Bovine lung CD4 ATF7IP + Isolation and in vitro culture of sub-type cells

Abstract

The application belongs to the technical field of T cell separation, and discloses yak lung CD4 ATF7IP + A method for separating and culturing subtype cells in vitro. The method accurately sorts target subtype cells by double verification in situ of tissues, adopts a strategy of "immunomagnetic bead pre-sorting + multi-parameter flow sorting", places the sorted cells in a specific culture medium (RPMI-1640 complete culture medium containing recombinant cytokines IL-2 and IL-7) for in vitro culture, and identifies the cultured cells by immunofluorescence technology to confirm the co-expression characteristics of CD4 and ATF7IP of the cells. The application improves the purity and efficiency of sorting of CD4 ATF7IP + subtype cells in yak lung, and is beneficial to the development and application of the subtype cells.

Description

Technical Field

[0001] This invention belongs to the field of T-cell isolation technology and relates to CD4 in yak lungs. ATF7IP + Methods for isolating and culturing subtype cells in vitro. Background Technology

[0002] yak ( Bos grunniens As a unique livestock species native to the Qinghai-Tibet Plateau, the T cell's unique high-altitude adaptability and immune mechanisms have always been a research hotspot. In immunological research, T cells, especially CD4+, are... + T cell subsets play a central role in the body's fight against infection and immune regulation. Currently, research on bovine or yak immune cells mainly focuses on the conventional CD4+. + On the isolation and functional analysis of T cells, regulatory T cells (Tregs) or Th1 / Th2 cells.

[0003] In cell separation technology, traditional methods often rely on density gradient centrifugation combined with non-specific immunomagnetic bead sorting. For example, after separating mononuclear cells using lymphocyte separation fluid, positive sorting is performed using anti-CD4 magnetic beads. However, this general method can only obtain mixed CD4. + The T cell population cannot be further distinguished into subtypes with different functions. In addition, in terms of in vitro culture, existing bovine T cell culture techniques mostly use universal culture media (such as RPMI-1640) and universal T cell stimulating factors (such as ConA or PHA), but these conditions can often only maintain the short-term survival of cells and are difficult to support the long-term expansion of specific subpopulations.

[0004] The aforementioned technologies have played an important role in routine immune cell research, but in studies targeting specific cell subpopulations in the unique organ of yak lungs, existing technologies have revealed the following significant limitations: (1) Lack of subtype-specific isolation methods: The lung microenvironment of yak is complex, and CD4 is involved in its isolation. + T cells may have multiple unknown or under-studied subtypes. Current technology relies solely on the CD4 single marker for sorting, which cannot distinguish between CD4+ subtypes. + The heterogeneity within T cells leads to a "mixed" cell population, making it impossible to target a specific subtype (such as CD4, which is the focus of this invention). ATF7IP + We will conduct precise research on the subtypes.

[0005] (2) Difficulty in in vitro culture: Due to a lack of understanding of the growth factor requirements of this specific subtype of cells, directly applying conventional T cell culture methods often results in poor cell viability, weak proliferation capacity, or loss of phenotype. In particular, for some cells with specific transcription factors (such as ATF7IP) expression, general culture conditions are difficult to maintain the stable expression of their characteristic markers, thus affecting the results of subsequent functional experiments.

[0006] (3) Limited verification methods: Existing studies often only use flow cytometry for sorting, lacking confirmation of the existence of the subtype in situ (RNA and protein levels) in tissues, resulting in "blindness" in the sorting target and increasing the risk of experimental failure.

[0007] Currently, there is no established research on CD4 in yak lungs. ATF7IP + The lack of specific isolation methods for this subtype of cells makes it difficult to isolate them efficiently and with high purity, severely hindering in-depth research on this subtype. Due to the unique characteristics of this cell subtype, there are no suitable in vitro culture conditions, making subsequent functional studies and applications difficult after isolation. Therefore, there is an urgent need to establish CD4+ culture methods for yak lung cells. ATF7IP + Methods for isolating and culturing subtype cells in vitro. Summary of the Invention

[0008] Based on the current state of technology, the purpose of this invention is to provide a highly efficient, high-purity, and specific method for isolating and culturing CD4 from yak lungs in vitro. ATF7IP + The subtype cell approach is beneficial for its development and application.

