells with epithelial cell medium and inoculate them into a new culture dish, generally subculturing at a ratio of 1:3.

[0059] 2.2 Cryopreserving

[0060] Resuspend the digested cells with cryopreservation solution. One plate of cells was resuspended with 2 mL of cryopreservation solution, and then transfer the suspension to cryopreservation tubes (1 mL of suspension was added to each cryopreservation tube) and place them in a programmable freezing container and leave them overnight in an ultra-low temperature freezer, and transfer them to liquid nitrogen for storage the next day.

[0061] 2.3 Resuscitating

[0062] Take out the cells from liquid nitrogen and quickly thaw them in a 37°C water bath, centrifuge at 300 g for 5 minutes to remove the supernatant, resuspend the precipitate with DMEM and centrifuge again at 300 g for 5 minutes, remove the supernatant and resuspend the precipitate with epithelial cell medium, inoculate on a culture dish and culture in a CO2 incubator.

[0063] Screening of organoid culture media for examples and comparative examples

[0064] Control Example 1 Organoid Medium: Advanced DMEM / F-12 basal medium, 1× N-2 additive, 1× B-27 additive, 1× GlutaMax, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 mM HEPES, 10 mM nicotinamide, 1 mM N-acetyl-L-cysteine, 50 ng / mL epidermal growth factor (EGF), 100 ng / mL Wnt3a protein, 100 ng / mL R-spondin1 protein, 100 ng / mL Noggin protein, 100 ng / mL insulin-like growth factor 1 (IGF-1), 100 ng / mL fibroblast growth factor 10 (FGF10).

[0065] Control Example 2 Medium: Control Example 1 + 3 μΜ CHIR99021,

[0066] Control Example 3 Medium: Control Example 1 + 5 μΜ A8301,

[0067] Control Example 4 Medium: Control Example 1 + 10 μΜ SB202190,

[0068] Control Example 5 Medium: Control Example 1 + 10 μΜ Y27632,

[0069] Control Example 6 Medium: Control Example 1 + 10 μΜ Y27632 + 5 μΜ A8301,

[0070] Control Example 7 Medium: Control Example 1 + 10 μΜ Y27632 + 3 μΜ CHIR99021,

[0071] Control Example 8 Medium: Control Example 1 + 10 μΜ Y27632 + 10 μΜ SB202190,

[0072] Control Example 9 Medium: Control Example 1 + 10 μΜ Y27632 + 5 μΜ A8301 + 10 μΜ SB202190,

[0073] Control Example 10 Medium: Control Example 1 + 3 μΜ CHIR99021 + 5 μΜ A8301,

[0074] Control Example 11 Medium: Control Example 1 + 3 μΜ CHIR99021 + 10 μΜ SB202190,

[0075] Control Example 12 Medium: Control Example 1 + 5 μΜ A8301 + 10 μΜ SB202190.

[0076] Example organoid culture medium: Advanced DMEM / F-12 basal medium, 1×N-2 supplement, 1×B-27 supplement, 1×GlutaMax, 100U / mL penicillin, 100μg / mL streptomycin, 10mM HEPES, 10mM nicotinamide, 1mM N-acetyl-L-cysteine, 50ng / mL epidermal growth factor (EGF), 100ng / mL Wnt3a protein, 100ng / mL R-spondin1 protein, 100ng / mL Noggin protein, 100ng / mL insulin-like growth factor 1 (IGF-1), 100ng / mL fibroblast growth factor 10 (FGF10), 3μΜ CHIR99021, 5μΜ A8301, 10μΜ SB202190, 10μΜ Y27632.

[0077] Experimental scheme: Referring to the in vitro construction method of Hu sheep rumen epithelial organoids in Example 3, the culture medium of the above-mentioned embodiments and comparative examples was used for organoid culture. The Hu sheep rumen epithelial cell suspension was mixed with matrix gel at a volume ratio of 1:4 and inoculated on a 24-well plate, 50 μL (about 2000 Hu sheep rumen epithelial cells) per well, incubated in a CO2 constant temperature incubator for more than 20 minutes to allow the matrix gel to fully solidify, and then 500 μL of organoid culture medium was added to each well, and the liquid was changed every 2 to 3 days. After 9 days of culture, the number of organoids formed was counted, and the organoid formation rate was calculated: organoid formation rate = (number of organoids per well / 2000) × 100%.

[0078] In Comparative Examples 1-12, some holes occasionally formed 1 to 2 organoids, and the average formation rate of Hu sheep rumen epithelial organoids was <0.1%. In the culture medium of the embodiment, cells gradually formed complex spherical structures from single cells. The formation rate of Hu sheep rumen epithelial organoids in the culture medium of the embodiment was >15%, and the formation rate of rumen epithelial cells induced by different generations of organoids had slight differences, but they were all stable at more than 15%.

