In-vitro transplantable thymus tissue slice, preparation method and application thereof, and method for constructing thymus chimeric mouse model

Abstract

The invention provides a thymus tissue slice capable of being transplanted in vitro, a preparation method and application and a method for constructing a thymus chimeric mouse model, the thymus tissue slice is placed on a mixed cellulose ester film of hemostatic sponge, a thymus tissue slice culture medium is added, and the thymus tissue slice is continuously cultured for 5-7 days in a cell incubator with the temperature of 37 DEG C and the CO2 content of 5% to obtain the thymus chimeric mouse model. The thymus tissue slice culture medium is replaced every day in the culture process, so that thymus cells in the cultured slice are removed, thymus tissue structures and stromal cells are reserved, and the transplantable thymus tissue slice is obtained. When the thymus chimeric mouse model is constructed, the sizes of thymus tissue slices can be adjusted according to different transplantation parts, HLA matching of donors and receptors is not needed, the model construction method is simple, the transplantation success rate is high, and the model construction method can be achieved only through a small number of thymus tissue slices.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue engineering, and specifically relates to an in vitro transplantable thymus tissue slice, a preparation method and application thereof, and a method for constructing a thymus chimeric mouse model. Background Art

[0002] The thymus is a central lymphoid organ in the human body that is closely related to the body's immune function. It can generate T lymphocytes and play an important immune function. With age, the thymus gradually shrinks and degenerates. But some people may have congenital athymus or dysplasia, such as DiGeorge syndrome. People with this disease are called congenital athymic patients, which can lead to dysplasia of multiple systems in the body and even death. There is currently no effective treatment for this disease. Using tissue slices for transplantation to construct a mouse model has positive significance for the treatment of patients with congenital athymus. Summary of the invention

[0003] In order to solve the problems in the prior art, the present invention provides an in vitro transplantable thymus tissue slice, a preparation method and application thereof, and a method for constructing a thymus chimeric mouse model.

[0004] The present invention solves the technical problem by adopting the following technical solutions:

[0005] The first object of the present invention is to provide a method for preparing an in vitro transplantable thymic tissue slice, comprising the following steps: placing the thymic tissue slice on a mixed cellulose ester membrane of a hemostatic sponge, adding a thymic tissue slice culture medium, and continuously culturing for 5-7 days in a cell culture incubator at 37°C and 5% CO2, replacing the thymic tissue slice culture medium daily during the culture process, so that the thymic cells in the cultured slice are removed, the thymic tissue structure and stromal cells are retained, and a transplantable thymic tissue slice is obtained.

[0006] Furthermore, a method for preparing an in vitro transplantable thymus tissue slice comprises the following steps:

[0007] 1) Prepare thymus tissue slice culture medium: including Hams' F-12 culture medium, heat-inactivated fetal bovine serum, HEPES buffer, L-glutamine, and penicillin-streptomycin;

[0008] 2) Forming tissue sections: cutting and / or shearing the animal thymus tissue into thymus tissue sections, and transferring the sections to a mixed cellulose ester membrane on a hemostatic sponge pre-moistened with the thymus tissue section culture medium in a sterile culture dish;

[0009] 3) Cultivating tissue slices: Place the sterile culture dish containing the thymic tissue slices in a cell culture incubator for culturing at 37°C and 5% CO2 for 5-7 days. During the culturing process, replace the thymic tissue slice culture medium daily and rinse the slices with the thymic tissue slice culture medium to remove the thymic cells in the slices while retaining the thymic tissue structure and stromal cells to obtain transplantable thymic tissue slices.

[0010] Furthermore, the tissue slices are prepared by shearing or slicing, and the slicing method forms a 0.5 mm thick and 5-10 mm in size. 2 The thymus tissue slices are cut into 1-5 mm 3 Thymus tissue sections.

[0011] Furthermore, the slicing method refers to removing the outer capsule and connective tissue of the animal thymus to be cut, and then directly dividing it into two thymic lobule slices, and using an animal soft tissue slicer to slice the thymic lobule into slices with a thickness of 0.5 mm and a size of 5-10 mm. 2 The thymus tissue small piece is then transferred to a mixed cellulose ester membrane on a hemostatic sponge pre-moistened with the thymus tissue slice culture medium in a sterile culture dish to obtain a thymus tissue slice.

