Special culture medium and kit for inducing hematopoietic stem cells to differentiate into T lymphocytes in vitro aiming at Foxn1 gene defect and application of special culture medium and kit

Abstract

The invention discloses a special culture medium for inducing hematopoietic stem cells to differentiate into T lymphocytes in vitro aiming at Foxn1 gene defects, a kit and application of the special culture medium and the kit, and relates to the technical field of cell culture. The simplified and efficient culture medium combination and culture process are included, the complex reagent preparation process is optimized into two independently packaged kits, the operation difficulty, pollution risk and cost input of organoid culture are remarkably reduced, additional matrix materials do not need to be added, and the stability of the cell growth state is facilitated. The culture medium is applied to hematopoietic stem cell culture and differentiation of T lymphocytes. The method is expected to be suitable for hematopoietic stem cells from marrow sources of other mammals including human beings, and the culture system is easy in source obtaining, low in cost, simple in operation process, short in induction time and beneficial to large-scale and industrialization.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, in particular to a special culture medium, a kit and application thereof for inducing hematopoietic stem cells to differentiate into T lymphocytes in vitro targeting Foxn1 gene deficiency. Background Art

[0002] The thymus and bone marrow are central immune organs for the development, differentiation, and maturation of human immune cells. Mature T and B cells reside in peripheral immune organs and respond to antigenic stimulation. The thymus is a crucial site for T cell maturation and the establishment of central tolerance. T cell differentiation is a coordinated process involving interactions with various thymocytes. T-cell lymphocytopenia occurs in the absence of the thymus. The thymus is composed of thymocytes and stromal cells. Stromal cells form the thymic tissue scaffold, promote the differentiation and proliferation of thymic epithelial cells, and support thymopoiesis and T lymphocyte maturation.

[0003] The master regulatory gene for thymic epithelial cell function is the Foxn1 gene. Foxn1 contributes to the structural integrity of the thymic environment and the generation of thymic epithelial cells by inhibiting the formation of thymic epithelial tubular cells and inducing the expression of key genes involved in the thymic epithelial cell program. Loss of Foxn1 function results in thymic hypoplasia and the absence of thymic stromal cells. Existing treatments include hematopoietic stem cell transplantation and allogeneic thymus transplantation. Hematopoietic stem cell transplantation rarely leads to fundamental clinical improvement and is prone to complications and even death. Although allogeneic thymus transplantation can restore T cell function in patients with Foxn1 deficiency, it is limited by the availability of donor tissue. Therefore, it is essential to develop models to study the mechanisms of thymic insufficiency and to develop treatments for thymic defects.

[0004] Nude mice are known to be Foxn1- / - BABL / c mice. The Foxn1 gene is involved in the development, differentiation, and maintenance of TECs during embryonic and postnatal life. Foxn1 deficiency leads to hypothymic function and the inability to develop and differentiate into naive T cells.

[0005] During the continuous differentiation of T cells, they can be divided into CD4 － CD8 － Double negative period, CD4 + CD8 + Double positive period and CD4 + CD8 － or CD4 －The CD8+ single-positive stage. The DN stage can be divided into DN1, DN2, DN3, and DN4 based on the expression of CD44 and CD25. The Notch 1 receptor plays a crucial role when T cells enter the DN1 stage; otherwise, the cells will differentiate into cells other than T cells, such as B cells and NK cells. Blockade of the DN2 stage can lead to the development of the lymphocyte pathway toward non-T cells. A key event occurring during the DN3 to DN4 stages is β-selection, which occurs during TCR rearrangement. The initiation of β-selection requires successful β-chain rearrangement and intracellular TCRβ, and CD3 expression is essential for the progression from DN3 to DN4. After TCRβ rearrangement is complete and expressed on the cell surface, the TCRα chain rearranges in DP cells, which then undergo positive and negative selection, ultimately differentiating into naive T cells.

[0006] OP9 stromal cells have been shown to be useful for inducing T cell differentiation in vitro. The Notch 1 signaling pathway influences mouse T cell development from the DN1 to DN4 stages, and potentially even the DN4 to ISP stages, and is crucial for T cell proliferation and differentiation during the β-selective stage. Previous studies have shown that OP9-DLL1 cells can induce some differentiation of hematopoietic stem and progenitor cells into CD4 and CD8 double-positive or single-positive T cells, but the efficiency of this differentiation is low.

[0007] In summary, treatments for thymic dysfunction (especially those caused by Foxn1 gene deletion) are still incomplete, and the induction of hematopoietic stem and progenitor cell differentiation into T lymphocytes in vitro faces numerous technical challenges, including high operational complexity, low efficiency, susceptibility to contamination and mix-up, high reagent preparation costs, operational difficulties, and difficulties in achieving large-scale and industrialized production. These issues urgently require researchers to seek new solutions to promote the advancement of thymic research and clinical applications. Summary of the Invention

[0008] To address the problems of the prior art, the present invention provides a specialized culture medium, kit, and application thereof for inducing the in vitro differentiation of hematopoietic stem cells into T lymphocytes in patients with Foxn1 gene deficiency. First, a simple and efficient culture medium combination and optimized culture method are designed. In the examples, mouse experiments demonstrate the directed differentiation of nude mouse hematopoietic stem cells into T lymphocytes. This method effectively simplifies the organoid culture process, improves experimental efficiency and results stability, and provides a more practical and economical in vitro model system for thymus development research, immunological mechanism exploration, and drug screening. This provides a reference for treatment directions for Foxn1 deficiency. The culture medium components of the differentiation system are simple and readily available, and the operation process is short and efficient, facilitating scale-up and industrialization.