[0009] To achieve the objective of this invention, the technical solution is as follows: 1. In situ dual verification: Before isolation, CD4 in yak lungs was verified at both the RNA and protein levels using fluorescence in situ hybridization and immunohistochemistry. ATF7IP + The presence and location of subtype cells ensure the authenticity of the target cells.

[0010] 2. Hierarchical Flow Cytometry Sorting: This method employs a strategy of "immunomagnetic bead pre-sorting + multi-parameter flow cytometry sorting." First, CD4 is enriched using immunomagnetic beads. + T cells are then analyzed by flow cytometry based on a specific combination of surface markers (live / dead dyes / CD45). + / CD3 + / CD4 + / CD127 + It can accurately sort out target subtype cells, greatly improving the purity and efficiency of sorting.

[0011] 3. In vitro culture and identification: The sorted cells were placed in a specific culture medium (RPMI-1640 complete medium containing recombinant cytokines IL-2 and IL-7) for in vitro culture, and the cultured cells were identified by immunofluorescence technology to confirm their co-expression characteristics of CD4 and ATF7IP.

[0012] Beneficial effects of this invention: 1. A hierarchical strategy of "immunomagnetic bead pre-sorting + multi-parameter flow cytometry sorting" is adopted, combined with a specific surface marker combination (CD45). + / CD3 + / CD4 + / CD127 + This significantly improves the sorting purity (up to 95% or more) and recovery rate of target cells, and effectively removes interference from other cell types.

[0013] 2. Optimize in vitro culture conditions; a specific combination of cytokines can support CD4. ATF7IP + The long-term survival and proliferation of the subtype cells in vitro, and the ability to maintain the stable expression of its characteristic marker (ATF7IP), ensured the reliability of subsequent experimental data.

[0014] 3. CD4 cells that can be stably cultured in vitro were obtained. ATF7IP + Cell lines or cell populations provide valuable in vitro cell models for studying the adaptive immune mechanisms of yaks on the plateau and the pathogenic mechanisms of lung-related infectious diseases, reducing reliance on large-scale live animal experiments and lowering research and development costs.

[0015] 4. A rigorous technical loop was constructed through dual verification at the tissue level using fluorescence in situ hybridization and immunohistochemistry, as well as reverse identification using immunofluorescence after sorting, ensuring the accuracy and authenticity of the obtained cell models.

[0016] The method described in this invention isolated and cultured high-purity CD4 from yak lungs. ATF7IP + The subtype cells provide direct experimental material for elucidating the role of the ATF7IP gene in the differentiation and functional regulation of T cell subsets, which will help advance research on ruminant immunology and high-altitude biology. Attached Figure Description

[0017] Figure 1 CD4 of yak lungs ATF7IP + Distribution of subtypes.

[0018] Figure 2 For yak CD4 ATF7IP +The marker gene for the subtype is ATF7IP.

[0019] Figure 3 For yak CD4 ATF7IP + In situ hybridization identification of subtypes.

[0020] Figure 4 For yak CD4 ATF7IP + Immunohistochemical identification of subtypes.

[0021] Figure 5 For yak CD4 ATF7IP + Subtype sorting by flow cytometry, where A is the experimental group and B is the negative control.

[0022] Figure 6 For yak CD4 ATF7IP + Immunofluorescence identification. Detailed Implementation

[0023] To better illustrate the present invention, the following embodiments are provided: Unless otherwise specified, all percentages mentioned below are volume percentages. Example 1

[0024] 1. Initial discovery of the yak CD4 ATF7IP+ subtype In single-cell transcriptome data from yak lungs, we analyzed CD4... + Dimensionality reduction clustering analysis was performed on T cells, resulting in 14 CD4 clusters. + T cell clusters ( Figure 1 Based on this, we annotated these cell clusters in detail and ultimately identified 11 CD4 species. + T cell subtypes, the most numerous of which is the CD4 ATF7IP+ subtype (6.57%, marker gene ATF7IP, ...). Figure 2 ).