[0079] Conclusion: The organoid culture medium with the simultaneous addition of CHIR99021, A8301, SB202190 and Y27632 achieved the successful culture of rumen epithelial organoids from Hu sheep. The addition of one or several components alone could not achieve the stable culture of rumen epithelial organoids from Hu sheep.

[0080] Example 3

[0081] This example illustrates the in vitro construction method of Hu sheep rumen epithelial organoids, and the specific steps are as follows:

[0082] 3.1 Cell digestion and counting

[0083] After removing the culture medium, wash the culture dish twice with PBS. Then add TrypLE and incubate in a CO₂ incubator for 10 - 15 minutes. Gently pipette and pass the digestion solution through a 40μm cell sieve, then transfer it to a centrifuge tube and centrifuge at 300g for 5 minutes. Remove the supernatant, resuspend the cells with organoid medium, take 18μL of the cell suspension, mix it with 2μL of trypan blue solution, and count the cells using a hemocytometer.

[0084] Preparation of organoid medium: Advanced DMEM / F-12 basal medium, 1×N-2 supplement, 1×B-27 supplement, 1×GlutaMax, 100U / mL penicillin, 100μg / mL streptomycin, 10mM HEPES, 10mM nicotinamide, 1mM N-acetyl-L-cysteine, 50ng / mL epidermal growth factor (EGF), 100ng / mL Wnt3a protein, 100ng / mL R-spondin1 protein, 100ng / mL Noggin protein, 100ng / mL insulin-like growth factor 1 (IGF-1), 100ng / mL fibroblast growth factor 10 (FGF10), 3μΜ CHIR99021, 5μΜ A8301, 10μΜ SB202190, 10μΜ Y27632.

[0085] 3.2 Seeding

[0086] Further dilute the cell suspension with organoid medium and mix it with Matrigel at a ratio of 1:4 to make the final cell concentration 40,000 cells / mL. Inoculate the cell suspension onto a 24-well plate, 50μL per well, and incubate in a CO₂ incubator for more than 20 minutes to allow the Matrigel to fully solidify. Then add 500μL of organoid medium to each well and change the medium every 2 - 3 days. Take pictures with ImageView software every day to record the growth status of the organoids. The morphological changes of the organoids during a 9-day culture period are as Figure 3 shown, and the cells gradually form complex spherical structures starting from single cells.

[0087] As Figure 4 and Figure 8 shown in B, measure the diameter of the organoids with ImageJ software and record the changes. As Figure 6 and Figure 8 shown in A, record the number of organoids in each well on the 9th day and calculate the organoid formation rate. (Note: Matrigel should be thawed on ice in advance and operated on ice throughout the process. The pipette tips required for the experiment should be pre-cooled at -20°C, and the 24-well plate should be preheated at 37°C for more than 1 hour. The organoid medium should be equilibrated to room temperature, and the medium change should be carried out slowly to prevent damage to the Matrigel).

[0088] Figure 3 and Figure 4It is illustrated that the edges of the organoids are clear after 9 days of culture, and the diameter can reach 250 μm.

[0089] Figure 6 It is illustrated that the formation rate of organoids induced by rumen epithelial cells from the 1st to 7th generations is stable above 15%.

[0090] Figure 8 A It is illustrated that removing epidermal growth factor (EGF), Noggin protein, Wnt3a protein, insulin-like growth factor 1 (IGF-1), and fibroblast growth factor 10 (FGF-10) will significantly reduce the organoid formation rate, and organoids cannot be formed after removing CHIR99021, A8301, SB202190, and Y27632.

[0091] Figure 8 B It is illustrated that removing epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and fibroblast growth factor 10 (FGF-10) will significantly reduce the organoid diameter.

[0092] 3.3 Passage

[0093] Take out the 24-well plate from the incubator, remove the organoid culture medium, add 1 mL of PBS and wash twice. Add 1 mL of Cell Recovery Solution to each well and incubate on ice for 2 hours until the Matrigel is completely dissolved. Transfer the liquid to a centrifuge tube and centrifuge at 300 g for 5 minutes. Remove the supernatant, resuspend the cells with TrypLE and incubate in a CO2 incubator for 30 minutes. Gently pipette to disperse the cells, pass through a 40 m cell sieve and transfer to a centrifuge tube, then centrifuge at 300 g for 5 minutes. Re-inoculate according to the descriptions in Methods 3.1 and 3.2. The passage results of the organoids are as Figure 7 shown. After continuous passage, the organoids maintain similar morphological characteristics with clear edges and similar diameters.