[0009] One of the purposes of the present invention is to provide a special culture medium for inducing the differentiation of hematopoietic stem cells into T lymphocytes in vitro for Foxn1 gene deficiency, which is composed of the following components in weight percentage: 1.5-2.5wt% B27, 25-35μmol / L ascorbyl phosphate magnesium sesquimagnesium salt hydrate, 0.8-1.2wt% antibiotics, 0.8-1.2wt% L-alanyl-L-glutamine, 4-6ng / mLrmFLT3-L, 4-6ng / mL rmIL-7, 9-11ng / mLrmSCF, and 0.04-0.06mM β-mercaptoethanol added to DMEM-F12 culture medium; experiments have confirmed that the culture medium can greatly maintain the growth stability and differentiation efficiency of T lymphocytes. When used, the culture medium must be changed every two days in the differentiation system, and the freshly prepared culture medium must be used up within one week.

[0010] For the technical solution described above, it is further preferred that the formula of the T lymphocyte differentiation medium, the most preferred ratio in the embodiment is as follows: 2wt% B27, 30μmol / L ascorbyl phosphate magnesium sesquimagnesium salt hydrate, 1wt% antibiotics, 1wt% L-alanyl-L-glutamine, 5ng / mL rmFLT3-L, 5ng / mL rmIL-7, 10ng / mL rmSCF and 0.05mM β-mercaptoethanol are added to DMEM-F12 medium.

[0011] For the technical solution described above, it is further preferred that, in addition to the T lymphocyte differentiation medium described above, a stromal cell culture medium is also included for use therewith; the stromal cell culture medium is mainly for the culture of stromal cells, and is an αMEM culture medium containing 9-12 wt% FBS; more preferably, an αMEM culture medium containing 10 wt% FBS.

[0012] A second object of the present invention is to provide a special kit for inducing the differentiation of hematopoietic stem cells into T lymphocytes in vitro for Foxn1 gene deficiency, which is used to induce hematopoietic stem cells to differentiate into T lymphocytes. The kit contains the reagents required for preparing the above-mentioned stromal cell culture medium and T lymphocyte differentiation medium.

[0013] A third object of the present invention is to provide a method for inducing the differentiation of hematopoietic stem cells into T lymphocytes in vitro using the above-mentioned specialized culture medium, comprising the following steps:

[0014] S1. OP9 cells should be transduced with enhanced green fluorescent protein (eGFP) using a lentiviral vector encoding the mouse DLL4 gene to obtain OP9-mDLL4 stromal cells. These cells should be cultured in a monolayer in stromal cell culture medium to obtain a monolayer of cells. LSK cells should be sorted from hematopoietic stem cells and stored in PBS at 4°C until use.

[0015] S2. Mixing the monolayer cells and the LSK cells in a ratio of 1-5:1 (more preferably 1-3:1, most preferably 1:1), spotting the mixture in a culture plate, adding T lymphocyte differentiation medium, and performing air-liquid interface culture;

[0016] S3. Perform flow cytometry sorting on the cells cultured at the air-liquid interface to obtain a population of CD4 and CD8 single-positive T lymphocytes.

[0017] For the technical solution described above, preferably, the hematopoietic stem cells are derived from the bone marrow of the femur and tibia of nude mice, the bone marrow is subjected to tissue digestion and dissociation to obtain a hematopoietic stem cell suspension, and then the Lin-cell suspension is sorted using a magnetic sorting technique, and the cells in the Lin-cell suspension are detected and the Lin - Sca-1 + c-Kit + (LSK) cell population.

[0018] For the technical solution described above, preferably, during the gas-liquid interface culture process in S3, samples are taken regularly, ground through a 40 μm cell sieve into a cell suspension, and then subjected to flow cytometry detection to obtain T lymphocyte differentiation status.

[0019] For the technical solution described above, it is further preferred that the cell suspension is ground into a 40 μm cell sieve, the cell pellet is extracted, and the suspension is resuspended, and TruStain FcX is added and pipetted to mix, and CD8-PE, TCRβ-PerCP-Cy5.5, CD25-PE-Cy7, CD44-BV510, CD4-BV570, CD117-BV605, CD3-APC, CD11c-AF700, CD45-APC-Cy7 antibodies are added after blocking, and the cells are incubated in the dark at 4°C for 20 minutes, and then flow cytometry buffer is added to terminate the incubation and wash the unbound antibodies. After centrifugation at 300g for 5 minutes, the supernatant is discarded, and after washing again, the cells are resuspended with a buffer containing DAPI for flow cytometry detection.

[0020] A fourth object of the present invention is to use the single-positive T lymphocyte population obtained by the above-mentioned culture method, specifically:

[0021] The above-mentioned single-positive T lymphocyte population can be further cultured to obtain a T lymphocyte population with multiple functions, specifically including any of the following technical features:

[0022] A. After receiving induced differentiation and negative selection, it differentiates into a population of T lymphocytes that has no immune rejection effect on specific antigens;

[0023] B. Co-culture with tumor cells to cultivate and differentiate tumor-infiltrating lymphocyte populations for specific tumor treatment;

[0024] C. Cultivate and differentiate T lymphocyte populations with immune functions, which are then delivered to immunodeficient individuals to supplement immune cells.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a method for inducing immunodeficient bone marrow hematopoietic stem cells to differentiate into T lymphocytes in vitro. This method addresses the problem that embryonic and postnatal thymic epithelial cells cannot develop, differentiate, and maintain due to Foxn1 gene deficiency. The development and differentiation of T lymphocytes can be achieved solely through in vitro induction.

