阅读说明：本技术 一种多细胞和人肝微粒体共培养方法、共培养培养基及其应用 (Co-culture method of multicellular and human liver microsome, co-culture medium and application thereof ) 是由 华辰凤 尚平平 史清照 赵俊伟 李翔 谢复炜 赵阁 秦亚琼 刘惠民 于 2021-09-02 设计创作，主要内容包括：本发明涉及一种多细胞和人肝微粒体共培养方法、共培养培养基及其应用。本发明提供了多细胞和人肝微粒体共培养方法,包括收集细胞、接种细胞、装载人肝微粒体以及共培养步骤,首次实现了Beas-2b、HUVEC、人肺巨噬细胞和人肝微粒体的体外共培养,为表征肺、血液循环、肝脏共同参与的暴露和代谢后再次损伤的研究奠定了基础。(The invention relates to a cocultivation method of multicellular and human liver microsome, a cocultivation medium and application thereof. The invention provides a method for co-culturing multicellular and human liver microsome, which comprises the steps of collecting cells, inoculating cells, loading human liver microsome and co-culturing, realizes the in-vitro co-culturing of Beas-2b, HUVEC, human lung macrophage and human liver microsome for the first time, and lays a foundation for representing the research of lung, blood circulation, exposure of liver participation and secondary injury after metabolism.)

1. A method for co-culturing multicellular and human liver microsomes is characterized by comprising the following steps:

1) preparing a Beas-2b cell suspension, a HUVEC cell suspension and a human lung macrophage suspension with a co-culture medium and the Beas-2b, the HUVEC and the human lung macrophages respectively;

the co-culture medium consists of a basic culture medium, fetal bovine serum, minocycline hydrochloride, penicillin and streptomycin, wherein the final concentration of the minocycline hydrochloride is 2-3 mu g/mL, the final concentration of the penicillin is 80-120U/mL, and the final concentration of the streptomycin is 80-120 mu g/mL; the basic culture medium is a DMEM culture medium;

2) the Beas-2b cell suspension was inoculated into a first Transwell chamber, the HUVEC cell suspension was inoculated into a second Transwell chamber, a mixture of human liver microsomes consisting of human liver microsomes, an NADPH regeneration system and the co-culture medium was loaded into a dialysis bag, and then the first Transwell chamber, the second Transwell chamber and the dialysis bag were co-cultured in a suspension of human lung macrophages.

2. The method of claim 1, wherein the final concentration of fetal bovine serum in the co-culture medium of step 1) is 8-12% by volume.

3. The method according to claim 1, wherein in step 2) the seeding concentration of both Beas-2b and HUVEC cells is 20 x 10⁴Per mL, human lung macrophage concentration of 5X 10⁴one/mL.

4. The method according to claim 1, wherein in step 3), the final concentration of human liver microsomes in the human liver microsome mixture is 1mg/mL, the final concentration of NADPH regenerating system in volume is 6%, and the final concentration of fetal bovine serum in volume is 8-12%.

5. The method of claim 1, wherein in step 3), the dialysis bag has a molecular weight cut-off of 3.5-5 KD.

6. Use of a method according to any one of claims 1 to 5 for the study of exposure to inhalable xenobiotics, metabolism, wherein the inhalable xenobiotics are introduced into a co-culture system.

7. The use of claim 6, wherein the use of Beas-2b cells infected with the inhalable exogenous material simulates the exposure and metabolic processes of the inhalable exogenous material through the co-participation of the lung, blood circulation and liver.

8. Use according to claim 7, wherein the inhalable foreign substance is a smoke, automobile exhaust, atmospheric PM2.5, pharmaceutical spray, toxic gas or aerosol.

9. A co-culture medium, which is characterized by consisting of a basal medium, fetal bovine serum, minocycline hydrochloride, penicillin and streptomycin, wherein the final concentration of the minocycline hydrochloride is 2-3 mug/mL, the final concentration of the penicillin is 80-120U/mL, and the final concentration of the streptomycin is 80-120 mug/mL; the basic culture medium is a DMEM culture medium.

