Triple co-culture cell model for immune function research as well as construction method and application of triple co-culture cell model

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to a triple co-culture cell model for immune function research as well as a construction method and application of the triple co-culture cell model. Based on HMEC-1, THP-1 and Jurkat cells, a triple co-culture cell model is constructed by adopting a Transwell three-dimensional construction mode, and a microenvironment in which T cells exist in human blood can be simulated. The obtained triple co-culture cell model is subjected to toxic attack by using mixed benzene, and the expression of CD3 on the surface of the Jurkat cell is not destroyed by co-culture when contamination is carried out for 24-48 hours, so that the TCR signal transduction function is reserved. After contamination is carried out for 24 hours, the expression of ICAM-1 of Jurkat cells of the triple co-culture cell model is up-regulated, and the regulation of an in-vivo immune microenvironment on the activation state of the T cells can be better reflected. The sensitivity of the triple co-culture cell model is higher than that of single culture, and the immune function can be better researched.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to a triple co-culture cell model for immune function research, its construction method, and its application. Background Technology

[0002] Immunotoxicity of chemical substances is a key focus in toxicological safety evaluation, and current toxicological safety evaluation methods are shifting from traditional toxicity testing systems based on whole-animal experiments to those primarily based on in vitro toxicity testing systems. T cells, as the core effector cells of the immune response, are widely involved in key physiological processes such as the recognition and clearance of external pathogens, the maintenance of immune system homeostasis, and the monitoring and clearance of abnormal self-cells (such as tumor cells or infected cells). In-depth research on the toxic effects of combined exposure to benzene, toluene, and xylene (benzene-toluene-xylene mixture, hereinafter referred to as BTX) on T cells and its potential targets is of great significance for revealing the key aspects of BTX's interference with the immune system.

[0003] Single-cell models are used to describe cellular behaviors such as adhesion, proliferation, and differentiation, and also to study the cytotoxicity of exogenous chemicals. However, single-cell models cannot simulate the complex interactions between cells in real tissues and lack regulatory networks such as cytokines and the matrix. Therefore, the role of single-cell models is diminishing, while cell co-culture technology can better simulate the in vivo environment, helping to observe interactions between cells and between cells and the culture environment, and to explore the toxic effects of substances. Developing structurally sound, functionally representative, and mechanistically sound cell co-culture models to replace animal models has become an important trend in toxicology and pharmacology research. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a triple co-culture cell model, its construction method and application, to simulate the interaction mechanism between Jurkat cells and other cells under physiological and pathological conditions, thereby more realistically revealing the effect of BXT on T cell damage and studying immune function.

[0005] This invention provides a method for constructing a triple co-culture cell model for immune function research, comprising the following steps:

[0006] HMEC-1 cells were seeded in the upper chamber of a Transwell co-culture plate, co-culture medium was added, and after the first culture and washing, a Transwell co-culture plate with HMEC-1 cells attached was obtained.

[0007] THP-1 cells were seeded in the upper chamber of the Transwell co-culture plate containing HMEC-1 cells, and the co-culture medium was added. Jurkat cells were seeded in the lower chamber, and the co-culture medium was added. Co-culture was then performed.

[0008] The co-culture medium includes RPMI-1640 basal medium, 10% v / v FBS, and 1% v / v penicillin-streptomycin antibiotics.

[0009] Preferably, the HMEC-1 cells, THP-1 cells, and Jurkat cells are all cells in the logarithmic growth phase.

[0010] Preferably, the seeding density of the HMEC-1 cells is 1×10⁻⁶. 5 The number of cells per mL is 1; the culture volume of the HMEC-1 cells is 1 mL.

[0011] Preferably, the seeding density of the THP-1 cells is 2 × 10⁻⁶. 5 The number of cells per mL was 1 mL; the culture volume of the THP-1 cells was 1 mL.

[0012] Preferably, the seeding density of the Jurkat cells is 4 × 10⁻⁶. 5 The number of Jurkat cells per mL was 2 mL.

[0013] Preferably, the first culture temperature is 37°C, the CO2 concentration is 5%, and the time is 24 hours.

[0014] Preferably, the co-culture temperature is 37°C, the CO2 concentration is 5%, and the time is ≥24h.

[0015] This invention provides a triple co-culture cell model obtained by the construction method described above.

[0016] This invention provides the application of the triple co-culture cell model described in the above technical solution in immune function research.

[0017] Preferably, the immune function study includes a study on the T-cell damage effect induced by exposure to mixed benzene; the mixed benzene includes benzene, toluene, and xylene.

[0018] Beneficial effects:

[0019] Jurkat cells, a cell line derived from human acute T-lymphoblastic leukemia, not only retain the key characteristics of normal T lymphocytes (T cell receptor signaling pathways, cytokine secretion lineages, and surface marker expression patterns), but also, due to their stable proliferative capacity and manipulability, have become a standard model for studying T cell immune response mechanisms. HMEC-1 cells, even after long-term passage culture, continuously express specific markers such as von Willebrand factor and platelet endothelial cell adhesion molecule-1, maintaining their typical cobblestone morphology and in vitro angiogenesis ability. Jurkat and HMEC-1 cells can form capillary-like structures, providing a vascular environment for T cell survival. The THP-1 cell line not only exhibits typical monocyte morphology but also retains important immune functions, including phagocytic activity, cytokine secretion, and the ability to respond to inflammatory stimuli, mimicking the mediating effects in immune and inflammatory responses. This invention utilizes HMEC-1 cells, THP-1 cells, and Jurkat cells to construct a triple co-culture cell model using the Transwell three-dimensional construction method. This model can simulate the microenvironment of T cells in human blood, enabling the study of immune function, especially the T cell damage effect caused by mixed benzene exposure. It has broad prospects for scientific research and translational applications. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 Jurkat cell viability under both single-culture and co-culture conditions is shown in the figure. n = 6;

[0022] Figure 2 The cytoskeleton and nuclear morphology of Jurkat cells under solitary and co-culture conditions;

