Cultivation method and application of immune organoids

Abstract

The present invention provides a method for culturing immune organoids and its application. The present invention cultures immune organoids by isolating lung-infiltrating lymph nodes and evaluates the level of mucosal immune response produced against influenza virus. By adding components such as BAFF and IL-2 to the cell culture medium and using the air-liquid interface method to three-dimensionally reconstruct immune cells derived from lung-infiltrating lymph nodes, immune organoids with a lymphoid structure are formed. These immune organoids can more realistically reflect the level of local immune response in the lungs and better retain various immune cells. At the same time, T cells are activated, enhancing their helper effect on B cells; the time for B cells to differentiate into plasma cells and mature is greatly shortened, and the efficiency of detecting and evaluating the immunogenicity of viral antigens is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to a method for culturing immune organoids and its application. Background Art

[0002] Organoid culture technology is a new culture method that has rapidly developed in recent years for generating living-organ or tissue-like structures, and is widely used in disease research and drug development. The culture of organoids is established based on an in vitro cell culture system. Similar to in vivo-derived tissues or organs, it can simulate the spatial distribution patterns and interconnections of various cell types in tissues and organs, has similar physiological functions to in vivo-differentiated organs, and can better simulate the occurrence process and physiological and pathological states of tissues and organs. Currently, relatively mature organoid culture methods have been applied to various organs such as the brain, lungs, liver, stomach, intestine, pancreas, and so on. However, there is currently no report on the method for culturing immune organoids derived from mucosal organs.

[0003] Lung mucosal immunity is crucial for resisting respiratory pathogens. By culturing immune organoids from lung-infiltrating lymph nodes, the evaluation efficiency of respiratory pathogens and related vaccine drugs can be greatly improved. Currently, for the evaluation of the immune response to influenza virus, it is mainly through infecting or immunizing animal models and then detecting the antibody levels in serum and the antigen-specific T cell responses in the spleen. The existing animal model evaluation system used requires about 1 month for the evaluation period, and needs to be completed in an experimental animal barrier, which has high requirements for operators, a long overall time-consuming, and high costs. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for culturing immune organoids and its application.

[0005] The concept of the present invention is as follows: By constructing an air-liquid culture model, immune cells derived from mucosal-infiltrating lymph nodes are connected together. Through the induction of multiple factors, a lymphoid organoid with a three-dimensional structure similar to germinal centers and follicular regions is formed and corresponding immune responses are generated. This culture method can retain various types of immune cells in the lymph nodes and more precisely simulate the local organ immune response level.

[0006] To achieve the object of the present invention, in a first aspect, the present invention provides a culture medium for in vitro culturing immune organoids, which comprises adding 2 mM GlutaMAX, 0.1 mM non-essential amino acids (1× non-essential amino acids), 1 mM sodium pyruvate (1× sodium pyruvate), 100 U / ml penicillin-streptomycin (1× penicillin-streptomycin), 100 μg / ml Normocin (1× Normocin), 10 μg / ml insulin, 5.5 μg / ml transferrin, 6.7 ng / ml sodium selenite, 2 μg / ml ethanolamine (1× insulin / selenium / transferrin cocktail, i.e., insulin-transferrin-selenium-ethanolamine mixture), as well as 1 μg / ml BAFF and 20 U / ml IL-2 to a complete culture medium.

[0007] In the present invention, the complete culture medium is RPMI1640 + 10% FBS.

[0008] The non-essential amino acids are composed of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the content of each component is 0.1 mM.

[0009] Normocin is an antibiotic mixture that can resist bacterial, fungal and mycoplasma contamination, and is purchased from Invivogen.

[0010] BAFF is B-cell Activating factor of the TNF family and is purchased from Biolegend.