10. The co-cultivation medium according to claim 9, wherein the final concentration of fetal bovine serum is 8-12% by volume.

Technical Field

The invention belongs to the technical field of in vitro multi-cell co-culture, and particularly relates to a co-culture method of multi-cell and human liver microsome, a co-culture medium and application thereof.

Background

Inhalable pollutants such as smoke, automobile exhaust and atmospheric PM2.5 can be inhaled from the mouth and the nose, and can reach tissues and organs of a human body through a blood circulation system after being exposed in the lung to damage the lung, the heart and other organs of the human body; wherein part of inhaled pollutants can enter blood circulation again to act on each organ to cause secondary injury after being metabolized and detoxified or activated by the liver. Currently, the in vitro cytotoxic effects on inhalable contaminants are mostly focused on a single cell or a single organ, or a single injury such as exposure injury or post-metabolic injury, but there is less research on characterizing lung, blood circulation, co-participating liver exposure and post-metabolic re-injury as a whole.

Human lung macrophages are used as innate immune cells to form a first line of defense against external injury, and play a key role in the occurrence, development, regression and tissue repair of pulmonary inflammatory reactions; beas-2b (Human Bronchial Epithelial Cells, Beas-2b) is an immortalized Human normal Bronchial Epithelial cell, has mesenchymal stem cell characteristics, and is widely used in vitro modeling research of respiratory system injury caused by automobile exhaust, atmospheric PM2.5 and the like; human Umbilical Vein Endothelial Cells (HUVECs) are one of vascular endothelial cells, and are widely used in vascular modeling, inflammation, pharmacology and tumor research, such as research on vascular injury caused by inhalable pollutants such as atmospheric PM2.5 and the like. The human liver microsome has complete I-phase metabolic enzymes (such as cytochrome P450, flavin monooxygenase, monoamine oxidase and the like), II-phase metabolic enzymes (such as glucuronyl transferase, sulfatase), esterase and the like, and can better simulate the metabolism of exogenous substances in vivo in vitro.

The realization of the co-culture of the three cells and the liver microsomes is the premise of research on characterization of the exposure and the secondary injury after the co-participation of the lung, the blood circulation and the liver. However, the existence positions of the three cells and the liver particles in the organism and the culture environment in-vitro culture are different, and different cells have different cell growth environments, so that the co-culture environment is determined according to the involved cells so as to ensure the independent and uniform co-culture existence form of the Beas-2b, the HUVEC, the human lung macrophage and the human liver particles is an urgent technical problem to be solved.

Disclosure of Invention

The invention aims to provide a method for co-culturing multiple cells and human liver microsomes, which can realize independent and unified co-culture of Beas-2b, HUVEC, human lung macrophages and human liver microsomes in vitro.

A second object of the invention is to provide the use of the above method.

The third purpose of the invention is to provide a co-culture medium.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for co-culturing multicellular and human liver microsomes comprises the following steps:

1) preparing a Beas-2b cell suspension, a HUVEC cell suspension and a human lung macrophage suspension with a co-culture medium and the Beas-2b, the HUVEC and the human lung macrophages respectively;

the co-culture medium consists of a basic culture medium, fetal bovine serum, minocycline hydrochloride, penicillin and streptomycin, wherein the final concentration of the minocycline hydrochloride is 2-3 mu g/mL, the final concentration of the penicillin is 80-120U/mL, and the final concentration of the streptomycin is 80-120 mu g/mL; the basic culture medium is a DMEM culture medium;

2) the Beas-2b cell suspension was inoculated into a first Transwell chamber, the HUVEC cell suspension was inoculated into a second Transwell chamber, a mixture of human liver microsomes consisting of human liver microsomes, an NADPH regeneration system and the co-culture medium was loaded into a dialysis bag, and then the first Transwell chamber, the second Transwell chamber and the dialysis bag were co-cultured in a suspension of human lung macrophages.