[0094] Example 4

[0095] Method for making frozen sections of rumen epithelial tissue and rumen epithelial organoids, the specific steps are as follows:

[0096] 4.1 Collection of organoids

[0097] Take out the 24-well plate from the incubator, remove the organoid culture medium, add 1 mL of PBS and wash twice. Add 1 mL of Cell Recovery Solution to each well and incubate on ice for 2 hours until the Matrigel is completely dissolved. Transfer the liquid to a centrifuge tube and centrifuge at 300 g for 5 minutes. After removing the supernatant, resuspend the organoids with PBS and centrifuge again at 300 g for 5 minutes. After removing the supernatant, the organoids are obtained.

[0098] 4.2 Fixation and dehydration

[0099] Resuspend the organoids with 4% paraformaldehyde solution and fix them overnight at 4°C. The next day, transfer the organoids to 15% sucrose solution and dehydrate them at 4°C for 24 hours. On the third day, transfer the organoids to 30% sucrose solution and dehydrate them at 4°C for 24 hours. (The fixation and embedding of rumen epithelial tissue directly start from this step.)

[0100] 4.3 Embedding

[0101] Transfer the dehydrated organoids to a mold and add OCT embedding medium. After placing it at -80°C for 10 minutes, the solidified embedding block can be removed from the mold.

[0102] 4.4 Sectioning

[0103] Use a cryostat to cut the embedding block into 10-μm-thick slices and adsorb them on adhesive glass slides. The slices can be stored at -20°C for a long time.

[0104] Example 5

[0105] The method for immunofluorescent staining of rumen epithelial cells grown in a 96-well plate is as follows:

[0106] 5.1 Remove the original culture medium and gently rinse it 3 times with PBS.

[0107] 5.2 Fix with 4% paraformaldehyde solution for 15 minutes, then rinse it 3 times with PBS, 5 minutes each time.

[0108] 5.3 Permeabilize at room temperature with 0.5% Triton X-100 (prepared with 10 mM PBS) for 20 minutes, then rinse it 3 times with PBS, 5 minutes each time.

[0109] 5.4 Dropwise add goat serum to block at room temperature for 30 minutes, then remove the blocking solution, add the diluted primary antibody (KRT14) and incubate overnight at 4°C.

[0110] 5.5 Remove the primary antibody, rinse it 3 times with PBS, 5 minutes each time, add the secondary antibody (Cy3) and incubate at 37°C for 1 hour.

[0111] 5.6 Remove the secondary antibody, rinse it 3 times with PBS, 5 minutes each time, stain with DAPI for 20 minutes, rinse it 3 times with PBS, 5 minutes each time, and observe under a fluorescence microscope. The results are as Figure 2 shown.

[0112] Figure 2 The immunofluorescence results of the rumen epithelial cells prove that the rumen epithelial cells can be labeled by the basal cell marker gene KRT14, indicating that the cells isolated in Example 1 are rumen epithelial basal cells.

[0113] Example 6

[0114] The method of slice immunofluorescence staining is as follows:

[0115] 6.1 Place the glass slide in a plastic box, add 0.01M sodium citrate to cover the slide, heat in a 95°C water bath for 5 minutes, and cool to room temperature naturally without opening the lid.

[0116] 6.2 Use forceps to pick out the glass slide and put it into a special plastic box for immunofluorescence. Add 0.2% Triton X-100 (prepared with 10mM PBS), wash on a horizontal shaker at room temperature for 5 minutes at 70 rpm, and repeat 3 times.

[0117] 6.3 Prepare PBB (add 5g bovine serum albumin and 500μL Triton X-100 to 1L PBS and dissolve thoroughly). Pick out the glass slide, dry the liquid around the sample as much as possible with absorbent paper, draw 3 circles around the sample in the same direction with an immunohistochemistry pen, add PBB-5% goat serum, and incubate in a humid box for 45 minutes.

[0118] 6.4 Aspirate the blocking solution, add the primary antibody (diluted with PBB), and place it in a humid box at 4°C overnight.

[0119] 6.5 Aspirate the primary antibody, wash on a horizontal shaker with PBB at room temperature for 10 minutes at 70 rpm, and repeat 3 times.

[0120] 6.6 Prepare PBB-secondary antibody / DAPI in the dark. After drying the liquid around the sample with absorbent paper, add PBB-secondary antibody / DAPI and incubate in the dark at room temperature for 1 hour.

[0121] 6.7 Aspirate PBB-secondary antibody / DAPI, wash on a horizontal shaker with PBB at room temperature for 5 minutes at 70 rpm, and repeat 5 times. Dry the liquid around the sample with absorbent paper, add 50% glycerol (prepared with 10mM PBS) dropwise to one side of the sample, gently cover the coverslip and ensure no bubbles are generated, fix the edge of the coverslip with nail polish, and observe under a fluorescence microscope. The immunofluorescence results of the frozen sections of rumen epithelial tissue and rumen epithelial organoids obtained according to the method described in Example 4 are as Figure 5 shown. KRT14 is used to label basal cells, IVL is used to label granular layer and stratum corneum cells, ZO-1 is used to label tight junction proteins between epithelial cells, and Ki 67 is used to label proliferating cells; among them, Figure A is rumen epithelial tissue, and Figure B is rumen epithelial organoids (scale bar: 100m).