[0011] In the present invention, the optimization of the components of the culture medium for in vitro culturing immune organoids is an improvement based on the components of the basic culture medium for organoid culture. 1) GlutaMAX is a commercial substitute for L-glutamine, which is not easily degraded and has less side effects on cells caused by its metabolites. 2) The addition of non-essential amino acids (glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, serine) is mainly to improve cell growth and activity. 3) The insulin-transferrin-selenium-ethanolamine mixture as a basic culture medium supplement can reduce the amount of fetal bovine serum used and is also suitable for subsequent co-culture of immune organoids and other tissue cells. 4) Penicillin-streptomycin is penicillin and streptomycin, and its addition is mainly for antibacterial. 5) B cells have BAFF receptors on their surface, and the addition of BAFF is very important for maintaining the survival, proliferation, antigen presentation and class switch recombination of B cells. 6) Low concentration of IL-2 can promote the proliferation of T cells.

[0012] In a second aspect, the present invention provides a method for culturing immune organoids, comprising the following steps:

[0013] (1) Take tissue-infiltrating lymph nodes (such as lung-infiltrating lymph nodes), cut them into small pieces, place them in a 100 μm filter screen for grinding, and rinse with complete medium until the grinding is thorough;

[0014] (2) Add the cell suspension passed through the filter screen above Ficoll, centrifuge at 400 g - 600 g for 30 min, and collect the cells in the buffy coat layer; Prepare complete medium plus, that is, add GlutaMAX (2 mM), 1× non-essential amino acids (0.1 mM), 1× sodium pyruvate (1 mM), 1× penicillin-streptomycin (100 U / ml), 1× Normocin (100 μg / ml), and 1× insulin-transferrin-selenium-ethanolamine mixture (insulin 10 μg / ml, transferrin 5.5 μg / ml, sodium selenate 6.7 ng / ml, ethanolamine 2 μg / ml) to the complete medium, which is the complete medium plus; Wash the cells with complete medium plus, count and resuspend to 1×10 7 -2×10 7 cells / ml to obtain a cell suspension;

[0015] (3) Prepare the above-mentioned one. Place a transwell with a membrane pore size of 0.4 μm in a (24-well) cell culture plate, add 0.5 ml of the prepared medium for in vitro culturing of immune organoids to each well, and add 100 μl of the cell suspension from step (2) to the transwell;

[0016] (4) Add vaccine drugs or antigens to the transwell, and place it in an incubator (37 °C, 5% CO2) for culturing;

[0017] (5) Rehydrate every 2 - 3 days, that is, supplement the medium for in vitro culturing of immune organoids to keep it above the liquid interface of the well plate;

[0018] (6) Change the medium every 7 days, use the medium for in vitro culturing of immune organoids to change the medium, take the supernatant for antibody level detection, and culture for 28 - 35 days in total. Immune cells grow in clusters, and the immune organoids of lung lymph nodes mature continuously, gradually forming aggregated cell clusters to form three-dimensional immune organoids with similar lymphoid structures.

[0019] Further, after taking the tissue-infiltrating lymph nodes in step (1), it also includes a step of pre-treating the tissue-infiltrating lymph nodes: After taking the tissue-infiltrating lymph nodes, place them in pre-cooled tissue preservation solution, and perform subsequent processing after 2 h.

[0020] Among them, the tissue preservation solution is: Ham's F12 medium + 100 U / ml penicillin-streptomycin + 100 μg / ml Normocin + 5 - 10 mM ROCK inhibitor Y27632.

[0021] Ham's F12 medium (product number: N3520-10X1L) was purchased from Sigma.

[0022] ROCK inhibitor Y27632 (product number: 1254 / 10) was purchased from Tocris.

[0023] In a third aspect, the present invention provides immune organoids cultured according to the described method.

[0024] In a fourth aspect, the present invention provides the use of the immune organoids as a tissue organ model in evaluating the immune response level of antigens or vaccine drugs.