The method of the invention provides three independent and uniform culture and co-incubation forms of cells and liver microsomes, and utilizes porous Transwell and dialysis bags, thereby not only isolating the cells from each other and directly contacting the cells with the liver microsomes, but also communicating the secreted or metabolized substances of the cells and the liver microsomes, and better simulating the metabolism of exogenous substances in vivo in vitro.

Preferably, the final concentration of fetal bovine serum in the co-culture medium of step 1) is 8-12% by volume. More preferably, the final concentration of fetal bovine serum is 10% by volume, the final concentration of minocycline hydrochloride is 2.5. mu.g/mL, the final concentration of penicillin is 100U/mL, and the final concentration of streptomycin is 100. mu.g/mL.

Preferably, in step 2), the seeding concentration of both Beas-2b and HUVEC cells is 20X 10⁴Per mL, human lung macrophage concentration of 5X 10⁴one/mL.

Preferably, in the step 3), the final concentration of the human liver microsomes in the human liver microsome mixture is 1mg/mL, the final volume concentration of the NADPH regenerating system is 6%, and the final volume concentration of the fetal calf serum is 8-12%. The NADPH regeneration system is prepared by mixing the solution A and the solution B according to the volume ratio of 5: 1.

Preferably, the molecular weight cut-off of the dialysis bag in the step 3) is 3.5-5 KD.

The application of the multicellular and human liver microsome co-culture method in the aspects of exposure and metabolism research of the inhalable exogenous substances introduces the inhalable exogenous substances into a co-culture system.

The co-culture method can be used for simulating the exposure of the inhalable exogenous substance to lung cells and the liver microsome metabolism, and simulating the process of exposing the exogenous substance (such as smoke), blood circulation and liver metabolism by breathing of a human body.

Preferably, the inhalable exogenous substance is used for infecting Beas-2b cells to simulate the exposure and metabolic processes of the inhalable substance which are commonly involved in lung, blood circulation and liver.

Preferably, the inhalable exogenous substance is smoke, automobile exhaust, atmospheric PM2.5, a medicinal spray, a toxic gas or an aerosol.

A co-culture medium, which consists of a basal medium, fetal bovine serum, minocycline hydrochloride, penicillin and streptomycin, wherein the final concentration of the minocycline hydrochloride is 2-3 mug/mL, the final concentration of the penicillin is 80-120U/mL, and the final concentration of the streptomycin is 80-120 mug/mL; the basic culture medium is a DMEM culture medium.

Preferably, the final concentration of fetal bovine serum is 8-12% by volume.

The invention provides a co-culture method of multicellular and human liver microsome, which realizes the co-culture of Beas-2b, HUVEC, human lung macrophage and human liver microsome in vitro for the first time and lays a foundation for representing the research of exposure and post-metabolism re-injury in which lung, blood circulation, liver and immune response participate together.

Drawings

FIG. 1 is a schematic diagram of a co-cultivation system constructed in example 1 of the present invention;

wherein, 1-Beas-2b Transwell chamber, 2-HUVEC Transwell chamber, 3-human liver corpuscle dialysis bag, 4-culture chamber, 5-human lung macrophage suspension, 6-chamber membrane;

FIG. 2 shows the cell morphology of Beas-2b cells in a common culture medium for Beas-2 b;

FIG. 3 shows the cell morphology of Beas-2b cells in HUVEC general medium;

FIG. 4 shows the cell morphology of Beas-2b cells in a medium commonly used for macrophages;

FIG. 5 shows the cell morphology of Beas-2b cells in the co-culture medium of the present invention;

FIG. 6 shows the cell morphology of macrophages in Beas-2b medium;

FIG. 7 is the cell morphology of macrophages in HUVEC general medium;

FIG. 8 shows the cell morphology of macrophages in a medium commonly used for macrophages;

FIG. 9 shows the cell morphology of macrophages in a co-culture medium according to the invention;

FIG. 10 shows the cell morphology of HUVEC in Beas-2b general medium;

FIG. 11 shows the cell morphology of HUVEC in the common culture medium for HUVEC;

FIG. 12 is the cell morphology of HUVEC in the medium commonly used for macrophages;

FIG. 13 is the cell morphology of HUVEC in the co-culture medium of the present invention;