[0012] Furthermore, the shearing method refers to removing the outer capsule and connective tissue of the animal thymus to be sheared, and then directly shearing it into 1-5 mm pieces with surgical scissors. 3 The thymus tissue small piece is then transferred to a mixed cellulose ester membrane on a hemostatic sponge pre-moistened with the thymus tissue slice culture medium in a sterile culture dish to obtain a thymus tissue slice.

[0013] Furthermore, the animal soft tissue slicer includes a groove formed by a bottom module and a middle module, and a blade. When slicing, the thymic lobule is placed in the groove formed by the bottom module and the middle module, and the blade passes between the bottom module and the middle module.

[0014] Furthermore, the thickness of the blade is 0.5 mm.

[0015] Furthermore, the thymus tissue slice culture medium includes 85% Hams' F-12 culture medium, 10% heat-inactivated fetal bovine serum, 25 mM HEPES buffer, 2 mM L-glutamine, and 1% penicillin-streptomycin.

[0016] Furthermore, after the thymus tissue slice culture medium is prepared, it is filtered through a 0.22 μm filter membrane before use.

[0017] Furthermore, the mixed cellulose ester membrane is a sterile hydrophilic membrane made of a biologically inert mixture of cellulose acetate and cellulose nitrate. When culturing the thymus tissue slices, a hemostatic sponge is placed in a culture dish, and the thymus tissue slices are placed on the mixed cellulose ester membrane and placed on the hemostatic sponge. The culture medium used is a thymus tissue slice culture medium.

[0018] Furthermore, when replacing the culture medium of the thymus tissue slices, firstly use a sterile pipette to suck out the original culture medium from the culture dish, then rinse the slices with new culture medium, and then replace with new culture medium.

[0019] The second object of the present invention is to provide an in vitro transplantable thymus tissue slice prepared by any of the above methods, wherein the transplantable thymus tissue slice refers to a thymus tissue slice cultured to the thymus cells (CD3 + ) are almost completely eliminated, leaving only dense reticular thymic epithelial cells (CK5 + ,CK8 + ) tissue sections.

[0020] The third object of the present invention is to provide a use of an in vitro transplantable thymus tissue slice in constructing a thymus chimeric mouse model.

[0021] The fourth object of the present invention is to provide a method for constructing a thymic chimeric mouse model, comprising the following steps: implanting a transplantable thymic tissue slice obtained by any of the above methods into the renal capsule, subcutaneous tissue or quadriceps muscle of a Balb / c-Nude athymic nude mouse to obtain a thymic chimeric mouse model, wherein the model does not require HLA matching of the donor and the recipient.

[0022] Furthermore, the thymic chimeric mouse model includes a mouse thymic chimeric mouse model constructed by allogeneic ectopic transplantation and a human thymic chimeric mouse model constructed by xenogeneic ectopic transplantation.

[0023] Furthermore, the transplantable thymus tissue slices obtained by the shearing method are used in a thymus chimeric mouse model of the renal capsule, and the transplantable thymus tissue slices obtained by the slicing method are used in a subcutaneous or quadriceps femoris thymus chimeric mouse model.

[0024] Furthermore, the transplant amount of the thymus tissue slices is 1 to 2 mg per mouse.

[0025] Compared with the prior art, the beneficial technical effects of the present invention are:

[0026] The mouse model of the present invention does not require HLA matching of the donor and the recipient when it is constructed, the model construction method is simple, the transplantation success rate is high, and only a small amount of tissue conditions are required to achieve it. In the present invention, the thymocytes are removed more thoroughly by configuring the culture medium.

[0027] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention is a schematic diagram of the process of preparing thymus tissue slices by the slicing method and shearing method.

[0029] Figure 2 These are photos of thymus tissue sections prepared in Examples 1 and 2 of the present invention.

[0030] Figure 3 These are photos of thymus tissue slices prepared in Example 1 and Example 2 of the present invention on the 0th, 3rd and 6th days of culture.

[0031] Figure 4 These are the hematoxylin-eosin (H&E) staining and immunohistochemistry display images of the thymus tissue sections prepared in Example 1 of the present invention on the 0th, 3rd and 6th days of culture.

[0032] Figure 5 These are the hematoxylin-eosin (H&E) staining and immunohistochemistry display images of the thymus tissue sections prepared in Example 2 of the present invention on the 0th, 3rd and 6th days of culture.

[0033] Figure 6 This is a peripheral blood flow analysis diagram of athymic nude mice transplanted via renal capsule in Example 4 of the present invention and a control group before and after surgery.