[0023] Figure 3 The cytoskeleton and nuclear morphology of HMEC-1 cells under single-culture and co-culture conditions;

[0024] Figure 4 The cytoskeleton and nuclear morphology of THP-1 cells under both solitary and co-culture conditions;

[0025] Figure 5 The survival rates of Jurkat cells under BTX-treated conditions in single and co-culture settings are expressed as follows: n=6, *: P<0.05;

[0026] Figure 6The expression of CD3 and ICAM-1 in Jurkat cells under BTX-treated, isolated, and co-cultured conditions is shown in Figure 1. A represents flow cytometry results; B represents statistical results of CD3 and ICAM-1 expression levels. Results are expressed as follows: n = 6, *: P < 0.05. Detailed Implementation

[0027] This invention provides a method for constructing a triple co-culture cell model for immune function research, comprising the following steps:

[0028] HMEC-1 cells were seeded in the upper chamber of a Transwell co-culture plate, co-culture medium was added, and after the first culture and washing, a Transwell co-culture plate with HMEC-1 cells attached was obtained.

[0029] THP-1 cells were seeded in the upper chamber of the Transwell co-culture plate containing HMEC-1 cells, and the co-culture medium was added. Jurkat cells were seeded in the lower chamber, and the co-culture medium was added. Co-culture was then performed.

[0030] The co-culture medium includes RPMI-1640 basal medium, 10% v / v FBS and 1% v / v penicillin-streptomycin (P / S).

[0031] In this invention, HMEC-1 cells are seeded in the upper chamber of a Transwell co-culture plate, co-culture medium is added, and after the first culture and washing, a Transwell co-culture plate with HMEC-1 cells attached is obtained.

[0032] As one embodiment, the seeding density of HMEC-1 cells in this invention is 1×10⁻⁶. 5 / mL. As one embodiment, the culture volume of HMEC-1 cells in this invention is 1mL. This invention limits the seeding density of the HMEC-1 cells; a seeding density higher than the density limited by this invention will lead to cell overgrowth, resulting in cell death due to lack of nutrients and excessive accumulation of metabolic substances, while a density lower than the density limited by this invention will restrict cell proliferation.

[0033] In one embodiment, the HMEC-1 cells described in this invention are 39th generation HMEC-1 cells. In another embodiment, the HMEC-1 cells described in this invention are HMEC-1 cells in the logarithmic growth phase. In another embodiment, this invention uses the original HMEC-1 cell culture medium to culture HMEC-1 cells to the logarithmic growth phase; the original HMEC-1 cell culture medium comprises 89% v / v RPMI-1640, 10% v / v FBS, and 1% v / v P / S. This invention does not have strict requirements on the method of culturing HMEC-1 cells to the logarithmic growth phase using the original HMEC-1 cell culture medium; conventional steps in the art are sufficient.

[0034] In one embodiment, the temperature of the first culture in this invention is 37°C. In one embodiment, the CO2 concentration of the first culture in this invention is 5%. In one embodiment, the culture time of the first culture in this invention is 24 hours.

[0035] HMEC-1 cells are the first immortalized microvascular endothelial cells. During long-term passage culture, these cells continuously express specific markers such as von Willebrand factor and platelet endothelial cell adhesion molecule-1, maintaining their typical cobblestone morphology and in vitro angiogenesis ability. During co-culture, they form capillary-like structures, providing a vascular environment for T cell survival.

[0036] In one embodiment, the cleaning process of the present invention utilizes PBS. In another embodiment, the cleaning process of the present invention is performed twice.

[0037] After obtaining the Transwell co-culture plate with HMEC-1 cells attached, the present invention seeded THP-1 cells into the upper chamber of the Transwell co-culture plate with HMEC-1 cells attached, added the co-culture medium, and seeded Jurkat cells into the lower chamber, added the co-culture medium, and then co-cultured them.

[0038] As one embodiment, the seeding density of THP-1 cells in this invention is 2 × 10⁻⁶. 5 / mL. As one embodiment, the culture volume of THP-1 cells in this invention is 1 mL. This invention limits the seeding density of the THP-1 cells; a seeding density higher than the density limited by this invention will lead to cell overgrowth, resulting in cell death due to lack of nutrients and excessive accumulation of metabolic substances, while a density lower than the density limited by this invention will restrict cell proliferation.

[0039] In one embodiment, the THP-1 cells described in this invention are 13th generation THP-1 cells. In another embodiment, the THP-1 cells described in this invention are THP-1 cells in the logarithmic growth phase. In another embodiment, this invention uses the original THP-1 cell culture medium to culture THP-1 cells to the logarithmic growth phase; the original THP-1 cell culture medium comprises 93% v / v ECM, 5% v / v FBS, 1% v / v ECGS, and 1% v / v P / S. This invention does not have strict requirements on the method of culturing THP-1 cells to the logarithmic growth phase using the original THP-1 cell culture medium; conventional steps in the art are sufficient.

[0040] THP-1 cells are a cell line derived from human acute monocytic leukemia. They not only exhibit typical monocytic morphology but also retain important immune functions, including phagocytic activity, cytokine secretion, and the ability to respond to inflammatory stimuli, mimicking mediating effects in immune and inflammatory responses. During co-culture, capillary-like structures are formed to provide a vascular environment for T cell survival.

[0041] As one embodiment, the seeding density of Jurkat cells in this invention is 4 × 10⁻⁶. 5 / mL. As one embodiment, the culture volume of Jurkat cells in this invention is 2mL. This invention limits the seeding density of the Jurkat cells; a seeding density higher than the density limited by this invention will lead to cell overgrowth, resulting in cell death due to lack of nutrients and excessive accumulation of metabolic substances, while a density lower than the density limited by this invention will restrict cell proliferation.

[0042] In one embodiment, the Jurkat cells described in this invention are 10th generation Jurkat cells. In another embodiment, the Jurkat cells described in this invention are Jurkat cells in the logarithmic growth phase. In another embodiment, this invention uses the original Jurkat cell culture medium to culture Jurkat cells to the logarithmic growth phase; the original Jurkat cell culture medium comprises 89% v / v RPMI-1640, 10% v / v FBS, and 1% v / v P / S. This invention does not have strict requirements regarding the method of culturing Jurkat cells to the logarithmic growth phase using the original Jurkat cell culture medium; conventional steps in the art are sufficient.