FIG. 14 is a comparative experiment of minocycline hydrochloride on co-culture systems containing human liver microsomes;

FIG. 15 is a schematic view of a co-culture/co-incubation system constructed in test example 3 of the present invention;

wherein, the device comprises a 1-Beas-2b Transwell chamber, a 2-HUVEC Transwell chamber, a 3-human liver corpuscle dialysis bag, a 4-culture chamber, a 40-culture chamber cover body, a 5-human lung macrophage suspension, a 6-chamber membrane, a 7-circulating pump, an 8-water bath device, a 9-air inlet, a 10-air outlet and an 11-three-way pipe.

Detailed Description

The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto; the various culture media, reagents, cell-based reagents and the like used in the examples and test examples are commercially available. Human liver microsomes, purchased from huizitankang biotechnology limited.

Example 1 Beas-2b, HUVEC, human Lung macrophages and human liver microsomes coculture method

This example provides a method for co-culturing Beas-2b, HUVEC, human lung macrophages and human liver microsomes, comprising the steps of:

1) preparing a co-culture medium;

sterile, volumetric final concentration 10% FBS (v/v) (Gbico, U.S. prime) sterile minocycline hydrochloride at a final concentration of 2.5. mu.g/mL, sterile penicillin at a final concentration of 100U/mL, and sterile streptomycin at a final concentration of 100. mu.g/mL in DMEM (Gbico, 11965092) medium solution was prepared.

2) Manufacturing a dialysis bag for loading human liver microsomes;

a dialysis bag with a U-shaped opening is formed by plastic packaging a commercial biotechnological regenerated cellulose membrane (3.5-5KD molecular weight cut-off, 10mm width and 60 ℃ temperature resistance limit) by a plastic packaging machine. And (3) observing whether the bag leaks by a finger-pressure method after sterile deionized water is added into the bag, and sequentially soaking the non-leaking dialysis bag in 95% alcohol, 75% alcohol, sterile deionized water and sterile deionized water for 10, 5 and 5min for later use.

3) Preparing a human liver microsome mixed solution for metabolism;

DMEM medium solutions of 10% FBS (v/v), 2.5. mu.g/mL sterile dimethylamine tetracycline hydrochloride, 100U/mL sterile penicillin, 100. mu.g/mL sterile streptomycin, 1mg/mL human liver microsomes (Virginian Taekang Biotech Co., Ltd.), and 6% (v/v) mixed solution of NADPH regenerating system A + B were prepared. It is prepared as before use.

4) Collecting the cells;

beas-2b, HUVEC and human lung macrophages to be passaged were collected and resuspended to 20X 10 final densities using coculture medium⁴one/mL, 20X 10⁴one/mL and 5X 10⁴Cell suspension per mL.

5) Inoculating cells;

20X 10 to⁴Beas-2b and 20X 10 of per mL⁴one/mL HUVEC was seeded into each of the 12-well and 6-well Transwell chambers and cell suspension was added in volumes of about 0.5mL and 1.5mL, respectively. The Beas-2b chamber and the HUVEC chamber were placed in a petri dish or a culture chamber where the fluids were in fluid communication with each other. Human lung macrophages were seeded into the petri dish or culture chamber and the liquid level of the added cell suspension submerged the membrane of the Transwell chamber and below the liquid level in the Transwell chamber.

6) Loading human liver microsomes;

the prepared human liver microsomes for metabolism are loaded into a sterile dialysis bag, and the volume is 1-2 mL. And the dialysis bag loaded with human liver microsomes was placed in a co-culture dish or culture chamber. The liquid level in the culture dish or the culture bin should be submerged above 2/3 which is the vertical height of the liquid in the dialysis bag.

The co-culture system constructed according to the above steps is shown in FIG. 1, and comprises a Beas-2b Transwell chamber 1, a HUVEC Transwell chamber 2, a dialysis bag 3 for human liver microparticles and a culture chamber 4; the culture chamber 4 comprises a lower main body and an upper cover body, a Beas-2b Transwell chamber 1 and a HUVEC Transwell chamber 2, wherein human liver microparticles are carried on the lower main body by a dialysis bag 3, a human lung macrophage suspension 5 is contained in the culture chamber 4, and the liquid level of the human lung macrophage suspension 5 is higher than that of a chamber membrane 6 of the Transwell chamber.