[0034] Figure 7 The peripheral blood mature CD3 of athymic nude mice transplanted with renal capsule in Example 4 of the present invention and the control group before and after surgery + TCR-β + T lymphocytes, CD4 + and CD8 + T cell ratio chart.

[0035] Figure 8 This is a hematoxylin-eosin (H&E) staining and immunohistochemistry display of the transplanted site of athymic nude mice transplanted via renal capsule 12 weeks after surgery in Example 4 of the present invention.

[0036] Fig. 9 Schematic diagram of the construction of mouse thymus chimeric mouse models with complete HLA matching and complete mismatching in Example 7 of the present invention.

[0037] Fig.10This is a peripheral blood flow analysis diagram of the thymic chimeric mouse model of HLA completely matched and completely mismatched mice and the control group before and after surgery in Example 7 of the present invention.

[0038] Fig.11 The peripheral blood mature CD3+ Thymus chimeric mouse model of HLA completely matched and completely mismatched mice and the control group before and after surgery were compared. + TCR-β + T lymphocytes, CD4 + and CD8 + T cell ratio chart.

[0039] Fig.12 This is the hematoxylin-eosin (H&E) staining and immunohistochemistry display of the transplantation site of the HLA completely matched and completely mismatched mouse thymic chimeric mouse model 12 weeks after surgery in Example 7 of the present invention.

[0040] Fig.13 The photograph of the human thymus tissue section cultured in vitro for 14 days obtained in Example 8 of the present invention and its hematoxylin-eosin (H&E) staining and immunohistochemistry display diagram.

[0041] Fig.14 This is a peripheral blood flow analysis diagram of the human thymus chimeric mouse model and the control group before and after surgery in Example 8 of the present invention.

[0042] Fig.15 This is a visual view, hematoxylin-eosin (H&E) staining and immunohistochemistry display of the transplantation site of the human thymus chimeric mouse model in Example 8 of the present invention 12 weeks after surgery. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope of protection that the present invention is intended to protect.

[0044] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0045] It should be noted that the materials selected in the embodiments of the present invention are as follows: surgical scissors and tweezers for collection and slicing are sterile disposable instruments; slicer blades are 0.5 mm thick sterile customized blades; culture dishes are 100 mm sterile, pyrogen-free polystyrene cell culture dishes; hemostatic sponges are absorbable gelatin sponges from Nanchang Hushida; mixed cellulose ester membranes are sterile hydrophilic membranes with a pore size of 5 μm; Ham's F-12 culture medium is selected from Gibco, 11765-054; heat-inactivated fetal bovine serum is selected from Sigma, F8318; HEPES buffer is selected from Procell, PB180325; L-glutamine is selected from Procell, PB180420; and penicillin-streptomycin is selected from Procell, PB180120.

[0046] Example 1 Preparation of thymus tissue slices by slicing method

[0047] Preparation of mouse thymus tissue sections using the sectioning method:

[0048] 1) Prepare thymus tissue slice culture medium:

[0049] The thymus tissue slice culture medium includes 85% Ham's F-12 culture medium, 10% heat-inactivated fetal bovine serum, 25mM HEPES buffer, 2mM L-glutamine, and 1% penicillin-streptomycin. After the culture medium is prepared, it is filtered using a 0.22 μm filter membrane in a Class A biosafety cabinet.

[0050] 2) Forming thymus tissue sections by sectioning method:

[0051] See attached Figure 1 , 2 , in a Class A biosafety cabinet, the thymus of a 6-day-old wild-type mouse was aseptically collected, and after removing the outer capsule and connective tissue, it was directly divided into two thymic lobules, and then the thymic lobules were cut into thymic tissue slices using an animal soft tissue slicer. The animal soft tissue slicer used in the embodiment of the present invention adopts the tissue slicer in patent CN202421160705.5, but the description of the animal soft tissue slicer used in the present invention is only for illustration and not for limitation, and all animal soft tissue slicers that can realize thymic tissue slices of the present invention can be used in the present invention.

[0052] During operation, the thymic lobule is placed in the groove formed by the bottom module and the middle module of the animal soft tissue slicer, and then the top module is gently covered on the thymic lobule to fix it. The operator inserts the blade into the gap between the middle and top modules to cut the thymic lobule in half, making a 0.5mm thick and 5-10mm large slice. 2After cutting each thymus tissue slice, remove the top module of the slicer and use forceps to transfer the two thymus tissue slices to the mixed cellulose ester membrane on the hemostatic sponge pre-moistened with thymus tissue culture medium in a sterile culture dish.