[0043] In one embodiment, the co-culturing temperature of the present invention is 37°C. In one embodiment, the CO2 concentration of the co-culturing medium of the present invention is 5%. In one embodiment, the co-culturing time of the present invention is ≥24h; in another embodiment, the co-culturing time of the present invention is 24-72h; in yet another embodiment, the co-culturing time of the present invention is 24-48h.

[0044] Jurkat cells, as a cell line derived from human acute T-lymphoblastic leukemia, not only retain the key characteristics of normal T lymphocytes (T cell receptor signaling pathway, cytokine secretion lineage, and surface marker expression patterns), but also have become a standard model for studying T cell immune response mechanisms due to their stable proliferation capacity and manipulability.

[0045] This invention provides a triple co-culture cell model obtained by the construction method described above. In the triple co-culture cell model established by this invention, Jurkat cells showed no significant difference in cell viability under both individual and co-culture conditions, and co-culture did not significantly affect the viability of Jurkat cells. By comparing the morphological characteristics of Jurkat cells, HMEC-1 cells, and THP-1 cells under individual and co-culture conditions, it was found that all three cell types maintained their typical morphological characteristics: Jurkat cells and THP-1 cells maintained spherical suspension growth, while HMEC-1 cells exhibited a cobblestone-like monolayer adherence. Furthermore, under co-culture conditions, no abnormal cytoskeleton depolymerization or nuclear condensation, fragmentation, or other nuclear morphological changes were observed in the three cell types, further confirming the successful construction of the co-culture model from a morphological perspective. Jurkat cells, HMEC-1 cells, and THP-1 cells exhibit significant differences in cell cycle distribution under co-culture and individual culture conditions. These cells may collectively regulate Jurkat cell proliferation and cell cycle progression through cytokine secretion. The co-culture system can simulate these physiological interactions, providing a more intuitive perspective for studying T cell behavior after injury. This invention establishes a triple co-culture cell model using Jurkat cells as T cells, HMEC-1 cells as vascular endothelial cells, and THP-1 cells as monocytes. This model can simulate the microenvironment of T cells in human blood, enabling the study of immune function, particularly the effects of benzene exposure on T cell damage.

[0046] Given the advantages of the triple co-culture cell model provided by this invention, its application in immune function research also falls within the scope of protection of this invention.

[0047] In one embodiment, the immune function study of the present invention includes a study on the T-cell damage effect induced by exposure to mixed benzene; the mixed benzene includes benzene, toluene, and xylene. In one embodiment, the concentration ratio of benzene, toluene, and xylene in the mixed benzene of the present invention is 1:1:1. In one embodiment, the sum of the concentrations of benzene, toluene, and xylene in the mixed benzene of the present invention is 0.4 mg / mL.

[0048] This invention utilizes a mixed benzene culture to challenge the aforementioned triple co-culture cell model. After 24–48 hours of exposure, co-culture did not disrupt CD3 expression on the Jurkat cell surface, preserving TCR signaling function. After 24 hours of exposure, ICAM-1 expression in the Jurkat cells of the triple co-culture cell model was upregulated, facilitating their migration to inflammatory sites and their immune function, thus better reflecting the regulation of T cell activation by the in vivo immune microenvironment. The triple co-culture cell model described in this invention exhibits higher sensitivity than single-cell culture and better reflects the epigenetic process of injury.

[0049] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a triple co-culture cell model for immune function research, its construction method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] 1. Main Instruments

[0052] Table 1. Main instruments and manufacturers used in this invention.

[0053]

[0054] 2. Main reagents

[0055] Table 2. Main reagents and manufacturers used in this invention.

[0056]

[0057]

[0058] 3. Solution preparation

[0059] (1) 600mg / mL benzene dilution: Take 3.43mL of 874.9mg / mL benzene stock solution, mix it with Jurkat cell culture medium and make up to 5mL. Prepare and use immediately.

[0060] (2) 600 mg / mL toluene dilution: Take 3.47 mL of 864.0 mg / mL toluene stock solution, mix it with Jurkat cell culture medium and make up to 5 mL. Prepare and use immediately.

[0061] (3) 600 mg / mL xylene dilution: Take 3.49 mL of 864.0 mg / mL benzene stock solution, mix it with Jurkat cell culture medium and make up to 5 mL. Prepare and use immediately.

[0062] (4) 600mg / mL BTX diluent: Mix 600mg / mL benzene, toluene and xylene diluents in a volume ratio of 1:1:1 to prepare BTX diluent, and use immediately after preparation.

[0063] (5) 0.4 mg / mL BTX treatment solution: Dilute 600 mg / mL BTX with Jurkat cell culture medium and DMSO to obtain a BTX treatment solution of 4.4 mg / mL. When treating, add the treatment solution at a volume ratio of 10:1 (cell suspension: treatment solution) to make the final BTX concentration 0.4 mg / mL and the final volume fraction of DMSO 0.1%. Prepare fresh before use.

[0064] 4. Co-cultured cells and culture medium

[0065] Jurkat cells (catalog number: CTCC-400-0075) were purchased from Zhejiang Meisen Cell Technology Co., Ltd.

[0066] HMEC-1 cells (catalog number: CTCC-001-0219) were purchased from Zhejiang Meisen Cell Technology Co., Ltd.

[0067] THP-1 cells were purchased from the American Type Culture Collection (ATCC) cell bank, Lot Number: 70043382.