[0025] 2. RNA scope fluorescence in situ hybridization verification of the CD4 ATF7IP+ subtype in yak lungs. (1) Paraffin embedding and sectioning of yak lungs Immediately after the yak is slaughtered, a sample is collected from a point 1 cm away from the apex of the lung. 3Lung tissue was extracted, surface blood was removed, and the tissue was rapidly fixed in 10 times its volume of 4% paraformaldehyde for 24 hours. After fixation, the tissue was removed and the target area was trimmed with a scalpel in a fume hood to ensure the tissue was flat. The trimmed tissue and label were placed in a dehydration box, which was then placed in a fully automated tissue dehydrator for gradient alcohol dehydration. The specific procedure was as follows: 75% ethanol soaking for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 2 hours, 95% ethanol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, anhydrous ethanol-xylene for 10 minutes, xylene I for 10 minutes, xylene II for 10 minutes, wax I for 1 hour, wax II for 1 hour, and wax III for 1 hour.

[0026] After dehydration, the tissue was placed in the HistoCore Arcadia fully automated tissue embedding machine for embedding. First, molten paraffin was injected into the embedding frame. Before the paraffin completely solidified, the tissue was removed from the dehydration chamber, placed in the embedding frame, and labeled accordingly. Then, the embedding frame was placed on a -6 ℃ cooling stage to cool. After the paraffin solidified, the paraffin block was removed and its shape trimmed. The trimmed paraffin block was then placed on a manual rotary microtome for sectioning, with a section thickness set to 6 μm. The sections were floated in a 40 ℃ water bath, flattened, retrieved with a glass slide, and placed in a 60 ℃ oven for 30 min. Finally, depending on the intended use, the sections were stored at -80 ℃ for later use.

[0027] (2) RNA scope fluorescence in situ hybridization To perform in situ detection of the CD4 ATF7IP+ subtype in lung tissue, we selected 6 μm thick paraffin sections of lung tissue as experimental material and performed in situ hybridization to detect the corresponding target (ATF7IP RNA) according to the instructions of the ViewRNA™ Tissue Fluorescence Assay kit.

[0028] (a) Preparation: Set the CO2 incubator to 40 ℃ and calibrate it with a thermometer; place the humidifier in the incubator in advance and preheat it at 40 ℃; turn on the water bath in advance and set it to two temperatures of 40 ℃ and 95 ℃ respectively for later use.

[0029] (b) Baking the slides: Place the slides on a slide holder and put them in an oven at 80 °C for 30 min.

[0030] (c) Dewaxing: Immerse the slides in xylene for 10 min, then immerse them in anhydrous ethanol-xylene (1:1) mixed solution for 10 min, shaking the slides up and down continuously during this time.

[0031] (d) Dehydration: The sections were soaked in anhydrous ethanol for 10 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 70% ethanol for 5 min, and finally soaked in PBS for 5 min × 3 times.

[0032] (e) Heat treatment: Prepare 1× Pretreatment Solution working solution in advance and preheat it to 95 ℃, using a thermometer to ensure temperature accuracy. Then, place the slide in a staining dish containing 1× Pretreatment Solution working solution and incubate in a water bath at 90-95 ℃ for 10 min. Rinse the slide with ultrapure water in the staining dish, continuously shaking the slide up and down, rinsing twice for 1 min each time.

[0033] (f) Draw hydrophobic circles: After gently wiping the area around the tissue with absorbent paper, draw circles about 2–3 mm away from the edge of the tissue block with an immunohistochemical pen, and then let it air dry for 3 min.

[0034] (g) Protease digestion: Preheat PBS to 40°C. Prepare a 1× protease working solution using the preheated PBS and vortex to mix. Add the prepared protease working solution dropwise into the hydrophobic zone of the slide, ensuring complete coverage of the tissue. Place the slide in a humidified chamber and incubate at 40°C in a CO2 incubator for 20 min. Observe under a bright-field microscope. Stop digestion when the cells in the tissue gradually become rounded. Subsequently, rinse the back of the slide with PBS to remove residual protease working solution, and place the slide in a staining dish. Wash with PBS for 1 min × 4 times, continuously shaking the slide up and down during the process.

[0035] (h) Fixation and crosslinking: Place the above slide in a staining dish containing 4% paraformaldehyde and fix at room temperature for 5 min. Then, rinse the back of the slide with PBS to remove formaldehyde residue. Next, place the slide in a staining dish and wash with PBS for 1 min × 3 times, shaking the slide up and down continuously during the process.