[0027] The present invention provides a culture medium combination for inducing nude mouse hematopoietic stem cells to differentiate into T lymphocytes in vitro. The advantages of the culture medium combination are also reflected in the culture process of human or other mammalian cells, effectively reducing the complexity of organoid culture, and facilitating improved experimental efficiency and consistency of results. The original more than ten reagents and dozens of preparation processes are simplified into two independently packaged prepared kits, greatly simplifying the operation process. At the same time, the contamination and confusion that may occur in the original preparation process are reduced, and the operation of the organoid culture process is simplified. When used to culture cells, no additional matrix material needs to be added, which saves costs and reduces the need to consider the impact of materials on cell growth. In addition, the culture medium combination is easily available, low-cost, simple to operate, and short induction time. The operator can complete the organoid culture process by following the operating steps of the kit, reducing the difficulty of manual operation and facilitating scale and industrialization.

[0028] The present invention provides an in vitro culture method for inducing nude mouse hematopoietic stem cells to differentiate into T lymphocytes, comprising: step 1, constructing and culturing stromal cells; step 2, obtaining nude mouse bone marrow-derived hematopoietic stem cells; step 3, constructing a T lymphocyte induction system; and step 4, inducing T lymphocyte differentiation. The culture method is highly similar to the thymus in normal BABL / c mice and can achieve the construction of a large number of mature T lymphocytes in a short period of time, effectively improving the stability of cell culture. The method has extremely high reference value for the development and differentiation of T lymphocytes in the human thymus, and is of great significance for studying the T cell maturation mechanism in the thymus, the pathological mechanism of thymic damage, the body's immune system repair, and tumor targeted therapy. The method can be used for large-scale thymic organoid culture, providing an ideal in vitro cell model for constructing a thymus biobank, studying the T cell maturation and differentiation mechanism, and developing thymus drugs.

[0029] In the present invention, although the experiments verified the effectiveness and superiority of the in vitro induction method based on the nude mouse model, based on the biological similarities and existing research results, it can be reasonably inferred and expected that it has universal beneficial effects, including that the thymic organoids cultured using the culture method provided by the present invention can be cultured for a long time, passaged or frozen for long-term storage.

[0030] The method described in the present invention is not only applicable to nude mouse bone marrow hematopoietic stem cells, but also has potential application value for hematopoietic stem cells derived from the bone marrow of other species including humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the construction, culture, and FACS sorting of OP9-mDLL4 stromal cells in Example 3. Figure A shows the cell morphology of OP9-DLL4 observed under bright field microscopy (left) and fluorescence microscopy (right); Figure B shows the qPCR verification of DLL4 mRNA expression in OP9-GFP cells and OP9-DLL4 cells. b P < 0.01 vs OP9-GFP cells; Panel C shows FACS sorting of cells overexpressing OP9-DLL4 signals;

[0032] Figure 2 Schematic diagram of the construction and growth of thymic organoids in Example 3, wherein Figure A shows the flow cytometry sorting of nude mouse LSK cells; Figure B shows the appearance of thymic organoids obtained by culture and differentiation of LSK cells under bright field microscopy (left) and fluorescence microscopy (right); Figures C to O show the immunofluorescence histochemical staining of thymic organoids for CD3 + T cells, CD4 + T cells and CD8 + T cells;

[0033] Figure 3 The thymus organoid culture system is used to induce the programmed differentiation of nude mouse bone marrow-derived hematopoietic stem cells into T lymphocytes,

[0034] A is the change in the proportion of DN cells, aP<0.05, bP<0.01 vs 1 week of the same type of cells;

[0035] B shows the changes in the ratio of CD3 hi TCRβhi and CD3 low TCRβlow in CD45+ cells;

[0036] C shows the changes in the ratio of CD4+ and CD8+ in CD3hi TCRβhi cells;

[0037] D is the change in the proportion of CD11c+ in CD45+ cells;

[0038] Figure 4 This is a diagram of the in vitro expansion of single positive cells during the programmed differentiation of nude mouse bone marrow-derived hematopoietic stem cells into T lymphocytes induced by the thymic organoid culture system in Example 3;

[0039] Figure 5 for Figure 4 Schematic diagram of CD4 SP T and CD8 SP T cell expansion after 7 days of culture;

[0040] Figure 6 CD4 + T cells and CD8 + Schematic diagram of ELISA analysis results after T cells were expanded and activated in vitro. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] For ease of understanding, the abbreviations or names mentioned below are first explained:

[0043] Glutamine Supplement (100X) is Gluta Max: a standardized cell culture medium formula containing L-glutamine in a stable dipeptide form;

[0044] DMEM-F12 medium: DMEM / F-12 medium without L-glutamine or HEPES. It is commonly commercially available and consists of a modified mixture of DMEM and Ham's F-12 medium in a 1:1 ratio. This modified DMEM / F-12 medium boasts a richer nutritional profile, including a wider variety of trace elements. It is widely used for culturing a variety of mammalian cells and is suitable for culturing mammalian cells in low serum concentrations and clonal density. DMEM / F-12 is also used as a basal medium for developing serum-free culture media. This product contains amino acids, vitamins, and inorganic salts necessary for various cell culture systems, but does not contain proteins or growth factors. Depending on the cell type, it requires the addition of 5-10% serum or serum-free supplements. It is sterilized by filtration through a 0.1μm filter, has a pH of 7.0-7.4, and contains 17.5mM D-glucose, 0.5mM sodium pyruvate, a phenol red indicator, and no L-glutamine or HEPES buffer.