[0068] All cells were cultured at 37°C, 5% CO2, and 70-80% humidity. The original culture medium was used for expansion and passage. For both co-culture and individual culture in well plates, each cell type was cultured in co-culture medium. The composition of the cell culture medium is as follows:

[0069] Jurkat cell culture medium: 89% v / v RPMI-1640 + 10% v / v FBS + 1% v / v P / S;

[0070] HMEC-1 cell original culture medium: 93% v / v ECM + 5% v / v FBS + 1% Endothelial Cell Growth Supplement (ECGS) + 1% v / v P / S;

[0071] THP-1 cell original culture medium: 89% v / v RPMI-1640 + 10% v / v FBS + 1% v / v P / S;

[0072] Co-culture medium: 89% v / v RPMI-1640 + 10% v / v FBS + 1% v / v P / S;

[0073] 5. Jurkat cell culture process

[0074] 5.1. Jurkat cell resuscitation

[0075] (1) Remove the cryovial from the liquid nitrogen and check for any damage;

[0076] (2) Immediately place it in a 37°C water bath and gently shake the cryovial continuously, being careful to avoid the water level exceeding the cap. Thaw it completely within 90 seconds. After surface disinfection with 75% ethanol, quickly transfer it into a 15mL centrifuge tube containing 3mL of the original culture medium in a clean bench and seal it with sealing film.

[0077] (3) Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 2 mL of Jurkat cell culture medium and resuspend the cells by pipetting.

[0078] (4) Inoculate the cell suspension into a T25 culture flask and add 3 mL of the original culture medium;

[0079] (5) Place the inoculated culture flasks in a cell culture incubator at a constant temperature of 37℃ and a concentration of 5% CO2 for incubation;

[0080] (6) Observe the proliferation status under a microscope after 24 hours, and decide whether to change the culture medium or perform passage treatment based on cell morphology and density.

[0081] 5.2. Jurkat cell passage

[0082] (1) Take a small amount of cell suspension for cell counting; the cell growth density reaches 1×10⁻⁶. 6 When the number of cells / mL reaches a certain level, passage can be prepared.

[0083] (2) Preheat the Jurkat cell culture medium in a 37℃ water bath;

[0084] (3) Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min;

[0085] (4) After centrifugation, discard the supernatant, add 2 mL of preheated original culture medium at 37℃, gently blow and mix the cell pellet thoroughly.

[0086] (5) The passage ratio can be determined according to the actual cell growth to ensure that the cell density in each flask is maintained at 5 × 10⁻⁶. 5 Cells / mL, seed cells into T25 culture flasks, and add the original culture medium to a final volume of 5mL;

[0087] (6) Place the culture flask in a 37°C, 5% CO2 incubator for static culture. Observe the growth the next day and perform subsequent cell medium replacement and cell passage according to the cell status.

[0088] 5.3. Jurkat cell cryopreservation

[0089] (1) Take a small amount of cell suspension for cell counting; the cell growth density reaches 1×10⁻⁶. 6 When the cell / mL ratio reaches a certain level, it is ready for cryopreservation.

[0090] (2) Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 min;

[0091] (3) After centrifugation, discard the supernatant and resuspend the cells in 1 mL of cryopreservation buffer (90% FBS + 10% DMSO), ensuring that the cell volume per tube is 1 × 10⁻⁶ cells. 6 cells / mL, sealed with sealing film;

[0092] (4) After sealing, place the cryovials into a programmable cooling box and put them into a -80°C freezer. After 24 hours, transfer the cryovials into a liquid nitrogen tank for long-term storage and complete the cell preservation registration.

[0093] 6. HMEC-1 cell culture

[0094] 6.1. HMEC-1 cell resuscitation

[0095] (1) Remove the cryovial from the liquid nitrogen and check for any damage;

[0096] (2) Quickly place the cryovial in a 37°C water bath and shake it frequently, being careful to avoid submerging the cap. Thaw it completely within 90 seconds, sterilize the surface with 75% ethanol, and then quickly transfer it into a 15mL centrifuge tube containing 3mL of the original culture medium in a clean bench. Seal the tube with sealing film.

[0097] (3) Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 2 mL of the original culture medium, mix well by pipetting, and then inoculate into a T25 culture flask. Add 3 mL of the original culture medium, and place the culture flask flat and shake the cells in an "∞" shape.

[0098] (4) Place the culture flask in a 37°C, 5% CO2 incubator for static culture. Observe the growth the next day and perform subsequent cell medium replacement and cell passage according to the cell status.

[0099] 6.2. HMEC-1 cell passage

[0100] When the cell density reaches 80% as observed under a microscope, it is ready for passage. The specific steps are as follows:

[0101] (1) Preheat the original culture medium and PBS in a 37℃ water bath;

[0102] (2) Discard the culture medium in the culture flask directly, wash twice with PBS, discard the PBS, and use a 1mL pipette to remove the remaining PBS in the culture flask.

[0103] (3) Add 3 mL of 0.25% trypsin, and quickly level the culture flask to spread the trypsin evenly;

[0104] (4) Observe the cell state under a microscope. After about 80% of the cells shrink and become round, immediately add 3 mL of the original culture medium, quickly mix the culture medium and trypsin, and stop the digestion.

[0105] (5) Aspirate the cell suspension, blow the bottom of the culture flask several times, transfer the cell suspension to a centrifuge tube, and centrifuge at 1000 rpm for 5 min.

[0106] (6) After centrifugation, discard the supernatant, add 2 mL of preheated original culture medium at 37℃, gently blow and mix the cell pellet thoroughly.

[0107] (7) The passage ratio can be determined according to the actual cell growth to ensure that the cell density in each flask is maintained at 5 × 10⁻⁶. 5 Cells / mL, seed cells into T25 culture flasks, add original culture medium to a final volume of 5mL, and shake the culture flasks in an "∞" shape to mix the cells;

[0108] (8) Place the culture flask in a 37°C, 5% CO2 incubator for static culture. Observe the growth the next day and perform subsequent cell medium replacement and cell passage according to the cell status.

[0109] 6.3. HMEC-1 cell cryopreservation

[0110] When the cell growth density reaches 80% as observed under a microscope, it is ready for cryopreservation. The specific steps are as follows:

[0111] (1) Discard the culture medium directly, wash twice with PBS and discard, and use a 1mL pipette to remove any remaining PBS from the culture flask. Add 3mL of 0.25% trypsin, and quickly level the culture flask to spread the trypsin evenly.

