Adenoid organ as well as culture medium and culture method thereof

Abstract

The invention provides an adenoid organ as well as a culture medium and a culture method thereof. The culture medium of the adenoid organ comprises an RPMI1640 culture medium, 10% of FBS, 1 * non-essential amino acid, 1 * penicillin-streptomycin, 1 * sodium pyruvate, 50 ng / ml amphotericin B, 20 [mu] g / ml gentamicin, 1 * insulin-selenium-transferrin compound and 0.5-1 [mu] g / ml recombinant human B cell activating factor BAFF are added into the RPMI1640 culture medium. By means of the method, the adenoid organoid derived from the immune cells can be successfully cultured, the cultured organoid contains B lymphocytes, T lymphocytes and macrophages at the same time and does not contain epithelial cells, various immune cells can be successfully maintained for 2 weeks, the immune environment of adenoid tissue can be truly simulated, and the culture cost is reduced. The model is a superexcellent model for in-vitro research of adenoid and related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid preparation, and relates to adenoid organoids and culture medium and culture method thereof. Background Art

[0002] Adenoids (also known as pharyngeal tonsils or proliferative bodies) are a group of lymphoid tissues located at the back of the pharyngeal vault, attached to the junction of the top and back walls of the nasopharynx, between the pharyngeal recesses on both sides, which is equivalent to the sphenoid body and the base of the occipital bone. Its structure is similar to that of the palatine adenoids, but the superficial layer is wrinkled and there are also lacunas, but with fewer branches. Its surface is covered with a layer of epithelium, which varies with age. Adenoids are part of the inner ring of pharyngeal lymphoids located at the entrance of the upper respiratory tract. They contain a large number of lymphoid follicles and diffuse immune cells, forming the first line of defense of the immune barrier of the digestive tract and respiratory tract. Repeated stimuli such as bacterial or viral infections can lead to pathological hyperplasia of lymphoid tissue in the tonsils and adenoids, which is called adenoid tonsil hypertrophy, and is the most common cause of obstructive sleep apnea (OSA) in children. If not diagnosed and treated in time, it will cause a series of serious complications, such as secretory otitis media, rhinosinusitis, maxillofacial developmental abnormalities (adenoid facies), growth retardation, neurocognitive damage, etc.

[0003] The immunological mechanism of adenoids is still unclear. Previous studies have suggested that the interaction between pathogenic microorganisms in the tonsil and adenoids region and the host mucosal immune system causes local immune disorders. In recent years, many studies have found that changes in the composition and function of lymphocytes in tonsil and adenoids tissue play an important role in this process. However, there is currently no in vitro model suitable for replicating human tonsil and adenoids for pathological research, and since experimental animal models cannot be used, the pathophysiological mechanism of tonsil and adenoids is difficult to study. Summary of the invention

[0004] Based on the above, the purpose of the present invention is to provide adenoids organoids and their culture medium and culture method. The adenoids organoids obtained by culture are identified to contain B lymphocytes, T lymphocytes and macrophages, but no epithelial cells, and can truly reflect the immune cell composition of the adenoids.

[0005] The technical solution adopted by the present invention to achieve the technical purpose is:

[0006] The present invention provides a culture medium for adenoid organoids, including RPMI1640 culture medium, to which 10% FBS, 1× non-essential amino acids, 1× penicillin-streptomycin, 1× sodium pyruvate, 50ng / ml amphotericin B, 20ug / ml gentamicin, 1× insulin-selenium-transferrin complex and 0.5-1μg / ml recombinant human B cell activating factor BAFF are added.

[0007] The present invention also provides a method for culturing adenoid organoids, comprising: using a gas-liquid interaction method in a cell culture chamber or directly culturing in a 96-well plate, wherein the culture medium in the chamber is RPMI1640 culture medium supplemented with 10% FBS, 1× non-essential amino acids, 1× penicillin-streptomycin, 1× sodium pyruvate, 50ng / ml amphotericin B, 20ug / ml gentamicin and 1× insulin-selenium-transferrin complex, and 0.5-1ug / ml recombinant human B cell activating factor BAFF is added to the complete culture medium under the chamber.

[0008] Preferably, when cultured directly in a 96-well plate, the final volume is 200ul, 1.5×10 6 Live cells / well, plated in 96-well ultra-low attachment plates.

[0009] Preferably, when the gas-liquid interaction method of the cell culture chamber is used for culture, 100 uL of the cell suspension is plated into the permeable membrane per well.

[0010] More preferably, the permeable membrane has a pore size of 0.4 μm.

[0011] More preferably, a 24-well sized polytetrafluoroethylene or polycarbonate membrane is placed in a standard 12-well plate; or a 96-well polycarbonate membrane plate with a single well receiving tray.

[0012] Preferably, adenoid cells for culture are obtained according to the following method: freshly isolated adenoid tissue is immersed in Ham's F12 medium containing Normocin, penicillin and streptomycin, immersed at 4°C for 1 hour and then rinsed, the tissue is placed in a small culture dish in a biological safety cabinet, Hank's solution is added, the tissue is cut into pieces, and then the tissue is crushed into single cells on a 100 μm cell sieve.