[0045] L-Alanyl-L-glutamine is used in cell culture applications as a stable alternative to L-glutamine, namely the glutamine supplement GlutaMAX TM , is a commercially available glutamine supplement.

[0046] OP9 cells containing mouse DLL4 and EGFP lentiviral plasmids (hereinafter referred to as OP9-DLL4-MOUSE stromal cells) were purchased from Saiku Biotechnology Co., Ltd.

[0047]

[0048] Example 1

[0049] This embodiment provides a culture medium combination for inducing nude mouse hematopoietic stem cells to differentiate into T lymphocytes in vitro, specifically:

[0050] A culture medium combination for inducing nude mouse hematopoietic stem cells to differentiate into T lymphocytes in vitro, comprising two culture media, wherein:

[0051] Stromal cell culture medium: αMEM medium containing 10 wt% FBS and 1 wt% penicillin-streptomycin solution, used for culturing stromal cells;

[0052] T lymphocyte differentiation medium: DMEM-F12 medium supplemented with 2wt% B27, 30μmol / L ascorbyl phosphate magnesium sesquimagnesium salt hydrate, 1wt% penicillin-streptomycin solution (100X), 1wt% glutamine supplement (100X), 5ng / mL rmFLT3-L, 5ng / mL rmIL-7, 10ng / mL rmSCF and 0.05mM β-mercaptoethanol for T lymphocyte differentiation.

[0053] Both culture media are sterile reagents and can be stored at 4°C for 7 days and at -20°C for 90 days after preparation.

[0054] OP9-DLL4 stromal cells were cultured as follows:

[0055] The received OP9-DLL4 stromal cells were karyotyped and tested for mycoplasma according to the GLP laboratory's standard operating procedures for cell characterization. Culture was initiated after passing the test. After the cells were stabilized in an incubator for 4 hours and adhered to the cell wall under a microscope, the culture medium in the culture flask was discarded, freshly prepared stromal cell culture medium was added, and the cells were cultured in an incubator. When the cells reached 80% confluence, the cells were passaged. The culture medium in the culture flask was discarded, and 3 ml of 1× PBS was added. The culture flask was gently shaken to allow the PBS to reach the cell surface, and the PBS was discarded. 3 ml of 0.25% trypsin solution was added, and the culture flask was gently shaken to ensure that the trypsin fully contacted the bottom of the culture flask. The trypsin was discarded, and the culture flask was placed in an incubator for digestion for 3 minutes. After digestion, the culture flask was removed and 5 ml of stromal cell culture medium was added to terminate the digestion. The cells at the bottom of the culture flask were pipetted off with a Pasteur pipette and collected in a 15 ml centrifuge tube. Centrifuge the tube at 300g for 5 minutes at 4°C, discard the supernatant, resuspend the cells in stromal cell culture medium, and plate them in a T75 culture flask for further culture. Observe cell growth regularly. When the cells have grown to 80% of the plating capacity, harvest the cells, resuspend them in cell freezing buffer, and store them in a -80°C freezer.

[0056] Example 2

[0057] and performing monolayer culture in the first culture medium to obtain a monolayer of cells;

[0058] This embodiment provides the above-mentioned culture medium combination for:

[0059] Step S1: Stromal cell construction and culture:

[0060] OP9 cells were transduced with enhanced green fluorescent protein (eGFP) using a lentiviral vector encoding the mouse DLL4 gene to obtain OP9-mDLL4 stromal cells ( Figure 1A), and performing monolayer culture in a stromal cell culture medium to obtain a monolayer of cells, specifically:

[0061] The top 5% of OP9-mDLL4 cells expressing GFP were sorted by flow cytometry (FACS). Figure 1 C), after passage in stromal cell culture medium and several weeks of culture, stable GFP expression was detected by flow cytometry, and mDLL4 gene expression was detected by qRT-PCR and DNA sequencing ( Figure 1 B) Only continue if there is no mycoplasma infection;

[0062] OP9-mDLL4 stromal cells were cultured and expanded in standard T75 or T150 tissue culture flasks containing stromal cell culture medium. When the cells reached 70-90% confluence, they were trypsinized twice a week for 5 minutes at 37°C. Splitting cells were routinely passaged at a cell:medium ratio of 1:10. Cells were cultured in a standard cell culture incubator at a humidified temperature of 37°C and 5% CO2.

[0063] OP9-mDLL4 stromal cells are expanded to the desired number by seeding them in multiple T75 or T150 culture flasks. Preferably, OP9-mDLL4 stromal cells are stored in the culture flasks for at least 2 days before co-culture to ensure that the trypsin treatment interval is >24 hours; preferably, the confluence of OP9-mDLL4 stromal cells must reach 70-90% on the day of co-culture.

[0064] Harvest OP9-mDLL4 stromal cells using trypsin (gently pipette cells to obtain a single-cell suspension. If visible clumps remain, filter through a 70 μm filter. Resuspend cells in T lymphocyte differentiation medium and count. Store the single-cell suspension on ice until needed for co-culture).