[0053] 3) Cultivation of thymus tissue slices:

[0054] The prepared mouse thymus tissue slices were placed in a cell culture incubator at 37°C and 5% CO2 for continuous culture for 5-7 days, and the thymus tissue slice culture medium was changed daily during the culture process. During the medium change process, the culture medium was first sucked out from the sterile culture dish using a sterile disposable pipette, and then fresh culture medium was sucked out to gently rinse each thymus tissue slice from the top of the slice in a flushing manner. During the culture period, a large number of thymocytes were flushed out of the thymus tissue slices or apoptosis occurred, while the thymus structure and thymus stromal cells were retained, and transplantable thymus tissue slices were obtained.

[0055] Example 2 Preparation of thymus tissue sections by shearing method

[0056] The difference from Example 1 is that the mouse thymus tissue slices were prepared by shearing method:

[0057] In a Class A biosafety cabinet, the thymus of 6-day-old wild-type mice was collected aseptically, and after removing the outer capsule and connective tissue, it was directly cut into 1-5 mm pieces using surgical scissors according to the transplantation requirements. 3 The thymus tissue pieces are then transferred with forceps to a mixed cellulose ester membrane on a hemostatic sponge pre-moistened with thymus tissue culture medium in a sterile culture dish to obtain thymus tissue sections.

[0058] Example 3 Determination of transplantable thymus tissue sections

[0059] The thymic tissue slices obtained in Example 1 and Example 2 are cultured in a thymic tissue slice culture medium until the thymocytes in the cultured slices are removed and the thymic tissue structure and stromal cells are retained, thereby obtaining a transplantable thymic tissue slice. There are two methods for determining whether a transplantable thymic tissue slice is formed: visual observation and histological detection method.

[0060] 1. Visual observation method

[0061] See attached Figure 3It can be seen that the mouse thymus tissue slices obtained in Example 1 and Example 2 are light red or pink on the day of preparation (culture day 0) due to residual blood in the thymus tissue. As the culture proceeds, the mouse thymus cells are continuously flushed out, and the color gradually becomes lighter. On the 4th to 6th day of culture, the mouse thymus tissue slices collapse and cling to the mixed cellulose ester membrane, and the color is light yellow, and some thinner sections become translucent. When the thymus tissue slices collapse and become translucent, transplantable thymus tissue slices are formed.

[0062] 2. Histological detection method

[0063] See attached Figure 4 , Attachment Figure 5 Table 1, the mouse thymus tissue sections obtained in Example 1 and Example 2 were stained with hematoxylin-eosin (H&E) and immunohistochemistry, and the thymic T lymphocytes were labeled with Anti-CD3 antibody; the thymic medullary epithelial cells were labeled with Anti-CK5 antibody; and the thymic cortex and medullary epithelial cells were simultaneously labeled with Anti-CK8 antibody. The observations were made on the 0th day, the 3rd day, and the 6th day of tissue culture. It can be seen that regardless of the slicing method or the shearing method, the thymic lymphocytes (CD3 + ), thymic epithelial cells (CK5 + ,CK8 + ) were evenly distributed throughout the thymus tissue, and the slice structure was intact; as the culture time prolonged, the mouse thymocytes were gradually eliminated, and the epithelial cells were retained in the thymus tissue. On the sixth day of culture, the mouse thymus tissue slices retained normal thymus structure, but the thymocytes (CD3 + ) are almost completely eliminated, leaving only dense reticular thymic epithelial cells (CK5 + ,CK8 + ), the thymus tissue slice at this time is a transplantable thymus tissue slice.

[0064] Table 1 Changes in the content of positive cells during slice culture

[0065]

[0066] Example 4: Verification of the effectiveness of thymus tissue slice transplantation into renal capsule

[0067] A 1-1.5 cm skin incision was made on the left abdomen of a Balb / c-Nude athymic nude mouse to expose the left kidney of the nude mouse and a small incision was made on the renal capsule to allow the thymus tissue slice to be inserted. The thymus tissue slice obtained in Example 2 was inserted into the incision and laid as flat as possible when it was determined to meet the transplantation conditions according to Example 3, and then the renal capsule was sutured with absorbable sutures to prevent the capsule from opening, and finally the skin was sutured again with absorbable sutures. The transplantation dose of the thymus tissue slice was 1-2 mg / mouse.