[0025] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects:

[0026] The present invention cultures immune organoids by isolating lung-infiltrating lymph nodes and evaluates the mucosal immune response level against influenza virus. On the one hand, it does not require the use of an animal barrier system, reducing the technical requirements for experimental operators; on the other hand, it can greatly shorten the time for detecting the immunogenicity of antigens such as influenza virus and vaccine drugs, improving the evaluation efficiency. Therefore, the preparation and culture of immune organoids derived from lymph nodes, especially lung lymph nodes, can efficiently evaluate the immune response of respiratory tract infection pathogens and vaccine drugs.

[0027] The present invention adds GlutaMAX (2 mM), 1× non-essential amino acids (0.1 mM), 1× sodium pyruvate (1 mM), 1× penicillin-streptomycin (100 U / ml), 1× Normocin (100 μg / ml), and 1× insulin-transferrin-selenium-ethanolamine mixture (insulin 10 μg / ml, transferrin 5.5 μg / ml, sodium selenite 6.7 ng / ml, ethanolamine 2 μg / ml), BAFF (1 μg / ml), IL-2 (20 U / ml), etc. to the cell culture medium, and uses the air-liquid method to three-dimensionally reconstruct immune cells derived from lung-infiltrating lymph nodes to form immune organoids with a similar lymphoid structure, truly reflecting the local immune response level of the lungs and better retaining various immune cells. Activated T cells while enhancing their helper effect on B cells; greatly shortening the time for B cells to differentiate into plasma cells and mature, and improving the efficiency of detecting and evaluating the immunogenicity of influenza virus antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is the technical flow chart for in vitro culturing of lung-infiltrating lymph node immune organoids in the preferred embodiment of the present invention.

[0029] Figure 2 This shows the formation of lung lymph node immune organoids in the preferred embodiment of the present invention. Morphological changes of lung lymph node immune organoids during the culturing process.

[0030] Figure 3 This shows the spatial characteristics of immunofluorescence staining of lung lymphoid immune organoids in the preferred embodiment of the present invention. The left is the immunofluorescence staining map of CD19 (B cell surface marker), and the right is the immunofluorescence staining map of DAPI (cell nucleus).

[0031] Figure 4 This is the flow cytometry staining map of the immune cell composition of lung lymphoid immune organoids in the preferred embodiment of the present invention. The immune cell composition is diverse, including CD4 + T cells, CD8 + T cells, B cells, natural killer (NK) cells, dendritic cells (DC), etc.

[0032] Figure 5 This shows that plasma cells are differentiated from B cells after culturing with antigens in the preferred embodiment of the present invention. During the culturing process of lymphoid immune organoids, flow cytometry was used to regularly detect the differentiation of B cells. Ctrl is the control group, and Stimuli is the antigen-stimulated group. The figure shows the flow cytometry diagrams of plasma cells (CD38++CD27++), B cells (CD38-CD27-), memory B cells (CD38-CD27+), pre-GC B cells (CD38+CD27-), and GC B cells (CD38+CD27+) at different time points.

[0033] Figure 6 This shows the differentiation of B cells during the culturing process of lung lymphoid immune organoids in the preferred embodiment of the present invention. The figure shows the statistical chart of the changing trends of the proportions of plasma cells (CD38++CD27++), B cells (CD38-CD27-), memory B cells (CD38-CD27+), pre-GC B cells (CD38+CD27-), and GC B cells (CD38+CD27+).

[0034] Figure 7It shows the change in the level of antigen-specific IgG during the culture of lung lymphoid immune organoids in the antigen immunogenicity evaluation process of a preferred embodiment of the present invention. At the 0th week (ctrl), 1st week (week 1), 2nd week (week 2), 3rd week (week 3), and 4th week (week 4) after antigen stimulation, the level of antigen-specific IgG in the supernatant of lung lymphoid immune organoids was detected by ELISA.