[0013] The present invention further provides adenoids organoids cultured by the above culture method, wherein the adenoids organoids simultaneously contain B lymphocytes, T lymphocytes and macrophages, but do not contain epithelial cells.

[0014] The beneficial effects of the present invention are:

[0015] The culture medium and culture method of the present invention can successfully culture adenoids organoids derived from immune cells. The cultured organoids contain B lymphocytes, T lymphocytes and macrophages at the same time, but do not contain epithelial cells. Various immune cells can be successfully maintained for 2 weeks, which can truly simulate the immune environment of adenoids and is an excellent model for in vitro research on adenoids and related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 These are bright field images of adenoid organoids cultured in the present invention, A. on the 6th day of culture; B. on the 14th day of culture; scale bar: 1000 μm;

[0017] Figure 2 The results of cell marker identification by immunofluorescence staining of adenoid organoids cultured for 6 days; scale bar: 100 μm;

[0018] Figure 3 The results of cell marker identification by immunofluorescence staining of adenoid organoids cultured for 14 days; scale bar: 100 μm. DETAILED DESCRIPTION

[0019] The present invention is described in detail below in conjunction with specific implementation methods. The following specific embodiments are helpful for those skilled in the art to further understand the present invention, but do not limit the present invention in any form.

[0020] Example

[0021] 1. Sample collection

[0022] Obtain the adenoids of children during surgery (Medical Ethics Approval Number: 2024-KYS-006), and soak them in Ham's F12 medium containing Normocin, penicillin, and streptomycin as soon as possible at 4°C for 1 hour for tissue sterilization. Then rinse the adenoids briefly with PBS and store them in an ice box or a refrigerator at 4°C.

[0023] 2. Tissue and Organoid Preparation

[0024] 1) In a biosafety cabinet, place the tissue in a small petri dish and add a little Hank's solution. Use ophthalmic scissors to cut into pieces of 2-5 mm. 3 The tissue was then crushed into single cells on a 100 μm cell sieve. After washing with complete medium (see Table 1), the cells were counted.

[0025] Table 1 Adenoid organoid culture medium components, suppliers and working concentrations

[0026] Reagents brand Part Number Working concentration RPMI1640 Punosa PM150110 - FBS Punosa 164210 10％ Nonessential amino acids, NEAA Punosa PB180424 1× Penicillin-Streptomycin Punosa PB180120 1× Sodium pyruvate Punosa PB180422 1× Amphotericin B Punosa PB180127 50ng / ml Gentamycin Punosa PB180124 20ug / ml Insulin / selenium / transferrin cocktail Source S450J7 1× BAFF Nearshore protein CU71 1μg / ml

[0027] 2) Transfer the single cell to a centrifuge tube, centrifuge at low speed (350g or about 1200 rpm) for 5 minutes, discard the supernatant, add freezing solution (10% DMSO + 90% FBS), divide into explosion-proof internal rotation cryotubes, transfer to a gradient cooling box, place in a -80°C ultra-low temperature refrigerator, and transfer the frozen cells to a liquid nitrogen tank overnight for storage.

[0028] 3) Thawing: Thaw the frozen cells into complete culture medium (see Table 1), count the cells, and adjust the cell concentration. The larger culture plate should have 6×10 cells per ml. 7 cells per ml in smaller culture plates. 7 cells, 7.5×10 6 / ml (96-well plate, 1.5×10 6 living cells).

[0029] 4) Plating: 100uL of cell suspension per well was plated onto a permeable membrane (0.4μm pore size, 24-well polycarbonate membrane) placed in a standard 12-well plate. The lower chamber consisted of complete medium (1ml for a 12-well plate) and 1μg ml -1 Recombinant human B-cell activating factor.

[0030] 5) Maintenance culture: Replenish the culture medium every other day by exchanging 30% of the volume with fresh organoid culture medium.

[0031] 3. Organoid Identification

[0032] The paraffin sections were prepared as follows. Adenoid tissue and organoids were fixed in 4% paraformaldehyde (PFA) for 48 hours. Then they were dehydrated with gradient ethanol, 70% EtOH for 1 hour, 80% Et0H for 1 hour, 90% Et0H for 1 hour, 95% Et0H for 1 hour, 100% EtOH twice for 1 hour each, xylene twice for 1 hour, and paraffin twice for 2 hours. The wax blocks were cut into 5um thickness using a paraffin slicer and baked at 65°C for 2 hours.