[0112] (2) Observe the cell state under a microscope. After about 80% of the cells shrink and become round, gently tap the outer wall of the culture container, immediately add 3 mL of the original culture medium, quickly mix the culture medium and trypsin, and stop digestion.

[0113] (3) Aspirate the cell suspension, blow the bottom of the culture flask several times, transfer the cell suspension to a centrifuge tube, and centrifuge at 1000 rpm for 5 min.

[0114] (4) After centrifugation, discard the supernatant and resuspend the cells in 1 mL of cryopreservation solution, ensuring that the cell volume in each tube is 1 × 10⁶ cells / mL. 6 cells / mL, sealed with sealing film;

[0115] (5) After sealing, place the cryopreservation tubes into a programmable cooling box and put them into a -80°C freezer. After 24 hours, transfer the cryopreservation tubes into a liquid nitrogen tank for long-term storage and complete the cell preservation registration.

[0116] 7. THP-1 cell culture

[0117] 7.1. THP-1 cell resuscitation

[0118] (1) Remove the cell cryopreservation tubes from the liquid nitrogen tank and check for any damage;

[0119] (2) Quickly place the cryovial in a 37°C water bath and shake it frequently, avoiding submerging the cap. Thaw it completely within 90 seconds. After sterilizing the surface of the cryovial with 75% ethanol, quickly transfer it into a 15mL centrifuge tube containing 3mL of the original culture medium in a clean bench and seal it with sealing film.

[0120] (3) Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 3 mL of THP-1 cell culture medium, gently pipette and mix well, and then inoculate into T25 culture flasks.

[0121] (4) Place the culture flask upright in a 37°C incubator for static culture. Observe the growth the next day and perform subsequent cell replenishment and cell passage operations according to the cell status.

[0122] 7.2. THP-1 cell passage

[0123] (1) Take a small amount of cell suspension for cell counting; the cell growth density reaches 1×10⁻⁶. 6 When the number of cells / mL reaches a certain level, passage can be prepared.

[0124] (2) Preheat the original culture medium in a 37℃ water bath;

[0125] (3) Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 min;

[0126] (4) After centrifugation, discard the supernatant, add 2 mL of preheated original culture medium at 37℃, gently blow and mix the cell pellet thoroughly.

[0127] (5) The passage ratio can be determined according to the actual cell growth to ensure that the cell density in each flask is maintained at 5 × 10⁻⁶. 5 Cells / mL, seed cells into T25 culture flasks, and add the original culture medium to a final volume of 5mL;

[0128] (6) Place the culture flask in a 37°C incubator for static culture. Observe the growth the next day and perform subsequent cell culture and cell passage operations according to the cell status.

[0129] 7.3. Cryopreservation of THP-1 cells

[0130] A small amount of cell suspension was taken for cell counting, and the cell growth density reached 1×10⁻⁶. 6 When the cell / mL ratio reaches a certain level, cryopreservation can be prepared. The specific steps are as follows:

[0131] (1) Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 min;

[0132] (2) After centrifugation, discard the supernatant and resuspend the cells in 1 mL of cryopreservation solution, ensuring that the cell volume per tube is 1 × 10⁻⁶. 6 cells / mL, sealed with sealing film;

[0133] (3) After sealing, place the cryovials into a programmable cooling box and put them into a -80°C freezer. After 24 hours, transfer the cryovials into a liquid nitrogen tank for long-term storage and complete the laboratory cell preservation registration.

[0134] 8. Cell Count

[0135] (1) Rinse the hemocytometer and special coverslip with 75% ethanol solution, drain on absorbent paper, and fully cover one side of the counting area with the coverslip.

[0136] (2) After centrifugation and removal of supernatant, add a small amount of original culture medium to the cell pellet and mix by pipetting. Take 100 μL of cell suspension into a 1.5 mL EP tube, add 200 μL of PBS and 10 μL of 0.4% trypan blue solution, and mix thoroughly by pipetting. Cells not stained by trypan blue are considered live cells.

[0137] (3) Take 10 μL of trypan blue stained cell suspension and slowly inject it into the counting area along the edge of the coverslip at a 45° angle, so that the liquid fills the entire counting area. Take an appropriate amount of suspension on the other side and perform the same operation. At this time, the area under the coverslip is filled with the cell sample to be counted.

[0138] (4) Wait a moment, then move the hemocytometer to the microscope and adjust it until the field of view is clear. Count the cells in the four large squares around the perimeter. For live cells that are pressed against the grid lines, count the cells on the two adjacent sides on the same side. Generally, the principle of "counting the upper line but not the lower line, counting the left line but not the right line" can be adopted. Do not count the cells on the other two sides. If cells are clustered, count them as one cell. When there are many cell clusters or the cells are unevenly distributed, the cell suspension to be counted should be mixed again, stained, and counted. If the cell density under the microscope is too high, the sample can be diluted before counting.

[0139] (5) Each cell sample was counted three times, and the average value was taken. After counting, the cell density was calculated. The cell density calculation formula is as follows: Cell density (cells / mL) = Total number of cells in 4 large grids × 10000.

[0140] Example 2

[0141] The specific steps for establishing the co-cultivation model are as follows:

[0142] (1) HMEC-1 cells in the logarithmic growth phase were taken and resuspended in the co-culture medium of Example 1 to obtain a cell density of 1×10⁻⁶. 5 HMEC-1 cell suspension of 1 cell / mL was seeded into the inner chamber of a Transwell co-culture plate. The seeding volume was 1 mL. The Transwell co-culture plate was placed in an incubator at 37°C and 5% CO2 and incubated for 24 h to allow the HMEC-1 cells to adhere to the plate.

[0143] (2) Remove the Transwell co-culture plate, wash the inner chamber of the Transwell co-culture plate twice with PBS, take THP-1 cells in the logarithmic growth phase, and resuspend them in the co-culture medium of Example 1 to obtain a cell density of 2 × 10⁻⁶ cells / mL. 5 THP-1 cell suspension of 1 cell / mL was seeded into the chamber of a Transwell co-culture plate at a seeding volume of 1 mL.