[0036] (i) Hybridization: Prepare Wash Buffer in advance and preheat Probe Set Diluent QT to 40 °C. Prepare Probe Set working solution at a probe to Probe Set Diluent QT volume ratio of 1:40. After thorough vortex mixing, drop the Probe Set working solution into the hydrophobic zone of the slide, ensuring complete tissue coverage. Then, place the slide in a humidified chamber and incubate at 40 °C in a CO2 incubator for 2 h. After incubation, wash the slide in a staining dish with Wash Buffer 2 min × 3 times, continuously shaking the slide up and down during the process.

[0037] The cell subtype target combinations detected in this experiment are detailed in Table 1. Positive controls (using GAPDH probes) and negative controls (using Probe Set Diluent QT) were also set up to verify the reliability of the experimental results.

[0038] Table 1. FISH detection of CD4 ATF7IP+ subtype-specific targets

[0039] (j) Signal Amplification: The Amplifier Diluent QF was brought to 40 °C. Pre-Amplifier working solution and Amplifier working solution were prepared separately and vortexed thoroughly. The slides were washed with fresh Wash Buffer for 2 min × 3 times, continuously agitating the slides during this process. The Pre-Amplifier working solution was added dropwise into the hydrophobic zone of the slide to ensure tissue coverage. The slides were then placed in a humidified chamber and incubated at 40 °C with CO2 for 30 min. Subsequently, the slides were washed again with Wash Buffer in a staining dish for 2 min × 3 times, continuously agitating the slides during this process. Next, the Amplifier working solution was added dropwise into the hydrophobic zone of the slide to cover the tissue. The slides were then placed in a humidified chamber again and incubated at 40 °C with CO2 for 30 min. Finally, the slides were washed again with Wash Buffer in a staining dish for 2 min × 3 times, continuously agitating the slides during this process.

[0040] (k) Adding fluorescent groups: Preheat Label Probe Diluent QF to 40 °C, prepare Label Probe working solutions (Type 1, Type 4, and Type 6), vortex to mix, wrap in aluminum foil, and store in the dark. Add the corresponding Label Probe working solution to the hydrophobic zone of the slide, ensuring complete coverage of the tissue. Place the slide in a humidified chamber and incubate at 40 °C in a CO2 incubator for 30 min. Next, wash the slide in a staining dish with Wash Buffer, washing 2 min × 3 times, then washing again for 10 min × 1 time, continuously shaking the slide up and down during the process. The total washing time should not exceed 30 min.

[0041] (l) Nucleus staining: Prepare the DAPI working solution, vortex to mix, wrap with aluminum foil, and store in the dark. Add the DAPI working solution to the hydrophobic zone of the slide, ensuring complete coverage of the tissue, and incubate in a dark room for 10 min. Then, rinse the back of the slide with PBS to remove excess DAPI, and wash the slide with PBS in a staining dish twice for 2 min, shaking the slide up and down continuously during the process.

[0042] (m) Mounting observation: Use a cotton swab dipped in a small amount of xylene to gently wipe away the hydrophobic ring on the slide, ensuring the slide surface is clean and free of residue. Then, apply an appropriate amount of anti-fluorescence quenching mounting medium to the tissue surface to mount the slide, cover it with a coverslip, and gently press to ensure a seal. After the mounting medium has completely cured, use a laser scanning confocal fluorescence microscope (ZEISS, LSM900) to acquire fluorescence images of the sample. Figure 3 ).

[0043] 3. Yak lung CD4 ATF7IP + Immunohistochemical verification of subtypes (1) Baking: The prepared 6 μm lung tissue paraffin sections were placed in a 60 ℃ oven for 1 h to promote tissue adhesion and remove excess paraffin.

[0044] (2) Dewaxing: The sections were placed in xylene twice for 20 min each time to remove the paraffin components from the tissue.

[0045] (3) Hydration: Immerse the sections in 100%, 95%, 70% and 50% ethanol solutions in sequence for 5 min each, gradually reducing the alcohol concentration to fully hydrate the tissue.