7) And (4) co-culturing.

According to the purpose of experiment, the test substance is added or not added into the independent space of the cells of the Beas-2b, the HUVEC, the human lung macrophage and/or the human liver microsome, and then the culture plate loaded with the Beas-2b, the HUVEC, the human lung macrophage and the human liver microsome is placed into an incubator for incubation and culture.

Example 2 Co-cultivation Medium

This example provides a medium consisting of a basal medium, fetal calf serum, minocycline hydrochloride, penicillin, and streptomycin, the basal medium being DMEM medium, the final concentration of fetal calf serum being 10% by volume, the final concentration of minocycline hydrochloride being 2.5 μ g/mL, the final concentration of penicillin being 100U/mL, and the final concentration of streptomycin being 100 μ g/mL. The culture medium can be applied to the co-culture of Beas-2b, HUVEC, human lung macrophage and human liver microsome.

Test example 1 cell morphology test of Co-culture Medium for Beas-2b, HUVEC, human Lung macrophages

The co-culture medium provided by the embodiment 2 of the invention is used for respectively culturing three cells of the Beas-2b, the HUVEC and the human lung macrophage, and the common culture medium of the Beas-2b, the HUVEC and the human lung macrophage is used as a control to compare the morphological change of the cells.

Selecting HUVEC, Beas-2b and human lung macrophage in exponential phase with good growth state, respectively preparing into cell suspension with different culture media (ECM complete culture medium, BEGM complete culture medium, DMEM complete culture medium, co-culture medium), and adjusting cell concentration to 5.0 × 10⁴one/mL. HUVEC, Beas-2b and human Lung megakarya in 12-well plates, 1.5mL per well, per mediumThe phagocytes were set in 3 duplicate wells. Cell culture plates at 37 ℃ 5% CO₂Incubation and culture are carried out in the incubator, and the cell morphology is observed by a microscope.

After 24h the cell morphology was observed under a microscope. Beas-2b, HUVEC, human lung macrophage commonly used medium is the complete medium corresponding to each cell in Table 1.

TABLE 1 three media commonly used for cells

The results of the experiments are shown in FIGS. 2-13, Beas-2b presents spindle shape in its usual culture medium, which is its normal cell morphology (FIG. 2); the cells appeared cobblestone in HUVEC common medium and changed morphology (fig. 3); fusiform form in macrophage common medium (FIG. 4) and in co-culture medium of the invention (FIG. 5) suggests that macrophage common medium and co-culture medium of the invention can maintain good cell morphology of the Beas-2b cells.

Human lung macrophages exhibited a rounded, clumpy-like growth morphology in their usual culture media, which is their normal cell growth morphology (fig. 8); the morphological parts of the cells were non-circular in shape and the morphological changes were observed in the usual culture media for Beas-2b and HUVEC (fig. 6, 7); the round cohesin-like growth morphology was present in the co-culture medium of the invention (fig. 9), suggesting that the co-culture medium of the invention can maintain good cell morphology of human lung macrophages.

HUVECs exhibited a cobblestone morphology under their usual media, which is their normal cell culture state (fig. 11); presents spindle shape under the common culture medium of Beas-2b, and the cell morphology changes (FIG. 10); the morphology of the paving cobblestones was exhibited in the macrophage common medium as well as in the inventive co-culture medium (fig. 12, fig. 13), suggesting that the macrophage common medium as well as in the inventive co-culture medium can maintain good cell morphology of HUVEC.

Comparing the components of the co-culture medium and the macrophage common culture medium, the co-culture medium has 2.5 mu g/mL more sterile minocycline hydrochloride component than the macrophage common culture medium, and the result shows that the 2.5 mu g/mL sterile minocycline hydrochloride does not influence the growth forms of the three cells. Finally, the inventive co-culture medium can maintain the co-growth of the three cells.