[0144] (3) Jurkat cells in the logarithmic growth phase were taken and resuspended in the co-culture medium of Example 1 to obtain a cell density of 4 × 10⁻⁶. 5 A Jurkat cell suspension of 1 cell / mL was seeded into the lower chamber of a Transwell co-culture plate at a seeding volume of 2 mL.

[0145] (4) After inoculating THP-1 cells and Jurkat cells into Tranwell co-culture plates, they were placed in a 37°C, 5% CO2 incubator for static culture and used for model verification or poisoning experiments.

[0146] Comparative Example 1

[0147] Jurkat cells cultured alone

[0148] Jurkat cells in the logarithmic growth phase were resuspended in the co-culture medium of Example 1 to obtain a cell density of 4 × 10⁻⁶ cells / year. 5 A Jurkat cell suspension of 2 mL / well was seeded into 6-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for subsequent experiments.

[0149] Comparative Example 2

[0150] HMEC-1 cells cultured alone

[0151] HMEC-1 cells in the logarithmic growth phase were taken and resuspended in the co-culture medium of Example 1 to obtain a cell density of 1×10⁻⁶. 5 HMEC-1 cell suspensions of 1 cell per mL were seeded into 12-well cell culture plates and cultured in an incubator at 37°C and 5% CO2 for subsequent experiments.

[0152] Comparative Example 3

[0153] THP-1 cells cultured alone

[0154] Logarithmic growth phase THP-1 cells were taken and resuspended in the co-culture medium of Example 1 to obtain a cell density of 2 × 10⁻⁶ cells / year. 5 THP-1 cell suspensions of 1 cell per mL were seeded into 12-well cell culture plates and cultured in an incubator at 37°C and 5% CO2 for subsequent experiments.

[0155] Test Example 1

[0156] Jurkat cell viability assay

[0157] In Example 1 (co-culture) and Comparative Example 1 (iso-culture), after culturing Jurkat cells for 24h, 48h, and 72h, 100 μL MTT solution was added to each well of the lower chamber of Example 1 and the 6-well plate of Comparative Example 1. The cells were incubated at 37℃ for 4h, followed by the addition of 1 mL Formazan lysis buffer for another 4h. The absorbance (OD) values ​​of the lower chamber and the 6-well plate were measured at 570nm using a microplate reader. The viability of Jurkat cells in the co-culture and iso-culture models was compared based on the measured OD values. The results are as follows: Figure 1 As shown.

[0158] according to Figure 1 As can be seen, at 24 h of culture, the OD values ​​for single-cell culture and co-culture were 0.47±0.03 and 0.5±0.05, respectively; at 48 h, they were 1.00±0.10 and 0.84±0.06, respectively; and at 72 h, they were 0.96±0.14 and 0.93±0.12, respectively. Compared with single-cell culture, there were no statistically significant differences in cell viability at each time point in the co-culture group (P>0.05), indicating that co-culture conditions did not significantly affect the cell viability of Jurkat cells, thus the co-culture model was initially successfully established.

[0159] Test Example 2

[0160] Fluorescent staining for observation of cytoskeleton and nuclear morphology

[0161] The dynamic reorganization of the cytoskeleton and the morphological characteristics of the cell nucleus are key indicators for assessing cellular functional status, directly reflecting physiological processes such as cell proliferation, migration, stress response, and cell death. Fluorescent staining was performed on cells cultured under different conditions to observe the cytoskeleton and nuclear morphology. The specific steps are as follows:

[0162] 1. Observation of cytoskeleton and nuclear morphology of Jurkat cells using fluorescent staining.

[0163] (1) Jurkat cells cultured alone (Comparative Example 1) and co-cultured (Example 1) were seeded onto glass slides coated with 0.1 mg / mL poly-L-lysine after 24 h, 48 h and 72 h respectively.

[0164] (2) Fix the cells on ice with 4% paraformaldehyde for 15 min and wash twice with PBS;

[0165] (3) Permeabilize the cells with 0.1% Triton X-100 for 10 min, and then immediately wash the cells twice with PBS;

[0166] (4) Add the microfilament red fluorescent probe solution to the slide at room temperature, incubate in the dark for 60 min, and then wash twice with immunostaining washing solution;

[0167] (5) Mount the slides using a mounting solution containing DAPI to resist fluorescence quenching;

[0168] (6) Confocal images were acquired using Zen software on a Zeiss LSM700 confocal scanning microscope system. The results are as follows: Figure 2 As shown, the scale bar is 20 μm.

[0169] 2. Observation of cytoskeleton and nuclear morphology of HMEC-1 cells by fluorescent staining.

[0170] (1) Place a 9 mm climbing sheet in a six-well plate, and then add HMEC-1 cell suspensions that were cultured separately (Comparative Example 2) and co-cultured (Example 1). After culturing for 48 h, 72 h and 96 h, remove the climbing sheet and gently wash the cells 1 to 2 times with cold PBS.

[0171] (2) Fix the cell smears on ice with 4% paraformaldehyde for 15 min and wash them three times with PBS;

[0172] (3) Permeabilize cells with 0.1% Triton X100 for 10 min, then wash with PBS 3 times;

[0173] (4) Add the microfilament red fluorescent probe solution to the slide at room temperature, incubate in the dark for 60 min, and then wash twice with immunostaining washing solution;

[0174] (5) Mount the slides using a mounting solution containing DAPI to resist fluorescence quenching;

[0175] (6) Confocal images were acquired using Zen software on the Zeiss LSM 700 confocal scanning microscope system. The results are as follows: Figure 3 As shown, the scale bar is 20 μm.

[0176] 3. Observation of cytoskeleton and nuclear morphology of THP-1 cells by fluorescent staining.

[0177] (1) THP-1 cells cultured alone (Comparative Example 3) and co-cultured (Example 1) were seeded on glass slides coated with 0.1 mg / mL poly-L-lysine after 24 h, 48 h and 72 h respectively.