[0035] Figure 8 It shows the activation status of T cells during the culture of lung lymphoid immune organoids in a preferred embodiment of the present invention. On the 12th, 19th, and 26th days of culture, the activation level (HLA-DR+CD38+) of T cells was detected. On the 12th and 19th days of culture, the proportion of activated T cells in the antigen-stimulated group was higher than that in the control group. On the 26th day of culture, no effective number of T cells could be detected in the control group without antigen stimulation, while a certain number of activated T cells could still be detected in the antigen-stimulated group.

[0036] Figure 9 It is the flow cytometry staining map of T cells in lung lymphoid immune organoids on the 28th day of culture in a preferred embodiment of the present invention. The left shows the proportion of T cells in the immune organoids in the control group without IL-2 stimulation in the medium, and the right shows the proportion of T cells in the immune organoids in the experimental group with IL-2 stimulation added to the medium. Figure 9 It shows the effect of adding or not adding IL-2 to the medium on the culture of immune organoids in a specific embodiment of the present invention. Detailed implementation manner

[0037] The present invention provides a medium and a culture method for in vitro culturing lung-infiltrating lymph node immune organoids.

[0038] This method uses lung-infiltrating lymph nodes for the culture of immune organoids, and the composition and differentiation of immune cells are relatively stable. Moreover, for the evaluation of the immunogenicity of pathogens, compared with using ordinary immune cells for in vitro stimulation, it can more accurately reflect the state of pulmonary immune response after respiratory virus infection.

[0039] 1. A relatively stable tissue preservation solution (Ham’s F12 medium + penicillin-streptomycin (100 U / ml) + Normocin (100 μg / ml) + ROCK inhibitor Y27632 (5 - 10 mM)) is used, where Y27632 can effectively inhibit ROCK and make the tissue state relatively stable.

[0040] 2. During the tissue culture process, add GlutaMAX, 1×nonessential aminoacids, 1×sodium pyruvate, 1×penicillin–streptomycin, 1×Normocin (InvivoGen), 1×insulin / selenium / transferrin cocktail (Gibco), BAFF (1 μg / ml), IL-2 (20 U / ml), etc. to the culture medium.

[0041] 3. Compared with using animal models, the experimental period is shorter, and there is no need to rely on animal barrier facilities. The culture method of lung-infiltrated lymph node immune organoids can activate various immune cells, effectively activate T cells and B cells, and rapidly differentiate into plasma cells to produce a large amount of antibodies. The time for plasma cell differentiation and maturation is shortened from 1 month to 2 weeks, effectively improving the evaluation efficiency of pathogen immune response.

[0042] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art, and the raw materials used are all commercially available products.

[0043] Example 1 Culture medium and culture method for in vitro culturing lung-infiltrated lymph node immune organoids

[0044] 1. Immediately after removing the lung-infiltrated lymph nodes, place them in pre-cooled tissue preservation solution (Ham’s F12 medium + penicillin-streptomycin (100 U / ml) + Normocin (100 μg / ml) + ROCK inhibitor Y27632 (5 - 10 mM)) and process them within 2 h.

[0045] 2. After rinsing the tissue with PBS, cut the tissue into small pieces with scissors, place them in a 100 μm filter screen for grinding, and continuously rinse with complete medium until the grinding is complete.

[0046] 3. Slowly add the filtered cell suspension on top of Ficoll. After centrifugation at 400 g - 600 g for 30 min, collect the cells in the buffy coat layer. Wash with complete medium (RPMI 1640 + 10% FBS) supplemented with GlutaMAX (2 mM), 1× non-essential amino acids (glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, serine at 0.1 mM each), 1× sodium pyruvate (1 mM), 1× penicillin-streptomycin (100 U / ml), 1× Normocin (100 μg / ml), and 1× insulin-transferrin-selenium-ethanolamine mixture (insulin 10 μg / ml, transferrin 5.5 μg / ml, sodium selenate 6.7 ng / ml, ethanolamine 2 μg / ml) (Gibco), then count and resuspend to 1.5×10 7 cells / ml for standby.