[0046] (4) Washing with ultrapure water: Soak the slices in ultrapure water for 10 min to further remove residual alcohol.

[0047] (5) Antigen heat retrieval (high-pressure retrieval): Place the slide in 1× citrate-sodium citrate buffer, heat the pressure cooker until at least a certain amount of steam is discharged from the exhaust pipe, and then close the pressure limiting valve. Start timing for 5 minutes when the pressure limiting valve rises, then turn off the power and allow the sample to cool naturally at room temperature.

[0048] (6) PBS washing: Immerse the slices in PBS for 10 min to remove buffer residue.

[0049] (7) Inactivation of endogenous peroxidase: Immerse the slides in endogenous peroxidase blocking solution and incubate at room temperature for 10 min, then rinse once with PBS to reduce non-specific staining.

[0050] (8) Permeation: Place the slices in permeation solution and incubate at room temperature for 10 min.

[0051] (9) PBS washing: Wash with PBS 3 times, 5 min each time.

[0052] (10) Blocking: Use blocking solution and incubate at room temperature for 1 h.

[0053] (11) Primary antibody incubation: Prepare the primary antibody in antibody dilution solution according to the appropriate dilution ratio and incubate overnight at 4 °C. At the same time, set up a negative control (using antibody dilution solution instead of primary antibody) to verify the reliability of the experimental results.

[0054] (12) PBS washing: Wash with PBS 4 times, 5 min each time.

[0055] (13) Secondary antibody incubation: Prepare the secondary antibody in the antibody dilution solution according to the appropriate dilution ratio and incubate at room temperature for 1 h.

[0056] (14) PBS washing: Wash with PBS 4 times, 5 min each time.

[0057] (15) DAB staining: Prepare DAB working solution and incubate in the dark for 20 min. During this period, observe the staining with a microscope to control the staining intensity.

[0058] (16) Hematoxylin counterstaining: After color development, rinse with ultrapure water, then place in hematoxylin solution for counterstaining for 5 min, followed by rinsing with ultrapure water to remove excess dye.

[0059] (17) Differentiation: Place the slices in 1% hydrochloric acid ethanol differentiation solution for 5 s to hydrate and control the counterstaining effect.

[0060] (18) Wash with ultrapure water: Rinse with ultrapure water for 5 min to remove residual differentiation solution.

[0061] (19) Dehydration: Place the slices in 95% ethanol and 100% ethanol in sequence for 5 min each.

[0062] (20) Mounting: Mount the slide using mounting adhesive and acquire images using a fully automated digital slide scanner (KFBIO). Figure 4 ).

[0063] 4. Yak CD4 ATF7IP + Subtype sorting and cultivation (1) Tissue dissociation: Immediately after slaughtering the yak, lung lobes were cut off, washed with HBSS to remove blood, and dried as much as possible. The lung tissue was completely immersed in the working solution of tissue preservation solution and transported back to the laboratory at 4 ℃ using ice packs. The lung tissue was washed three times with HBSS containing a mixture of 5% penicillin-streptomycin-amphoteric B to remove the bronchi and surface tissue. The remaining tissue was cut into appropriate sizes and transferred into gentleMACS™ C Tubes dissociation tubes. The tubes were placed in a tissue dissociator and the lung tissue pre-dissociation program was run (m_lung_01 program was used in this embodiment). The enzyme mixture shown in Table 2 was added for digestion, and the tubes were incubated in a 37 ℃ water bath for 40 min, with the centrifuge tubes shaken vigorously every 5 min. Over-digestion (more than 60 min) will lead to a decrease in cell yield. After digestion, the dissociation tubes were placed in the tissue dissociator again and the lung tissue final dissociation program was run (m_lung_02 program was used in this embodiment) to obtain a cell suspension.

[0064] Table 2 Preparation of Lung Tissue Dissociation Enzyme Mixture

[0065] (2) Lysis of red blood cells: The cell suspension was filtered through 70 μm and 40 μm cell sieves. The cell sieves were rinsed with pre-chilled HBSS, and the cell suspension was collected. The suspension was centrifuged at 500 × g for 5 min, and the supernatant was discarded. The cell pellet was resuspended in three volumes of pre-chilled red blood cell lysis buffer, gently pipetted to mix, and allowed to stand at room temperature for 2 min. The suspension was then centrifuged at 500 × g for 5 min, and the supernatant was discarded. This process was repeated once, and then cell counting was performed. After counting, the cells were centrifuged at 500 × g for 5 min.