[0068] Flow cytometric analysis of peripheral blood of athymic nude mice transplanted with thymus tissue sections and renal capsule:

[0069] The tail vein blood of athymic Balb / c-Nude mice transplanted with thymic tissue slices was collected before renal capsule transplantation (week 0) and at weeks 4, 8, and 12 after transplantation for flow cytometry analysis. At the same time, the sham operation model of normal mice without transplanted thymic tissue slices was collected as the control group.

[0070] To analyze newly emigrated lymphocytes and the proportion of CD4 and CD8 T cells, the following combination was used: anti-mouse CD45 BV510 (Biolegend); anti-mouse CD3 BUV395 (BD); anti-mouse CD4 PE-Cy7 (Biolegend); anti-mouse CD8 Percp Cy5.5 (Biolegend); anti-mouse TCR-β APC (Biolegend); L / D APC-Cy7 (Invitrogen).

[0071] See attached Figure 6 , Attachment Figure 7 It can be seen that mature CD3 + TCR-β + The proportion of T lymphocytes was higher than that of the control group, and the proportion gradually increased, and was significantly higher than that of the control group at the 8th and 12th weeks after transplantation. + and CD8 + T cells maintain a normal ratio. Therefore, allogeneic heterotopic transplanted thymus tissue slices can reconstruct the central immunity of athymic nude mice.

[0072] Histological analysis of the transplanted site of thymic tissue sections transplanted into the renal capsule of athymic nude mice:

[0073] At 12 weeks after renal capsule transplantation, tissues from the transplanted sites were collected for hematoxylin-eosin (H&E) staining and immunohistochemistry. Anti-CD3 antibody was used to label thymic T lymphocytes; Anti-CK5 antibody was used to label thymic medullary epithelial cells; and Anti-CK8 antibody was used to label both thymic cortical and medullary epithelial cells.

[0074] See attached Figure 8 It can be seen that normal thymus structure (H&E), surviving T cells (CD3 + ), surviving thymic epithelial cells (CK5 + ,CK8 + ), showing that mouse thymic tissue can regenerate the thymus at the transplant site, indicating that renal capsule transplantation can provide an immune microenvironment for mouse thymic tissue and support the development of T lymphocytes.

[0075] Example 5: Verification of the effectiveness of subcutaneous transplantation of thymus tissue slices

[0076] A 1-1.5 cm skin incision was made on the back of a Balb / c-Nude athymic nude mouse to expose the subcutaneous tissue of the back of the nude mouse, and a small incision was formed to allow the thymus tissue slice to be inserted. The thymus tissue slice obtained in Example 1 was inserted into the incision and laid as flat as possible when it was determined to meet the transplantation conditions according to Example 3, and then the back skin was sutured with absorbable sutures to prevent the skin from opening. The transplantation dose of the thymus tissue slice was 1-2 mg / mouse.

[0077] Example 6 Verification of the effectiveness of thymus tissue slice transplantation into quadriceps femoris

[0078] A 1-1.5 cm skin incision was made at the quadriceps muscle of the right hind limb of a Balb / c-Nude athymic nude mouse, the fascia was opened to expose the muscle, and the muscle tissue was bluntly separated to form a pocket between the muscle fibers. The nude mouse thymus tissue slice obtained in Example 1 was laid flat in the pocket when it was judged to meet the transplantation conditions according to Example 3, so that it was completely wrapped by the muscle, and then the muscle was sutured with absorbable sutures to prevent the muscle from reopening, and finally the skin was sutured again with absorbable sutures. The transplantation dose of the thymus tissue slice was 1-2 mg / mouse.

[0079] Example 7: Transplantation of thymus tissue sections into renal capsules shows that transplantation does not require HLA matching of donor and recipient

[0080] A 1-1.5 cm skin incision was made on the left abdomen of CD45.2 Balb / c-Nude athymic nude mice to expose the left kidney of the nude mice and a small incision was made on the renal capsule to allow the thymus tissue slice to be inserted. The thymus of 6-day-old 45.1 Balb / c wild-type mice or the thymus of CD45.1 C57BL / 6J wild-type mice was collected. The thymus tissue slice obtained by Example 2 was inserted into the incision and laid as flat as possible when it was judged to meet the transplantation conditions by Example 3, and then the renal capsule was sutured with absorbable sutures to prevent the capsule from opening, and finally the skin was sutured again with absorbable sutures. The transplantation dose of the thymus tissue slice was 1-2 mg / mouse.