[0047] 4. Prepare the medium for in vitro culture of immune organoids. Use forceps to place a 0.4-μm chamber (transwell) into a 24-well cell culture plate. Add 0.5 ml of the prepared medium for in vitro culture of immune organoids to each well, and add 100 μl of cell suspension into the chamber.

[0048] Among them, the medium for in vitro culture of immune organoids is: add 2 mM GlutaMAX, 0.1 mM non-essential amino acids (1× non-essential amino acids), 1 mM sodium pyruvate (1× sodium pyruvate), 100 U / ml penicillin-streptomycin (1× penicillin-streptomycin), 100 μg / ml Normocin (1× Normocin), and 10 μg / ml insulin, 5.5 μg / ml transferrin, 6.7 ng / ml sodium selenate, 2 μg / ml ethanolamine (1× insulin / selenium / transferrin cocktail, i.e., insulin-transferrin-selenium-ethanolamine mixture), as well as 1 μg / ml BAFF and 20 U / ml IL-2 to the complete medium.

[0049] 5. Add antigens such as vaccine drugs into the chamber, and place it in a 37°C, 5% CO2 incubator for culture.

[0050] 6. Rehydrate every 2 - 3 days, supplement the medium for in vitro culture of immune organoids, and keep it above the liquid interface of the well plate.

[0051] 7. Change the medium every 7 days. Use the medium for in vitro culture of immune organoids to change the medium, collect the supernatant for detection of antibody levels, and detect B cell differentiation and T cell activation degree.

[0052] The present invention mainly applies lymphoid immune organs to the evaluation of the immune response to influenza virus, truly reflecting the local immune response level in the lungs and shortening the process of plasma cell differentiation and maturation. The technical process is shown in Figure 1 .

[0053] For the culture of immune organs of lymph nodes derived from other tissues and organs, this method can be referred to, but the response to antigen immunogenicity may vary due to different sources.

[0054] Example 2 Detection of the immunogenicity of influenza virus by pulmonary lymphoid immune organs

[0055] After the treatment of steps 1-4 in Example 1, influenza virus H1N1 (A / Puerto Rico / 8 / 1934) was added to the chamber, and the MOI was adjusted to 1. The immune cells grew in clusters, and the pulmonary lymph node immune organs matured continuously, gradually forming aggregated cell clusters, forming three-dimensional immune organs with similar lymphoid structures ( Figure 2 , Figure 3 ). At the same time, the immune cell composition of the pulmonary lymph node immune organs also showed diversification, including CD4+ T cells (CD3+CD4+), CD8 + T cells (CD3+CD8+), B cells (CD19+), natural killer cells (CD56+CD3-), dendritic cells (CD11c+), etc. ( Figure 4 ).

[0056] After culturing the lymphoid immune organs for 1-2 weeks, B cells can differentiate into a large number of plasma cells (CD38++CD27++) (Note: ++ represents strongly positive) ( Figure 5 ). On the 7th and 12th days of culture, B cells in the culture chambers stimulated with antigen can differentiate into a relatively high proportion of plasma cells (CD38++CD27++: 40.7% on the 7th day, 44.0% on the 12th day). Plasma cells in the culture chambers stimulated with antigen can be maintained for 4-5 weeks, while only a small number of plasma cells can be differentiated in the control wells and maintained for about two weeks.

[0057] During the culture of the lymphoid immune organs, flow cytometry was used to regularly detect the differentiation of B cells ( Figure 6 ). The proportion of plasma cells (CD38++CD27++) showed a trend of increasing first and then decreasing, accompanied by the decrease of B cells (CD38-CD27-), memory B cells (CD38-CD27+), and the increase of pre-GC B cells (CD38+CD27-), GC B cells (CD38+CD27+).

[0058] The IgG level against influenza A virus in the culture supernatant was detected using an ELISA kit ( Figure 7 ). It can be seen that at 2 weeks of culture, the IgG level increased significantly and remained at a relatively high level within 4 weeks.