[0033] The immunofluorescence staining method is as follows. The paraffin sections were soaked in xylene twice for 3 minutes and in 100% Et0H twice for 3 minutes each. The sections were rehydrated with 95% Et0H for 3 minutes, 70% EtOH for 3 minutes, and 50% EtOH for 3 minutes, and then antigen retrieval was performed in a microwave oven with sodium citrate buffer (10mM sodium citrate and 0.05% Tween 20). The samples were washed twice for 5 minutes in TBS containing 0.025% Tx-100 and blocked with blocking solution (PBS containing 5% BSA) for 2 hours at room temperature. Incubate at 4°C overnight with primary antibodies diluted in PBS containing 5% BSA. Wash 3 times with PBST for 5 minutes, and then incubate at room temperature for 40 minutes with secondary antibodies diluted in TBS containing 5% BSA. After washing the samples with PBST 3 times for 5 minutes, incubate the samples with Hoechst solution diluted in PBS. Wash twice with PBS. Examine the slides under a fluorescence microscope.

[0034] Figure 2 This is the result of cell marker identification by immunofluorescence staining of adenoids organoids cultured for 6 days. In the figure above, blue fluorescence DAPI represents the cell nucleus, green fluorescence marks CD3-positive T lymphocytes, and red fluorescence marks CD19-positive B lymphocytes. The results show that CD3- or CD19-positive cells were detected in the organoids at the same time, indicating that the organoids cultured for 6 days contain both T lymphocytes and B lymphocytes. In the figure below, blue fluorescence DAPI represents the cell nucleus, green fluorescence marks CD11B-positive macrophages, and red fluorescence marks E-CADHERIN-positive epithelial cells. The results show that only CD11B-positive cells were detected in the organoids, but no E-CADHERIN-positive cells, indicating that the organoids cultured for 6 days contain macrophages but not epithelial cells. The above results show that adenoids organoids derived from immune cells were successfully cultured.

[0035] Figure 3This is the result of the identification of cell markers by immunofluorescence staining of adenoids organoids cultured for 14 days. In the figure above, blue fluorescence DAPI represents the cell nucleus, green fluorescence marks CD3-positive T lymphocytes, and red fluorescence marks CD19-positive B lymphocytes. The results show that CD3- or CD19-positive cells were detected in the organoids at the same time, indicating that the organoids cultured for 14 days successfully retained T lymphocytes and B lymphocytes. In the figure below, blue fluorescence DAPI represents the cell nucleus, green fluorescence marks CD11B-positive macrophages, and red fluorescence marks E-CADHERIN-positive epithelial cells. The results show that only CD11B-positive cells were detected in the organoids, but no E-CADHERIN-positive cells, indicating that the organoids cultured for 14 days successfully retained macrophages but not epithelial cells. The above results show that adenoids organoids derived from immune cells can be successfully cultured for 14 days.

[0036] In summary, the culture medium and culture method of the present invention can successfully culture adenoids organoids derived from immune cells. The cultured organoids contain B lymphocytes, T lymphocytes and macrophages at the same time, but do not contain epithelial cells. In addition, various immune cells can be successfully maintained for 2 weeks, which can truly simulate the immune environment of adenoids and is an excellent model for in vitro research on adenoids and related diseases.

[0037] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A culture medium for adenoid organoids, comprising RPMI1640 culture medium, to which 10% FBS, 1× non-essential amino acids, 1× penicillin-streptomycin, 1× sodium pyruvate, 50 ng / ml amphotericin B, 20 ug / ml gentamicin, 1× insulin-selenium-transferrin complex and 0.5-1 μg / ml recombinant human B cell activating factor BAFF are added.

2. Methods for culturing adenoid organoids, including: The cells were cultured using the gas-liquid interaction method in a cell culture chamber or directly in a 96-well plate. The culture medium in the chamber was RPMI1640 medium supplemented with 10% FBS, 1× non-essential amino acids, 1× penicillin-streptomycin, 1× sodium pyruvate, 50ng / ml amphotericin B, 20ug / ml gentamicin and 1× insulin-selenium-transferrin complex. The complete culture medium at the bottom of the chamber was supplemented with 0.5-1ug / ml recombinant human B cell activating factor BAFF.

3. The culture method according to claim 2, characterized in that When cultured directly in a 96-well plate, the final volume was 200ul, 1.5×10 6 Live cells / well, plated in 96-well ultra-low attachment plates.

4. The culture method according to claim 2, characterized in that When using the gas-liquid interaction method of the cell culture chamber, 100uL of cell suspension is plated into the permeable membrane per well.

5. The culture method according to claim 4, characterized in that The permeable membrane has a pore size of 0.4 μm.

6. The culture method according to claim 4, characterized in that: Place 24-well PTFE or polycarbonate membranes in standard 12-well plates; or 96-well polycarbonate membrane plates with a single-well receiving tray.

7. The culture method according to claim 2, characterized in that: Adenoid cells for culture were obtained as follows: freshly isolated adenoid tissue was immersed in Ham's F12 medium containing Normocin, penicillin and streptomycin, and then rinsed after immersion at 4°C for 1 hour. The tissue was placed in a small culture dish in a biosafety cabinet, Hank's solution was added, the tissue was chopped, and then the tissue was crushed into single cells on a 100 μm cell sieve.

8. Adenoids organoids cultured by the culture method according to any one of claims 2 to 7, wherein the adenoids organoids simultaneously contain B lymphocytes, T lymphocytes and macrophages, but do not contain epithelial cells.

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