[0065] Furthermore, in step S1, preparing the spare LSK cells includes two steps:

[0066] (1) Obtaining bone marrow-derived hematopoietic stem cells from the femur and tibia of nude mice, the steps are as follows:

[0067] ① After disinfection, fix the mouse, separate the femur and tibia of the hind limb, and separate the bone marrow into a flow cytometry buffer (1*PBS containing 2mM EDTA and 0.5wt% BSA) to obtain a cell tissue suspension, specifically:

[0068] After irradiating the clean bench with ultraviolet light for 30 minutes, surgical instruments disinfected with 75% alcohol and a foam board padded with aluminum foil were placed in the clean bench and an alcohol lamp was lit. Four- to eight-week-old healthy nude mice were anesthetized with 5% pentobarbital and sacrificed by cervical dislocation. After soaking in 75% alcohol for 5 minutes, they were immediately removed and wiped clean with sterile gauze. Reducing the interval between death and dissection can help improve bone marrow cell survival. A 5mL syringe needle was used to penetrate the mouse's paw pad and the mouse was placed supinely on a foam board padded with aluminum foil. First, a small Y-shaped incision was made above the urethral opening. Curved-tip scissors were inserted into the incision and gently opened and closed to separate the skin and subcutaneous tissue. This prevented the testicles and other reproductive organs from being pulled out when dissecting the lower body of the male mouse. Using forceps, the skin at the incision was grasped. Straight-tip scissors were used to cut upward along the ventral midline and then downward along the anterior aspect of the hind limb, completely separating the hind limb from the skin except for the sole of the foot. Cut the quadriceps muscle at the proximal end of the femur to expose the anterior femur; use scissors to press against the posterior femur and cut the hamstring muscle at the knee joint to expose the hip joint. Fold the thigh toward the hip joint and directly cut the femoral head and acetabulum to obtain the hind limb.

[0069] Hyperextend the ankle joint. Use ophthalmic scissors to first cut the anterior tibial tendon and Achilles tendon at the ankle joint, then cut the distal tibia in the gap between the ankle joint and the ankle joint. Use forceps to secure the tibial head. Use another pair of forceps to grasp the tendon and flip the muscle over the knee joint. Hyperextend the knee joint and use forceps to grasp the femur and twist it against the direction of joint movement. This will dislocate the tibia.

[0070] Use forceps to clamp the femur with the front side facing outward and the proximal end (femoral head end) facing downward and folded forward, so that the femoral head can be completely freed; then pull the quadriceps and hamstring muscles. If they are difficult to free by pulling, cut them directly without damaging the bone marrow cavity.

[0071] Immediately transfer the sample to a culture dish containing 10 mL of pre-chilled PBS. Grasp the bone with forceps and gently shake it to remove any residual surface tissue. After removing the condyle and epiphysis, insert a 1 mL syringe needle into the medullary cavity with an appropriate amount of flow cytometry buffer. Gently flush out the bone marrow until the cavity turns white or light pink. The bones of mice under six weeks old are more fragile, so the needle can be inserted directly without removing the epiphysis.

[0072] ② Tissue digestion and dissociation to obtain hematopoietic stem cell suspension, specifically:

[0073] Filter the cell tissue suspension through a 70μm mesh into a 50mL centrifuge tube and centrifuge at 300g and 4°C for 5 minutes. Remove the supernatant and resuspend the bone marrow tissue in 5mL of red blood cell lysis buffer. Let it stand at room temperature for 5 minutes. Add 30mL of pre-chilled buffer (4°C flow buffer) to terminate lysis. Centrifuge at 400g for 5 minutes at 4°C and discard the supernatant. Resuspend the cells in 5mL of pre-chilled buffer, filter through a 40μm mesh into a 50mL centrifuge tube, and count the cells to obtain a hematopoietic stem cell suspension.

[0074] (2) Isolation of LSK cells from bone marrow-derived hematopoietic stem cells:

[0075] ① Use magnetic separation technology (MACS) to separate Lin- cell suspension, specifically:

[0076] The hematopoietic stem cell suspension was centrifuged at 4°C and 300g for 10 min, the supernatant was removed, and the suspension was centrifuged every 10 min. 7 Resuspend the cells in 40 μL of flow cytometry buffer and 7 The cells were stained with 10 μL of Biotin-Antibody Cocktail, mixed thoroughly, and incubated at 4°C in the dark for 10 min;

[0077] Every 10 7 30 μL of flow cytometry buffer and 20 μL of Anti-Biotin MicroBeads were added to the cells, mixed thoroughly, and incubated at 4°C in the dark for 15 min;

[0078] Every 10 7 The cells were washed by adding 1 mL of flow cytometry buffer, centrifuged at 300 g for 10 min at 4°C, and the supernatant was removed;

[0079] Resuspend to contain 10 7 Cells, the volume of flow buffer doubles with the number of cells, and then the cell suspension to be magnetized is obtained by sieving with a 40 μm mesh;

[0080] Rinse the magnetic column three times with 500 μL of flow cytometry buffer, waiting for the residual liquid to drain completely before proceeding to the next step. After rinsing the magnetic column, add 500 μL of the cell suspension to be magnetized to the magnetic column. The Lin-cell suspension flows out from the bottom of the magnetic column. When the residual liquid is almost drained, add more cell suspension to be magnetized until the process is complete.