[0059] The method of the present invention is also beneficial to the maintenance of the activity of T cells. During the culture process, the change in the proportion of HLA-DR+CD38+ is relatively small ( Figure 8 ). In the culture wells with antigen stimulation added, T cells can maintain good activity, and their survival time can be maintained for about 4 weeks. This has a promoting effect on the function of B cells.

[0060] The present invention further investigated the effects of adding or not adding IL-2 to the culture medium on the culture of immune organs, as follows:

[0061] When IL-2 is not added to the culture medium, the maintenance time of T cell activity is relatively short and it is difficult to continue to maintain after 4 weeks. When a low dose of IL-2 is added, the maintenance and functional response of T cells are more persistent. On the 28th day of immune organ culture, the number and proportion of T cells in the control group without IL-2 treatment decreased significantly ( Figure 9 ).

[0062] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A method for culturing immune organoids, characterized in that, It includes the following steps: (1) Take tissue-infiltrating lymph nodes, cut them into small pieces, place them in a 100-μm filter screen for grinding, and rinse with complete medium until the grinding is thorough; (2) Add the cell suspension that has passed through the filter above the Ficoll, centrifuge at 400 g - 600 g for 30 min, and collect the cells in the buffy coat layer; Prepare complete medium plus, that is, add 2 mM GlutaMAX, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 U / ml penicillin-streptomycin, 100 μg / ml Normocin, 10 μg / ml insulin, 5.5 μg / ml transferrin, 6.7 ng / ml sodium selenite, and 2 μg / ml ethanolamine to the complete medium, which is the complete medium plus; After washing the cells with the complete medium plus, count and resuspend them to 1×10 7 -2×10 7 cells / ml to obtain a cell suspension; Among them, the complete medium is RPMI 1640 + 10% FBS; The non-essential amino acids are composed of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the content of each component is 0.1 mM; (3) Prepare a medium for culturing immune organoids in vitro. Place a chamber with a membrane pore size of 0.4 μm in a cell culture plate, add 0.5 ml of the prepared medium for culturing immune organoids in vitro to each well, and add 100 μl of the cell suspension in step (2) to the chamber; (4) Add an antigen or an attenuated vaccine to the chamber and place it in an incubator for culturing; (5) Rehydrate every 2 - 3 days, that is, supplement the medium for culturing immune organoids in vitro to keep it above the liquid interface of the well plate; (6) Change the medium every 7 days. Use the medium for culturing immune organoids in vitro to change the medium, take the supernatant for detecting the antibody level, and co-culture for 28 - 35 days; The medium for culturing immune organoids in vitro is prepared by adding 2 mM GlutaMAX, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 U / ml penicillin-streptomycin, 100 μg / ml Normocin and 10 μg / ml insulin, 5.5 μg / ml transferrin, 6.7 ng / ml sodium selenite, 2 μg / ml aminoethanol, as well as 1 μg / ml BAFF and 20 U / ml IL-2 to the complete medium; Among them, the complete medium is RPMI 1640 + 10% FBS; The non-essential amino acids are composed of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the content of each component is 0.1 mM; BAFF is B cell activating factor; The tissue-infiltrating lymph node is a lung-infiltrating lymph node.

2. The method according to claim 1, wherein After taking the tissue-infiltrating lymph node in step (1), it further includes a step of pre-treating the tissue-infiltrating lymph node: After taking the tissue-infiltrating lymph node, place it in a pre-cooled tissue preservation solution, and perform subsequent processing after 2 h; Among them, the tissue preservation solution is: Ham’s F12 medium + 100 U / ml penicillin-streptomycin + 100 μg / ml Normocin + 5 - 10 mM ROCK inhibitor Y27632.

3. The immune organoids obtained by culturing according to the method described in claim 1 or 2.

4. The application of the immune organoids described in claim 3 as a tissue organ model in evaluating the immune response level of an antigen or a vaccine drug.

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