[0178] (2) Fix the cells on ice with 4% paraformaldehyde for 15 min and wash twice with PBS;

[0179] (3) Permeabilize the cells with 0.1% Triton X-100 for 10 min, and then immediately wash the cells twice with PBS;

[0180] (4) Add the microfilament red fluorescent probe solution to the slide at room temperature, incubate in the dark for 60 min, and then wash twice with immunostaining washing solution;

[0181] (5) Mount the slides using a mounting solution containing DAPI to resist fluorescence quenching;

[0182] (6) Confocal images were acquired using Zen software on the Zeiss LSM 700 confocal scanning microscope system. The results are as follows: Figure 4 As shown, the scale bar is 20 μm.

[0183] according to Figures 2-4 As can be seen, compared with the three cell types cultured alone for 24h, 48h and 72h, the three cell types maintained their typical morphological characteristics: Jurkat cells and THP-1 cells maintained spherical suspension growth, HMEC-1 cells showed cobblestone-like monolayer adhesion, and the cytoskeleton and nuclear morphology of the three co-cultured cells did not change significantly. No abnormal cytoskeleton depolymerization or nuclear condensation, fragmentation or other nuclear morphological changes were observed, further confirming the successful construction of the co-culture model from the perspective of cell morphology.

[0184] Test Example 3

[0185] Cell cycle detection

[0186] The cell cycle process is finely regulated by multiple intercellular signaling pathways, including multi-level information integration involving soluble factors, adhesion molecules, and the extracellular matrix. The specific steps for cell cycle detection under different culture methods are as follows:

[0187] (1) After culturing Jurkat cells, HMEC-1 cells, and THP-1 cells individually (Comparative Examples 1-3) and co-cultured (Example 1) for 24h, 48h, and 72h, respectively, 1×10⁻⁶ cells were collected. 6 Centrifuge each cell at 300g for 5 minutes, precipitate the cells and discard the supernatant, then wash once with 1mL PBS;

[0188] (2) Add 1 mL of 70% ethanol pre-cooled in an ice bath and mix well. Fix at 4°C for 1 h and centrifuge at 300 g for 5 min to precipitate the cells.

[0189] (3) Add PBS pre-cooled in an ice bath to resuspend the cells and centrifuge again at 300g for 5min;

[0190] (4) Add 0.5 mL of PI staining solution (0.5 mL of staining buffer + 25 μL of PI staining solution (20X) + 10 μL of RNase A (50X)) to each tube of cell sample to resuspend the cells, and incubate at 37°C in the dark for 30 min.

[0191] (5) Red fluorescence was detected by flow cytometer at an excitation wavelength of 561 nm, and cell cycle analysis was performed using FlowJo V10.10.0 software. The results are shown in Table 3.

[0192] Table 3 Cell cycle distribution under single-culture and co-culture conditions ( n=3)

[0193]

[0194] As shown in Table 3, co-culture conditions significantly affected the cell cycle progression of the three cell types. Compared with monoculture, Jurkat cells showed an increased proportion of cells in G1 and S phases (a decreased proportion in G2 phase, P<0.05) after 24 h of co-culture, while at 48 h and 72 h, a trend of decreasing G1 phase and increasing S and G2 phases was observed (significant difference at 72 h, P<0.05). HMEC-1 cells showed a decreased proportion of cells in G1 and S phases (P<0.05) and an increased proportion in G2 phase (P>0.05) after 24 h of co-culture, but at 48 h and 72 h, an increase in G1 and S phases and a decrease in G2 phase. THP-1 cells showed an increased proportion of cells in G1 phase (a decrease in S and G2 phases, P<0.05) after 24 h of co-culture, while at 48 h and 72 h, a decrease in G1 phase and an increase in S phase (with a continuous decrease in G2 phase). Jurkat cells, HMEC-1 cells, and THP-1 cells exhibited significant differences in cell cycle distribution under co-culture and individual culture conditions, suggesting that Jurkat cells, HMEC-1 cells, and THP-1 cells may jointly regulate T cell proliferation and cell cycle progress through the secretion of cytokines. The co-culture system can simulate these physiological interactions, providing a more intuitive perspective for studying the behavior of T cells after injury.

[0195] Test Example 4

[0196] Validation of the co-cultivation model

[0197] 1. After Jurkat cells in Comparative Example 1 (cultured alone) and Example 1 (co-cultured) were cultured for 24 h, 100 μL and 200 μL of 4.4 mg / mL BTX treatment solution were added to the upper and lower chambers of Transwell and the 6-well plate, respectively, to make the final concentration of BTX 0.4 mg / mL.

[0198] 2. Place the Transwell co-culture plates and 6-well plates in an incubator and incubate for 24 h and 48 h, and then perform subsequent index testing.

[0199] 3. Jurkat cell viability detection after BTX exposure

[0200] (1) After 24h and 48h of exposure, 100μL MTT solution was added to each well of the lower chamber and the 6-well plate, and incubated at 37℃ for 4h.

[0201] (2) Add 1 mL of Formazan solution and continue incubation for 4 h;

[0202] (3) The absorbance (OD value) of the lower chamber and the 6-well plate was detected by an ELISA reader at 570 nm.

[0203] (4) Calculate cell viability using the following formula: Cell viability (%) = (OD-treated group - OD-free group) / (OD control group - OD-free group) × 100%, and the result is as follows: Figure 5 As shown.

[0204] according to Figure 5 It can be seen that at 24 h of exposure, the cell survival rate in the monoculture group was 81.99% ± 5.49%, while that in the co-culture group was 78.28% ± 10.3%, with no statistically significant difference between the two groups. After 48 h of exposure, the survival rate in the monoculture group increased to 88.08% ± 2.04%, while that in the co-culture group decreased to 76.26% ± 3.49%, and the difference between the two groups was statistically significant (P < 0.05). This result indicates that under 48 h of exposure conditions, the co-culture environment can enhance the toxic effect of BTX on Jurkat cells.