[0081] Furthermore, in order to improve the cell purity, the obtained Lin-cell suspension can be added to the magnetic column again for purification.

[0082] ② Detect cells in the Lin- cell suspension and sort LSK cells, specifically:

[0083] Collect the Lin-cell suspension obtained by MACS and transfer 18 μl of the suspension to a 500 μl centrifuge tube. Add 2 μl of 0.4 wt% trypan blue staining solution, mix thoroughly, and incubate for 3 minutes. (To prevent adhesion of the smaller volume of trypan blue staining solution to the wall, add a small volume of reagent to the larger volume.) Count 10 μl of the mixture. Dead cells will be observed to be enlarged, appearing blue and dull; live cells maintain normal morphology and are unstained. The mixture is centrifuged at 300 g for 10 minutes at 4°C. The supernatant is removed and the tube is resuspended in 100 μl of flow cytometry buffer. 3 μl of TruStain FcX is added and mixed thoroughly by pipetting. Block the tube in the dark for 5 minutes. Then, 2 μl of Sca-1-FITC antibody and 5 μl of c-kit-APC antibody are added. Incubate on ice in the dark for 20 minutes, resuspend the tube in 1 mL of pre-chilled buffer, and centrifuge at 300 g for 5 minutes at 4°C to remove the residual liquid. After repeated washing, add 500 μl flow cytometry buffer to resuspend, filter with a 40 μm mesh, and then sort on the machine. The sorting temperature is controlled at 4°C and the time is controlled within 40 min.

[0084] The sorted cells were added to a centrifuge tube containing 1 mL of lymphocyte differentiation medium, and then centrifuged at 300 g for 5 min at 4 ° C and the supernatant was removed. The cell pellet was the target cell LSK cell ( Figure 2 A).

[0085] Furthermore, the specific process of gas-liquid interface cultivation in step S2 includes two steps:

[0086] (1) Construction of T lymphocyte differentiation system

[0087] In this example, a 6-well culture plate was prepared and 1 mL of T lymphocyte differentiation medium was added to each well. Using small blunt forceps, a single transwell chamber was inserted into each well, ensuring that the bottom membrane of the transwell chamber was in contact with the culture medium. The chamber was then placed in a biosafety cabinet until ready for use.

[0088] LSK cells and stromal cells (the total number of cells in each organoid system is at least 1*10 5) After mixing in a 1:1 ratio, add 5 μl of culture medium for each system to obtain a viscous cell slurry, and drop the cell slurry onto the top surface of the transwell (Note: ① In actual operation, the cell slurry is very thick. To avoid losing cells stuck to the sides of the pipette tip, use a pipette to pipette up and down the culture medium to wet the tip before transferring the cell slurry. ② Be careful not to overflow the culture medium onto the top surface of the transwell. ③ Because the cell slurry is very viscous, adjusting its volume may be challenging. Cells required for multiple differentiation systems can be combined into a single tube and the supernatant carefully removed using a vacuum flask and a glass pipette. For example, after vortexing the test tube at medium-low speed to resuspend the cell clumps, add 10 μl of T lymphocyte differentiation medium and gently pipette to mix the cell clumps. This makes it easier to measure the exact volume with a pipette. Then, add enough T lymphocyte differentiation medium to obtain a final volume of 60 μl (equivalent to 12 systems). Measure the required volume when using).

[0089] Use tweezers to remove the transwell chamber from the culture medium and place it on the edge of the well to allow it to drain slightly before inoculating the cell cluster. A single transwell corresponds to one system. Pipette 5 μl of cell slurry and gently release it into the middle of the membrane at the bottom of the transwell chamber to release the cell slurry. The cell slurry will form a small sphere on the surface of the transwell chamber.

[0090] In this example, 12 systems were inoculated to increase T cell production. A maximum of 12 systems can be placed in a six-well transwell plate. If a 12-well transwell plate is used, a maximum of one system can be placed in each well, and a maximum of 12 systems can be placed in a plate. (It should be noted that a maximum of two systems can be placed in a single transwell chamber, approximately 1 cm apart and as close to the center of the culture tank as possible). Use tweezers to gently return the transwell chamber to the well containing culture medium. The culture medium should not directly contact the upper surface of the cell organoid.

[0091] (2) Replace T lymphocyte differentiation medium every 2 days

[0092] Tilt the culture plate so that the culture medium flows to one side of the well, but be careful not to let the culture medium flow onto the surface of the transwell chamber;

[0093] Use a pipette to aspirate the culture medium from around the transwell chamber, leaving 100-200 μl of culture medium under the transwell chamber to prevent it from drying out;

[0094] Using a 1 mL pipette tip, add 1 mL of fresh T lymphocyte differentiation medium preheated to 37°C between the transwell chamber and the well, allowing the medium to spread under the transwell chamber.