[0066] (3) Magnetic bead washing: Vortex to resuspend the magnetic beads. Take the required amount of magnetic beads into a 1.5 mL centrifuge tube, add 1 mL of sorting buffer, centrifuge at 10000 × g for 1 min, and discard the supernatant. Add 1 mL of sorting buffer again, repeat the washing process once, and then resuspend the magnetic beads with the same volume of sorting buffer as before. If washing 20 μL of magnetic beads, use 20 μL of sorting buffer for resuspending.

[0067] (4) Magnetic bead sorting: After centrifugation, discard the supernatant, resuspend the cells in magnetic bead sorting buffer, and adjust the cell density to 1 × 10⁻⁶. 8 cells / mL. Prepare 100 μL of cell suspension (approximately 1 × 10⁻⁶ cells / mL). 8Add 10 cells to the bottom of a sterile flow cytometry tube, add 2 μL of Biotin-Antibody Mix, mix well, and incubate at 4 °C for 10 min. When adding the cell suspension, ensure that the cells are added from the bottom of the flow cytometry tube, avoiding adding them along the tube wall. After incubation, add 20 μL of washed Streptavidin Magnetic Beads, mix well, and incubate at 4 °C for 10 min. If more cells need to be sorted, the amounts of Biotin-Antibody Mix and Streptavidin Magnetic Beads can be increased proportionally. After incubation, add 2.5 mL of sorting buffer to the flow cytometry tube and gently mix 5 times with a pipette (avoid vigorous shaking or inverting). Place the flow cytometry tube containing cells on a magnetic rack and let it stand for 5 min. Gently pour the cell suspension into a sterile centrifuge tube (ensuring the flow cytometry tube remains on the magnetic rack at all times), and centrifuge at 500 × g for 5 min. After centrifugation, discard the supernatant and collect the cells.

[0068] (5) Flow cytometry sorting: based on yak CD4 ATF7IP + The top 10 marker genes of the subtype were selected, and appropriate surface marker genes were used for flow cytometry sorting. Figure 5 Next, the sorted cells were subjected to immunofluorescence identification. First, the cells were resuspended in HBSS (without Tris buffer and protein) to prepare a single-cell suspension and cell counting was performed. Based on the counting results, the amounts of dye and antibody to be used were determined. Zombie Violet™ working solution was diluted 1:1000 in HBSS. 10 6 -10 7 Each cell was resuspended in 100 μL of diluted Zombie Violet™ solution (final chromosome volume 100 μL, e.g., 50 μL cell suspension + 50 μL dye mixture). Cells were incubated at room temperature in the dark for 30 min (this step is for staining with cell viability dyes; the buffer system must not contain FBS / BSA). After staining, the cells were washed with 2 mL of FASC buffer, centrifuged at 500 × g for 5 min, and the supernatant was discarded. Then, CD4+ was added. ATF7IP + The subtype sorting antibody mixture should have a final chromosome volume of 100 μL (e.g., 50 μL cell suspension + 50 μL antibody mixture). After thorough mixing, incubate on ice in the dark for 30 min (or at room temperature in the dark for 15 min). Gently tap the tube once during staining to ensure thorough mixing. CD4 ATF7IP +The subtype sorting antibodies were grouped as shown in Table 3, including experimental and control groups (blank control, single-positive control, and FMO control). After incubation, the cells were centrifuged again at 500 × g for 5 min, and the supernatant was discarded. The cells were resuspended in 1 mL FASC Buffer, centrifuged at 500 × g for 5 min, and the supernatant was discarded. Finally, the cells were resuspended at 10... 6 1 cell / 500 μL FASC Buffer, stored on ice in the dark until flow cytometry is performed.