[0081] Preparation of thymic chimeric mouse model: A 1-1.5 cm skin incision was made on the left abdomen of CD45.2 Balb / c-Nude athymic nude mice to expose the left kidney of the nude mice and a small incision was made on the renal capsule to allow the thymic tissue slices to be inserted. The transplantable thymic tissue slices of the completely matched group and the completely mismatched group were inserted into the incisions of different athymic nude mice and spread as flat as possible, and then the renal capsule was sutured with absorbable sutures to prevent the capsule from opening, and finally the skin was sutured again with absorbable sutures. The transplantation dose of thymic tissue slices was 1-2 mg / mouse.

[0082] See attached Fig. 9 According to the method of this example, HLA completely matched and completely mismatched thymic chimeric mouse models were constructed.

[0083] Completely matched group: The thymus of 6-day-old CD45.1 Balb / c wild-type mice was harvested to form transplantable thymic tissue slices, which were then implanted into the renal capsule of CD45.2 Balb / c-Nude athymic nude mice.

[0084] Completely mismatched group: The thymus of 6-day-old CD45.1 C57BL / 6J wild-type mice was harvested to form transplantable thymic tissue slices, which were then implanted into the renal capsule of CD45.2 Balb / c-Nude athymic nude mice.

[0085] The preparation method of the transplantable thymus tissue slices of the completely matched group and the completely mismatched group refers to Example 2, and the transplantable thymus tissue slices are judged according to the method of Example 3.

[0086] Peripheral blood flow analysis:

[0087] To analyze newly emigrated thymocytes and the proportion of CD4 and CD8 T cells, the following combination was used: anti-mouse CD45.2 BV510 (Biolegend); anti-mouse CD45.1 PE (Biolegend); anti-mouse CD3 BUV395 (BD); anti-mouse CD4 PE-Cy7 (Biolegend); anti-mouse CD8 Percp Cy5.5 (Biolegend); anti-mouse TCR-β APC (Biolegend); L / D APC-Cy7 (Invitrogen).

[0088] The tail vein blood of athymic CD45.2 Balb / c-Nude mice was collected before transplantation (week 0) and at weeks 4, 8, and 12 after transplantation in the fully matched group and the fully mismatched group for flow cytometry analysis. At the same time, the sham operation group with thymus tissue sections of normal non-transplanted mice was used as the control group.

[0089] See attached Fig.10 ,11 Compared with the control group, mature CD3 + TCR-β + CD45.2 + T lymphocytes, and the proportion gradually increased, and was significantly higher than that of the control group 12 weeks after transplantation. + and CD8 + T cells maintained a normal ratio. Therefore, this result shows that mouse thymus tissue slice transplantation does not require HLA matching of the donor and recipient, and thymus tissue slice allogeneic ectopic transplantation can successfully reconstruct central immunity.

[0090] Histological analysis:

[0091] At 12 weeks after the CD45.2 Balb / c-Nude athymic nude mouse renal capsule transplantation in the fully matched group and the fully mismatched group, the transplantation site tissues were collected for hematoxylin-eosin (H&E) staining and immunohistochemistry detection. Anti-CD3 antibody was used to label thymic T lymphocytes; Anti-CK5 antibody was used to label thymic medullary epithelial cells; and Anti-CK8 antibody was used to label both thymic cortical and medullary epithelial cells.

[0092] See attached Fig.12 It can be seen that normal thymus structure (H&E), surviving T cells (CD3 + ), surviving thymic epithelial cells (CK5 + ,CK8 + ), indicating that the thymic tissues of mice in both the fully matched and fully mismatched groups survived at the transplantation site and formed thymus, providing an immune microenvironment for the development of T cells in athymic nude mice.

[0093] Example 8 Transplantation of human thymus tissue slices to construct a human thymus chimeric mouse model

[0094] The human thymus tissue slices were placed in a cell culture incubator at 37°C and 5% CO2 for continuous culture for 14 days, and the thymus tissue slice culture medium was changed daily during the culture process. During the liquid replacement process, the culture medium was first sucked out from the sterile culture dish using a sterile disposable pipette, and then fresh culture medium was sucked out to rinse each thymus tissue slice slightly from the upper part of the slice. During the culture period, a large number of thymocytes were flushed out or apoptotic from the thymus tissue slices, while the thymus structure and thymus stromal cells were preserved, and a transplantable thymus tissue slice was obtained when it was judged to meet the transplantation conditions according to Example 3.