[0095] Furthermore, in step S2, during the gas-liquid interface culture process, samples can be taken regularly (e.g., weekly) and ground into a cell suspension through a 40 μm cell sieve, and then subjected to flow cytometry analysis to obtain T lymphocyte differentiation status, specifically:

[0096] Aspirate the medium from the cell suspension and add a Figure 2 B) Add 100 μl of preheated trypsin to the upper surface, incubate in a 37°C incubator for 5 min, then remove and add 1 mL of stromal cell culture medium to terminate digestion;

[0097] Then, the cells were centrifuged at 4°C and 300 g for 5 min and the supernatant was removed to obtain a cell pellet, which was then resuspended in 100 μl of PBS to obtain a pelleted cell suspension;

[0098] The precipitated cell suspension was divided equally into six centrifuge tubes, and 5 μl of TruStain FcX was added to each tube, mixed by pipetting, and blocked for 5 minutes;

[0099] After blocking, CD8-PE, TCRβ-PerCP-Cy5.5, CD25-PE-Cy7, CD44-BV510, CD4-BV570, CD117-BV605, CD3-APC, CD11c-AF700, and CD45-APC-Cy7 antibodies were added in sequence and incubated in the dark at 4°C for 20 minutes. Then, flow cytometry buffer was added to terminate the incubation and wash the unbound antibodies. After centrifugation at 300g for 5 minutes, the supernatant was discarded. After washing again, the cells were resuspended to the volume of the machine with a buffer containing DAPI (obtained by diluting with flow cytometry buffer according to the DAPI instructions) and subjected to flow cytometry detection to obtain the differentiation of T lymphocytes ( Figure 2 C to O).

[0100] like Figure 3As shown, during the programmed differentiation of LSK cells into T lymphocytes, the continuous differentiation of T cells can be divided into the CD4-CD8- double negative (DN) stage, the CD4+CD8+ double positive (DP) stage, and the CD4+CD8- or CD4-CD8+ single positive (SP) stage based on the expression of CD44 and CD25 on the cell surface. The DN stage can be divided into the DN1 stage (CD44+CD25-), DN2 stage (CD44+CD25+), DN3 stage (CD44-CD25+), and DN4 stage (CD44-CD25-). When T cells enter the DN1 stage, the Notch 1 receptor plays a crucial role; otherwise, the cells will differentiate into cells other than T cells, such as B cells and NK cells [2-4]. Blockade of the DN2 stage can lead to the development of the lymphocyte pathway towards non-T cells (such as myeloid cells and NK cells). A key event that occurs during the DN3 to DN4 stage is β-selection, which is the process of TCR rearrangement. The initiation of β-selection requires the successful rearrangement of β chains and the presence of intracellular TCRβ, while CD3 expression is essential for the progression from DN3 to DN4. After TCRβ rearrangement is complete and expressed on the cell surface, TCRα chains rearrange in DP cells, which then undergo positive and negative selection, ultimately differentiating into naive T cells

[10] .

[0101] The differentiation of T cells in thymic organoids at the corresponding time points was obtained by multi-color staining of thymic organoids with flow cytometry antibodies. The flow cytometry results showed that a large number of DN1 cells were detected in the first week, indicating that Notch 1 receptors had been activated in large quantities and HSPC-directed T cells were differentiating. Since the second week of culture, the proportion of T cells in the DN1 stage has continued to decrease from about 70wt%, especially in the fourth and fifth weeks, when the ratio of DN1 to DN cells was about 20wt%; at the same time, the DN4 stage cells continued to increase from about 18wt% to between 50wt% and 60wt%, indicating that T cells have completed the differentiation from DN1 to DN4 ( Figure 3 To demonstrate that T cells have completed β selection from DN3 to DN4, we analyzed the expression of CD3 and TCRβ in CD45+ T cells. We found that after three weeks of culture, the ratio of CD3hiTCRβhi cells increased and the ratio of CD3lowTCRβlow cells decreased, indicating that T cells in thymic organoids have completed β selection ( Figure 3B). Analysis of CD4 and CD8 expression in CD3hiTCRβhi cells revealed that the ratio of CD4+CD8+ cells gradually increased after the second week, reaching a peak at the fourth week, and the ratio of CD4+CD8+ cells also increased simultaneously, indicating that T cells in thymic organoids may have completed positive selection ( Figure 3 C). At the same time, a certain proportion of CD4+ or CD8+ SPT cells were observed in the CD3hiTCRβhi cell population, and immunofluorescence staining of thymic organoids showed CD4+ and CD8+ T cells surrounding OP9-DLL4 stromal cells, indicating that T cells in the thymic organoid system may have completed negative selection. Since negative selection requires the participation of dendritic cells, DCs cells in CD45+ cells were analyzed ( Figure 3 D) CD11c expression was observed throughout the culture system, confirming the potential for negative selection in the thymic organoid system. These results demonstrate that the OP9-DLL4 thymic organoid culture system is capable of directing the differentiation of nude mouse HSPCs into SP T cells.

[0102] Furthermore, CD4 and CD8 SP T cells were isolated from the thymic organoid system cultured for 5 weeks and incubated with CFSE flow cytometry antibodies and then cultured in expansion medium. Flow cytometry was performed on days 0, 1, 3, 5, and 7 of culture using flow cytometry antibodies CD25-PE / Cy7 and 7-AAD. Figure 4 ) and found that SP T cells expanded significantly while upregulating the activation marker CD25. The results of the test after 7 days of culture showed that CD4 SP T cells had expanded to the sixth generation of cells ( Figure 5 A), CD8 SP T cells have been expanded to the fifth generation of cells ( Figure 5 B) demonstrates that naive T cells differentiated in the organoid system can be expanded in large quantities in vitro.

[0103] Furthermore, CD4+ and CD8+ T cells were stimulated with PMA, and the supernatants were collected and analyzed by ELISA for IL-2 ( Figure 6 A), IFN-γ ( Figure 6 B), TNF-α ( Figure 6 The results showed that both CD4+ and CD8+ SP T cells could be expanded in large quantities in vitro and activated by PMA to secrete large amounts of IL-2, IFN-γ, and TNF-α, proving that the SP T cells differentiated in the thymic organoid system have immune response function.