[0069] Table 3 CD4 ATF7IP + Subtype sorting antibody grouping

[0070] (6) Cell collection and expansion culture after sorting: First, prepare 2 mL of T cell receiving medium (78% RPMI 1640 + 20% FBS + 2% penicillin-streptomycin-amphoteric B mixture) for receiving sorted cells. Wash the non-TC treated 24-well plates twice with HBSS, then add 3 μg / mL of Anti-CD3 antibody at 500 μL / well and incubate at 37℃ for 2 h (or overnight at 4℃). After discarding the unbound antibody solution, wash the coated wells twice with HBSS for later use. Next, the sorted CD4 ATF7IP+ subtype cells were expanded and cultured using T cell expansion medium containing IL-2 and IL-7 (85.9868% RPMI 1640 + 10% FBS + 2% penicillin-streptomycin-amphotericidal B mixture + 1% L-Alanyl-L-Glutamine + 1% Sodium Pyruvate + 0.01% Mycoplasma removal reagent + 0.003% Anti-CD28 + 0.0001% hIL-2 + 0.0001% hIL-7). The cell density was adjusted to 10⁶ cells / mL for seeding, and the cells were cultured continuously at 37 ℃ and 5% CO₂ for 5 days. If the medium turned yellow on day 3, the medium should be replaced promptly (either by partially replacing the medium or by adding 1 mL of fresh medium), or the cells could be separated at a 1:1 ratio (no medium replacement required, just add fresh medium, but anti-CD3 coating should be done beforehand).

[0071] (7) CD4 ATF7IP +Subtype cryopreservation: Collect CD4 ATF7IP+ subtype cell suspension, centrifuge at 500 × g for 5 min, discard the supernatant, and retain the cell pellet. Resuspend the cell pellet using Cell-Vive™ CD Low DMSO Cryopreservation and transfer it to cryovials. Cryovials should be immediately transferred to a −80 °C freezer for storage. After 24–36 h, the cryovials can be transferred to liquid nitrogen for long-term storage. Each 1 mL of Cell-Vive™ CD Low DMSO Cryopreservation can preserve 5 × 10⁶ cells. 6 Up to 5 × 10 7 Each cell.

[0072] (8) CD4 ATF7IP + Subtype resuscitation: Remove the cryovials containing cells from the liquid nitrogen container and immediately place them in a 37°C water bath, gently agitating until only a small amount of ice remains. Transfer the cell suspension to centrifuge tubes, add 1 mL of RPMI-1640, mix gently, centrifuge at 500 × g for 5 min, discard the supernatant, and retain the cell pellet. Finally, resuspend the cells to the desired density using T-cell expansion medium and seed them.

[0073] 5. Yak CD4 ATF7IP + Immunofluorescence identification of subtypes The immunofluorescence procedure for the CD4 ATF7IP+ subtype is as follows: Collect approximately 1 × 10⁻⁶ cells / mL. 6 Transfer each cell to a 2 mL centrifuge tube, gently resuspend in 2 mL PBS, centrifuge at 800 × g for 5 minutes, and discard the supernatant. To thoroughly remove serum interference, repeat the washing steps twice. Add an appropriate amount of 4% paraformaldehyde fixative, fix at room temperature for 15 minutes, then resuspend the cells in PBS, centrifuge at 800 × g for 5 minutes, and discard the supernatant. Add immunostaining permeabilization buffer and incubate at room temperature for 10 minutes. Resuspend in PBST, centrifuge at 800 × g for 5 minutes, and discard the supernatant. Add immunostaining blocking buffer and block at room temperature for 30 minutes. Centrifuge and discard the blocking buffer, add ATF7IP polyclonal antibody (1:500), and incubate overnight at 4 °C. Wash twice with PBST, then add goat anti-rabbit IgG secondary antibody (1:500) and incubate at room temperature in the dark for 2 hours. After washing twice with PBST, DAPI staining solution was added, and the cells were stained at room temperature in the dark for 8 minutes. After washing twice with PBST, the cell suspension was added to the adhesive slide, and the slide was air-dried at room temperature in the dark. Finally, the slide was mounted with anti-fluorescence quenching mounting medium.

[0074] Combination Figure 6 The results showed that this invention successfully obtained CD4 ATF7IP from yak lungs.+ Subtype cells.