[0095] The thymus tissue slices before the transplantation surgery in this example were observed, and it was found that the cultured thymus tissue slices were translucent yellow or brown in color and adhered closely to the mixed cellulose ester membrane.

[0096] Human thymus tissue slices were transplanted into the left kidney capsule / right hind limb quadriceps muscle / back subcutaneous tissue of Balb / c-Nude athymic nude mice at a transplantation dose of 1-2 mg / mouse, so that they were completely wrapped by the muscles.

[0097] Histological analysis before renal capsule transplantation surgery:

[0098] Hematoxylin-eosin (H&E) staining and immunohistochemistry were performed on thymic tissue sections before transplantation surgery, showing that Anti-CD3 antibody labeled thymic T lymphocytes; Anti-CK5 antibody labeled thymic medullary epithelial cells; Anti-CK14 antibody labeled thymic medullary epithelial cells; Anti-Pan-CK antibody labeled thymic epithelial cells and Hassall bodies.

[0099] See attached Fig.13 , it can be seen that the human thymus tissue sections before surgery were stained after 14 days of in vitro culture. The hematoxylin-eosin (H&E) stained sections showed that the normal thymic lobule structure was retained, and the cortical and medullary regions could be clearly distinguished; lymphocytes were largely removed, and the reticular thymic epithelial cells increased significantly; there were at least 2 Hassall bodies with clear structures under a single field of view under the microscope. Immunohistochemistry showed that >80% of the area had no thymic lymphocytes; CK5 staining was scattered throughout the thymic tissue; CK14 staining was scattered throughout the thymic tissue, showing a "lace-like" appearance; Pan-CK (keratin AE1 / AE3) positive areas were scattered throughout the tissue. The presence of Hassall bodies and intact nuclei and successful CK14 staining indicate that normal, transplantable thymus tissue sections have been cultured.

[0100] Peripheral blood flow analysis:

[0101] Blood from the tail vein of mice transplanted into the left renal capsule of Balb / c-Nude athymic nude mice was collected before transplantation (week 0) and at weeks 4, 8, and 12 after transplantation for flow cytometry analysis. At the same time, thymus tissue sections of normal non-transplanted mice were collected as a sham operation control group.

[0102] To analyze newly emigrated thymocytes and the proportion of CD4 and CD8 T cells, the following combination was used: anti-mouse CD45 BV510 (Biolegend); anti-mouse CD3 BUV395 (BD); anti-mouse CD4 PE-Cy7 (Biolegend); anti-mouse CD8 Percp Cy5.5 (Biolegend); anti-mouse TCR-β APC (Biolegend); L / D APC-Cy7 (Invitrogen).

[0103] See attached Fig.14 In the 4th week after renal capsule transplantation, mature CD3 + TCR-β + The proportion of T lymphocytes was higher than that of the control group, and the proportion gradually increased, and was significantly higher than that of the control group at the 8th and 12th weeks after transplantation. + and CD8 + T cells maintained a normal ratio. Therefore, transplanted human thymus tissue slices were able to reconstruct the central immunity of athymic nude mice and successfully construct a chimeric human thymus mouse model.

[0104] Histological analysis after renal capsule transplantation surgery:

[0105] At 12 weeks after the renal capsule transplantation, tissues from the transplanted sites were collected for hematoxylin-eosin (H&E) staining and immunohistochemistry. Anti-CD3 antibody was used to label T lymphocytes reconstituted in the thymus; Anti-CD4 antibody was used to label mouse CD4 reconstituted in the thymus. + T lymphocytes; Anti-CD8 antibody labels mouse CD8 reconstituted in the thymus + T lymphocytes; Anti-CK5 antibody labels thymic medullary epithelial cells; Anti-CK14 antibody labels thymic medullary epithelial cells; Anti-Pan-CK antibody labels thymic epithelial cells and Hassall bodies.

[0106] See attached Fig.15 It can be seen that 12 weeks after transplantation, although some areas of the thymus tissue sections were fatty, the normal thymus structure was retained (H&E). Immunohistochemistry showed that the surviving thymic epithelial cells (CK5 + ,CK14 + 、Pan-CK + ) remained intact, indicating that human thymus tissue sections could still develop normally in the mouse kidney capsule. The tissue structure also contained reconstituted mouse lymphocytes (CD3 + 、CD4 + 、CD8 +) fully demonstrates that the transplantable human thymus tissue chip can provide an immune microenvironment for T cell development in athymic nude mice, allowing nude mice to use human thymus tissue slices to initiate the development and maturation of T cells, thereby restoring the development of T cells in nude mice.