[0205] 4. Expression of CD3 and ICAM-1 in Jurkat cells after BTX exposure

[0206] Cluster of differentiation 3 (CD3) is a transmembrane protein complex on the surface of T cells. As a key component of the T-cell receptor (TCR), it plays a central role in TCR signaling, T cell activation, and immune regulation, and is essential for maintaining normal T cell function. Intercellular adhesion molecule-1 (ICAM-1) is an important member of the immunoglobulin superfamily, primarily mediating adhesion between leukocytes and vascular endothelial cells, and participating in the recruitment and migration of immune cells during inflammation. Under physiological conditions, resting T cells typically show low or almost no ICAM-1 expression. Upregulation of ICAM-1 expression is an adaptive response of T cells to toxic stimuli, facilitating their migration to inflammatory sites and exerting immune function. Based on this principle, the expression levels of CD3 and ICAM-1 in Jurkat cells after BTX exposure were detected. The specific steps are as follows:

[0207] (1) Cell counts were performed 24 hours and 48 hours after exposure to the virus, and 1×10⁻⁶ cells were collected. 6 A cell suspension of individual cells;

[0208] (2) Centrifuge at 300g for 5 minutes and discard the supernatant;

[0209] (3) Add 1 mL of PBS to resuspend, centrifuge at 300 g for 5 min, and discard the supernatant;

[0210] (4) Prepare an antibody mixture by adding 1 μL of CD3 and 1 μL of LICAM1 primary antibody to 100 μL of PBS for each sample;

[0211] (4) Add 100 μL of primary antibody mixture to each sample and incubate at 4°C in the dark for 30 min;

[0212] (5) After adding 1 mL of PBS to stop staining, centrifuge at 300 g for 5 min and discard the supernatant;

[0213] (6) Add 1 μL of Goat Anti-Mouse IgG H&L (AF647) H&L and 1 μL of Goat Anti-Rabbit IgG H&L (Cy3) secondary antibody to 1 mL of PBS for each sample to prepare an antibody mixture;

[0214] (7) Add 1 mL of secondary antibody mixture to each sample and incubate at 4°C in the dark for 30 min;

[0215] (8) After adding 1 mL of PBS to stop staining, centrifuge at 300 g for 5 min and discard the supernatant;

[0216] (9) After resuspending in 500 μL PBS, the sample was analyzed by flow cytometry.

[0217] (10) Flow cytometry was used to detect CD3 at an excitation wavelength of 640 nm and ICAM-1 at 561 nm. The results are as follows: Figure 6 As shown.

[0218] according to Figure 6 As shown in Figure A, the CD3 expression level of Jurkat cells was 93.16% ± 4.08% in Jurkat cells cultured alone and 93.61% ± 3.49% in co-cultured Jurkat cells at 24 h of exposure, with no statistically significant difference (P > 0.05). At 48 h of exposure, the CD3 expression level was 2.36% ± 0.2% in Jurkat cells cultured alone and 2.09% ± 0.1% in co-cultured Jurkat cells, with no statistically significant difference (P > 0.05). This demonstrates that co-culture did not disrupt CD3 expression on the surface of Jurkat cells, preserving TCR signaling function.

[0219] according to Figure 6 As shown in Figure B, the ICAM-1 expression level in Jurkat cells was 4.95% ± 0.71% in monocultured Jurkat cells and 8.59% ± 1.54% in co-cultured Jurkat cells at 24 h of exposure (P < 0.05); at 48 h of exposure, the expression level was 1.16% ± 0.12% in monocultured Jurkat cells and 1.4% ± 0.5% in co-cultured Jurkat cells, with no statistically significant difference (P > 0.05). After 24 h of exposure, the co-culture model better reflects the regulation of T cell activation status by the in vivo immune microenvironment.

[0220] As can be seen from the above, the triple co-culture cell model provided by this invention can simulate the complex cell interaction environment of human respiratory tissue after exposure to harmful factors such as heavy metals. It not only closely resembles the real physiological and anatomical structure in terms of structure, but also more comprehensively reproduces the multi-cell synergistic toxic response caused by heavy metal exposure at the functional level, and can be used to study respiration.

[0221] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a triple co-culture cell model for immune function research, characterized in that, Includes the following steps: HMEC-1 cells were seeded in the upper chamber of a Transwell co-culture plate, co-culture medium was added, and after the first culture and washing, a Transwell co-culture plate with HMEC-1 cells attached was obtained. THP-1 cells were seeded in the upper chamber of the Transwell co-culture plate containing HMEC-1 cells, and the co-culture medium was added. Jurkat cells were seeded in the lower chamber, and the co-culture medium was added. Co-culture was then performed. The co-culture medium includes RPMI-1640 basal medium, 10% v / v FBS, and 1% v / v penicillin-streptomycin antibiotics.

2. The construction method according to claim 1, characterized in that, The HMEC-1 cells, THP-1 cells, and Jurkat cells are all cells in the logarithmic growth phase.

3. The construction method according to claim 1 or 2, characterized in that, The HMEC-1 cells were seeded at a density of 1×10⁻⁶. 5 The number of cells per mL is 1; the culture volume of the HMEC-1 cells is 1 mL.

4. The construction method according to claim 1 or 2, characterized in that, The seeding density of the THP-1 cells was 2 × 10⁻⁶. 5 The number of cells per mL was 1 mL; the culture volume of the THP-1 cells was 1 mL.

5. The construction method according to claim 1 or 2, characterized in that, The seeding density of the Jurkat cells was 4 × 10⁶. 5 The number of Jurkat cells per mL was 2 mL.

6. The construction method according to claim 1, characterized in that, The first culture was carried out at a temperature of 37°C, a CO2 concentration of 5%, and for 24 hours.

7. The construction method according to claim 1, characterized in that, The co-culture temperature was 37℃, the CO2 concentration was 5%, and the time was ≥24h.

8. The triple co-culture cell model obtained by the construction method according to any one of claims 1 to 7.

9. The application of the triple co-culture cell model as described in claim 8 in immune function research.

10. The application according to claim 9, characterized in that, The immune function study includes a study on the T cell damage effect induced by exposure to mixed benzene; the mixed benzene includes benzene, toluene, and xylene.

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