Claims

1. A CD4+ ion from yak lung ATF7IP + A method for the specific isolation and in vitro culture of subtype cells, characterized in that, This can be achieved through the following method: (1) In situ dual verification: Before isolation, CD4 in yak lungs was first verified at both the RNA and protein levels using fluorescence in situ hybridization and immunohistochemistry. ATF7IP + The presence and location of subtype cells; (2) Hierarchical flow cytometry sorting: The strategy of "immunomagnetic bead pre-sorting + multi-parameter flow cytometry sorting" is adopted. First, CD4 is enriched using immunomagnetic beads. + T cells were then sorted into target subtypes using flow cytometry based on a specific combination of surface markers; the specific combination of surface markers was selected from live / dead dyes and CD45. + CD3 + CD4 + and CD127 + The combination; (3) In vitro culture: The sorted subtype cells were placed in a specific culture medium for in vitro culture, and the cultured cells were identified by immunofluorescence technology to confirm their co-expression characteristics of CD4 and ATF7IP; the specific culture medium was selected from RPMI-1640 complete culture medium containing recombinant cytokines IL-2 and IL-7.

2. The yak lung CD4 as described in claim 1 ATF7IP + A method for the specific isolation and in vitro culture of subtype cells, characterized in that, The hierarchical flow sorting method described in step (2) is as follows: First, CD4 was enriched using immunomagnetic beads. + T cells: (a) Tissue dissociation: The yak lung lobes are pretreated: blood, bronchi and surface tissue are removed, an enzyme mixture is added for digestion, and after digestion, the tissue is run on a tissue dissociator to obtain cell fluid; (b) Lysis of red blood cells: Filter the cell slurry from step (a) using a cell sieve, collect the cell suspension, centrifuge, resuspend the cell pellet in red blood cell lysis buffer, gently pipette to mix, let stand at room temperature, centrifuge, and discard the supernatant; then perform cell counting, and centrifuge after counting is complete; (c) Cleaning of magnetic beads: Vortex the magnetic beads to resuspend them, take the required amount of magnetic beads into a centrifuge tube, add sorting buffer, centrifuge, and discard the supernatant; add sorting buffer to resuspend the magnetic beads. (d) Magnetic bead sorting: Resuspend the cell pellet obtained in step (b) in magnetic bead sorting buffer and adjust the cell density; then add it to the bottom of a sterile flow cytometry tube and incubate; after incubation, add sorting buffer to the flow cytometry tube, mix well, place on a magnetic rack and let stand, centrifuge and discard the supernatant to collect the cells; Then flow sorting is performed: based on yak CD4... ATF7IP + The top 10 marker genes of the subtype were selected using live / dead dyes and CD45. + CD3 + CD4 + and CD127 + Flow cytometry sorting of surface marker genes; The in vitro culture method described in step (3) is as follows: All percentages mentioned below are volume percentages. (a) Cell collection and expansion culture after sorting: Prepare cell receiving medium: 78% RPMI 1640 + 20% FBS + 2% penicillin-streptomycin-amphoteric acid B mixture for receiving sorted cells; coat untreated culture plates with Anti-CD3 antibody, incubate, discard unbound antibody solution, and wash the coated wells with HBSS; subsequently use T cell expansion medium containing IL-2 and IL-7: 85.9868% RPMI 1640 + 10% FBS + 2% penicillin-streptomycin-amphoteric acid B mixture + 1% L-Alanyl-L-Glutamine + 1% Sodium Pyruvate + 0.01% Mycoplasma removal reagent + 0.003% Anti-CD28 + 0.0001% hIL-2 + 0.0001% hIL-7, for sorted CD4 cells. ATF7IP + Subtype cells were expanded and cultured. (b) CD4 ATF7IP + Subtype cryopreservation: CD4 collection ATF7IP + The subtype cell suspension was centrifuged, the supernatant was discarded, and the cell pellet was retained; the cell pellet was resuspended in cell cryopreservation medium containing DMSO and transferred to cryovials; (c)CD4 ATF7IP + Subtype resuscitation: Remove the cryopreservation tube containing cells from the liquid nitrogen tank, transfer the cell suspension to a centrifuge tube, add RPMI 1640, mix gently, centrifuge and discard the supernatant, retaining the cell pellet. Finally, resuspend the cells to the required density using T cell expansion medium and seed them.

Images