[0104] Example 3

[0105] This embodiment provides a method for preparing a T lymphocyte population (or T lymphocyte derivative) with multiple functions.

[0106] The T lymphocytes obtained in Example 2 above are a single-positive T lymphocyte population. Continuing to culture can obtain a T lymphocyte population with multiple functions, such as:

[0107] (1) After receiving induced differentiation and negative selection, they differentiate into a population of T lymphocytes that have no immune rejection effect on specific antigens;

[0108] (2) Co-culture with tumor cells to culture and differentiate tumor-infiltrating lymphocyte populations for specific tumor treatment;

[0109] (3) Cultivate and differentiate T lymphocyte populations with immune functions for delivery to immunodeficient individuals to supplement immune cells.

[0110] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is limited by the accompanying embodiments and their equivalents.

Claims

1. A special culture medium for inducing the differentiation of hematopoietic stem cells into T lymphocytes in vitro targeting Foxn1 gene deficiency, characterized by: The invention is composed of the following components by weight percentage: 1.5-2.5wt% B27, 25-35μmol / L ascorbyl phosphate magnesium sesquimagnesium salt hydrate, 0.8-1.2wt% antibiotics, 0.8-1.2wt% L-alanyl-L-glutamine, 4-6ng / mLrmFLT3-L, 4-6ng / mL rmIL-7, 9-11ng / mL rmSCF, and 0.04-0.06mM β-mercaptoethanol are added to DMEM-F12 culture medium.

2. The specialized culture medium according to claim 1, wherein: The formula and proportions of the T lymphocyte differentiation medium are as follows: 2 wt% B27, 30 μmol / L ascorbyl phosphate magnesium sesquimagnesium salt hydrate, 1 wt% antibiotics, 1 wt% L-alanyl-L-glutamine, 5 ng / mL rmFLT3-L, 5 ng / mL rmIL-7, 10 ng / mL rmSCF, and 0.05 mM β-mercaptoethanol were added to DMEM-F12 medium.

3. The specialized culture medium according to claim 1, wherein: In addition to the T lymphocyte differentiation medium described above, the medium also includes a stromal cell culture medium used therewith; the stromal cell culture medium is an αMEM culture medium containing 9-12 wt% FBS.

4. A special kit for inducing the differentiation of hematopoietic stem cells into T lymphocytes in vitro for Foxn1 gene deficiency, the kit comprising reagents required for preparing the stromal cell culture medium and T lymphocyte differentiation medium according to claim 3.

5. A method for inducing hematopoietic stem cells to differentiate into T lymphocytes in vitro using the dedicated culture medium according to claim 3, comprising the following steps: S1. OP9 cells were transduced with enhanced green fluorescent protein using a lentiviral vector encoding the mouse DLL4 gene to obtain OP9-mDLL4 stromal cells, which were then cultured in a monolayer in stromal cell culture medium to obtain monolayer cells, and LSK cells were sorted from hematopoietic stem cells. S2. After mixing the monolayer cells and the LSK cells at a ratio of 1-5:1, the cells were spotted on a culture plate, and T lymphocyte differentiation medium was added for air-liquid interface culture; S3. Perform flow cytometry sorting on the cells cultured at the air-liquid interface to obtain a population of CD4 and CD8 single-positive T lymphocytes.

6. The method according to claim 5, characterized in that: The hematopoietic stem cells are derived from the bone marrow of the femur and tibia of nude mice. The bone marrow is digested and dissociated to obtain a hematopoietic stem cell suspension. Then, a magnetic separation technique is used to separate the Lin-cell suspension. After detecting the cells in the Lin-cell suspension and separating the Lin - Sca-1 + c-Kit + (LSK) cell population.

7. The method according to claim 5, characterized in that: During the gas-liquid interface culture process in S3, samples were taken regularly and ground through a 40 μm cell sieve into a cell suspension, which was then subjected to flow cytometry testing to obtain the differentiation of T lymphocytes.

8. The method according to claim 5, characterized in that: The cell suspension was ground through a 40 μm cell sieve, and the cell pellet was extracted and resuspended into a suspension. TruStain FcX was added and pipetted to mix. After blocking, CD8-PE, TCRβ-PerCP-Cy5.5, CD25-PE-Cy7, CD44-BV510, CD4-BV570, CD117-BV605, CD3-APC, CD11c-AF700, and CD45-APC-Cy7 antibodies were added. The cells were incubated in the dark at 4°C for 20 minutes, and then flow cytometry buffer was added to terminate the incubation and wash the unbound antibodies. After centrifugation and washing, the cells were resuspended with a buffer containing DAPI for flow cytometry detection.

9. Use of the single-positive T lymphocyte population obtained by culture using the method of claim 5.

10. The application according to claim 9, characterized in that: The application is to further culture the above-mentioned single-positive T lymphocyte population to obtain a T lymphocyte population with multiple functions, including any of the following characteristics: A. After receiving induced differentiation and negative selection, it differentiates into a population of T lymphocytes that has no immune rejection effect on specific antigens; B. Co-culture with tumor cells to cultivate and differentiate tumor-infiltrating lymphocyte populations for specific tumor treatment; C. Cultivate and differentiate T lymphocyte populations with immune functions, which are then delivered to immunodeficient individuals to supplement immune cells.

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