[0107] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0108] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A method for preparing an in vitro transplantable thymus tissue slice, characterized in that: The method comprises the following steps: placing thymic tissue slices on a mixed cellulose ester membrane of a hemostatic sponge, adding thymic tissue slice culture medium, and continuously culturing for 5-7 days in a cell culture incubator at 37°C and 5% CO2, replacing the thymic tissue slice culture medium daily during the culture process, so that the thymic cells in the cultured slices are cleared and the thymic tissue structure and stromal cells are retained, thereby obtaining transplantable thymic tissue slices.

2. The method for preparing an in vitro transplantable thymus tissue slice according to claim 1, characterized in that: The following steps are involved: 1) Prepare thymus tissue slice culture medium: including Ham's F-12 medium, heat-inactivated fetal bovine serum, HEPES buffer, L-glutamine, and penicillin-streptomycin; 2) Forming tissue sections: cutting and / or shearing the animal thymus tissue into thymus tissue sections, and transferring the sections to a mixed cellulose ester membrane on a hemostatic sponge pre-moistened with the thymus tissue section culture medium in a sterile culture dish; 3) Cultivating tissue slices: Place the sterile culture dish containing the thymic tissue slices in a cell culture incubator for culturing at 37°C and 5% CO2 for 5-7 days. During the culturing process, replace the thymic tissue slice culture medium daily and rinse the slices with the thymic tissue slice culture medium to remove the thymic cells in the slices while retaining the thymic tissue structure and stromal cells to obtain transplantable thymic tissue slices.

3. A method for preparing an in vitro transplantable thymus tissue slice according to claim 1 or 2, characterized in that: The tissue slices are prepared by shearing or slicing, and the slicing method forms a 0.5 mm thick and 5-10 mm in size. 2 The thymus tissue slices are cut into 1-5 mm 3 Thymus tissue sections.

4. The method for preparing an in vitro transplantable thymus tissue slice according to claim 3, characterized in that: The slicing method refers to removing the outer capsule and connective tissue of the animal thymus to be cut, and then directly dividing it into two thymic lobule slices, and using an animal soft tissue slicer to slice the thymic lobule into slices with a thickness of 0.5 mm and a size of 5-10 mm. 2 The thymus tissue small piece is then transferred to a mixed cellulose ester membrane on a hemostatic sponge pre-moistened with the thymus tissue slice culture medium in a sterile culture dish to obtain a thymus tissue slice.

5. The method for preparing an in vitro transplantable thymus tissue slice according to claim 3, characterized in that: The shearing method refers to removing the outer capsule and connective tissue of the animal thymus to be sheared, and then directly shearing it into 1-5 mm 3 The thymus tissue small piece is then transferred to a mixed cellulose ester membrane on a hemostatic sponge pre-moistened with the thymus tissue slice culture medium in a sterile culture dish to obtain a thymus tissue slice.

6. A method for preparing an in vitro transplantable thymus tissue slice according to claim 1 or 2, characterized in that: The thymus tissue slice culture medium includes 85% Ham's F-12 culture medium, 10% heat-inactivated fetal bovine serum, 25 mM HEPES buffer, 2 mM L-glutamine, and 1% penicillin-streptomycin.

7. An in vitro transplantable thymus tissue slice prepared by the method according to any one of claims 1 to 5, wherein the transplantable thymus tissue slice is cultured until the thymocytes (CD3 + ) are almost completely eliminated, leaving only dense reticular thymic epithelial cells (CK5 + ,CK8 + ) tissue sections.

8. Use of the in vitro transplantable thymus tissue slice according to claim 7 in constructing a thymic chimeric mouse model.

9. A method for constructing a thymic chimeric mouse model, characterized in that: The method comprises the following steps: implanting the transplantable thymus tissue slice obtained by the method according to any one of claims 1 to 6 into the renal capsule, subcutaneous tissue or quadriceps muscle of a Balb / c-Nude athymic nude mouse to obtain a thymus chimeric mouse model, wherein the model does not require HLA matching of the donor and the recipient.

10. A method for constructing a thymic chimeric mouse model according to claim 9, characterized in that: The thymic chimeric mouse model includes a mouse thymic chimeric mouse model constructed by allogeneic heterotopic transplantation and a human thymic chimeric mouse model constructed by xenogeneic heterotopic transplantation.

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