[0122] According to Figure 5It can be seen that the rumen epithelial organoids have a similar morphological structure to the rumen epithelial tissue. Their epithelial polarity is inward, and the cells inside the organoids are positive for IVL, indicating that the internal cells are keratinized cells. The outer layer cells of the organoids are positive for KRT14 and Ki67, indicating that the outer layer cells of the organoids are basal cells with proliferative ability. At the same time, the whole organoids are positive for ZO-1, indicating that the organoids are all composed of epithelial cells.

[0123] In summary, the present invention provides a medium for rumen epithelial organoids of Hu sheep, and a method for in vitro construction of rumen epithelial organoids using this medium. The organoid medium used in this method can achieve a formation rate of more than 15% for rumen epithelial organoids, and the organoids constructed by this method retain a structure similar to that of rumen epithelial tissue.

[0124] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. The same or similar parts among the embodiments can be referred to each other. For the device disclosed in the embodiments, since it corresponds to the method disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the description of the method part.

[0125] The above has introduced in detail the medium for rumen epithelial organoids of Hu sheep, the method for in vitro construction of epithelial organoids and their applications provided by the present invention. Specific examples are used herein to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. An in vitro construction method of rumen epithelial organoids of Hu sheep, characterized in that The method includes the following steps: (1) Sampling: Take the rumen of Hu sheep, rinse it with PBS containing four antibiotics and strip the muscle layer, and store it in DMEM containing four antibiotics; the four antibiotics are penicillin, streptomycin, gentamicin, and amphotericin B. (2) Isolation of rumen epithelial cells: Divide the epithelial tissue into small pieces, wash it repeatedly with PBS containing four antibiotics, add the washed small pieces of epithelial tissue to trypsin-EDTA digestive solution and digest it by shaking in a 37°C water bath. Stop digestion and collect the cells after the round epithelial cells are digested. (3) Culture of rumen epithelial cells: The isolated cells are suspended in the medium for Hu sheep rumen epithelial cells and then cultured in a CO2 constant temperature incubator. The rumen epithelial cells can be passaged and cryopreserved. (4) Construction of rumen organoids: Digest the cells from the culture dish and suspend them in Matrigel, then inoculate them on a 24-well plate, add the medium for Hu sheep rumen epithelial organoids, and culture them in a CO2 constant temperature incubator. Change the medium every 2 - 3 days, and Hu sheep rumen epithelial organoids can be obtained after 9 days of culture. Among them, the medium for Hu sheep rumen epithelial cells includes: DMEM, fetal bovine serum, penicillin, streptomycin, insulin, epidermal growth factor (EGF), and Y-27632. The medium for Hu sheep rumen epithelial organoids includes: Advanced DMEM / F-12 basal medium, N-2 supplement, B-27 supplement, GlutaMax, penicillin, streptomycin, HEPES, nicotinamide, N-acetyl-L-cysteine, epidermal growth factor (EGF), Wnt3a protein, R-spondin1 protein, Noggin protein, insulin-like growth factor 1 (IGF-1), fibroblast growth factor 10 (FGF10), CHIR99021, A8301, SB202190, Y27632.

2. The in vitro construction method according to claim 1, characterized in that: The PBS containing four antibiotics in step (1) refers to a 10 mM PBS solution containing 1000 U / mL penicillin, 1 mg / mL streptomycin, 500 mg / mL gentamicin, and 25 mg / mL amphotericin B; the DMEM containing four antibiotics in step (1) refers to a DMEM medium containing 1000 U / mL penicillin, 1 mg / mL streptomycin, 500 mg / mL gentamicin, and 25 mg / mL amphotericin B.

3. The in vitro construction method according to claim 1, characterized in that: The termination of digestion in step (2) is to add DMEM containing 20% fetal bovine serum with the same volume as the digestive solution.

4. The in vitro construction method according to claim 1, characterized in that: In step (3), passage is carried out at a ratio of 1:3 every 4 - 5 days. After resuspending the cells with the cryopreservation solution, they can be stored in liquid nitrogen for a long time; the formulation of the cryopreservation solution is: 90% fetal bovine serum and 10% dimethyl sulfoxide mixed evenly.

5. The in vitro construction method according to claim 1, characterized in that: In step (4), the volume ratio of cells to Matrigel during inoculation is 1:4.

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