Test example 2 comparative test of minocycline hydrochloride to co-culture system containing human liver microsomes

Human liver origin is difficult to guarantee sterility, and when human liver microsomes are prepared, sterile liver microparticles can hardly be obtained. In the co-culture process, if not controlled, the pollution source in the human liver microsome can affect the sterile environment of the cells, and the normal growth of the cells is difficult to maintain.

A medium solution was prepared consisting of BEGM BulletKit with final concentrations of 100U/mL penicillin and 100U/mL streptomycin. Culture medium solutions containing 25 donor human liver microsomes were prepared, respectively, in which the culture medium solution contained only 25 donor human liver microsomes (control group) and erythromycin solution (Shenzhen Zike Biotech Co., Ltd.; erythromycin group), 25mg/L chloramphenicol (Bilun Sp., chloramphenicol group) and 2.5mg/L dimethylamine tetracycline hydrochloride (Bilun Sp., dimethylamine Tetracycline hydrochloride group) at a final concentration of 200 mg/L.

The Beas-2b suspension (5X 10) in exponential growth phase is added⁴one/mL) were inoculated in a 6-well plate, after the cells were attached to the wall, cell supernatants were removed, five solutions were added respectively for culturing Beas-2b cells, which were designated as a medium group (a), a control group (b), an erythromycin group (c), a chloramphenicol group (d), and a minocycline hydrochloride group (e), and cell morphology was observed after 48 hours, as shown in fig. 4.

As can be seen from FIG. 14, no well-growing cells were observed in the control group (b), the erythromycin group (c), and the chloramphenicol group (d), and a contaminated state was observed; the minocycline hydrochloride group (e) was found to grow well in cells, was in a non-contaminating state, and was in consistent morphology with the cells of the medium group (a), suggesting that the minocycline hydrochloride group may inhibit the source of contamination in the experimental liver microsomes without affecting the growth of the cells.

In this experimental example, the application of minocycline hydrochloride was described by using the culture medium BEGM BulletKit as an example, and the properties of the culture medium consisting of DMEM medium, fetal calf serum, penicillin and streptomycin according to the present invention were substantially the same as those of the above-described culture medium group.

Test example 3 cytotoxicity of cigarette Smoke in Beas-2b, HUVEC, human Lung macrophages and human liver microsomes

The cytotoxicity of cigarette smoke under the co-incubation of Beas-2b, HUVEC, human lung macrophage and human liver microsome is inspected by using a co-culture/co-incubation system as shown in figure 15, the system comprises a Beas-2b Transwell chamber 1, a HUVEC Transwell chamber 2, a dialysis bag for human liver microsome 3 and a culture bin 4, the culture bin 4 comprises a culture bin main body and a culture bin cover body 40, the culture bin main body is divided into an inner tank and an outer tank, the outer tank can be injected with water, and the incubation is carried out at 37 ℃; the inner tank can be used for placing a small chamber, a dialysis bag and human lung macrophage suspension 5, the height of the human lung macrophage suspension 5 in the inner tank is higher than that of a small chamber membrane 6, the liquid in the inner tank realizes circulating flow by using a circulating pump 7, and the liquid is changed by using a three-way pipe 11; the outer tank is connected with a water bath device 8 to realize the control of the incubation temperature in the inner tank. Each chamber is provided with an air inlet 9 and an air outlet 10 to achieve contamination and to maintain pressure balance in the chamber.

The experimental procedure was as follows:

1) the co-culture/co-incubation device (culture chamber 4) is connected with a circulating pump and a water bath pump, so that the connection is ensured to be watertight.

2) The water temperature in the water bath pump is adjusted to be 37 ℃, and the water bath pump injects circulating water with the temperature of 37 ℃ into the outer tank of the co-culture/co-incubation device.

3) Ultraviolet sterilizing co-culture/co-incubation device for 30min, adding 75% alcohol into the inner tank of the co-culture/co-incubation device, filling, and opening circulating pump 7 during filling to ensure that the whole pipeline contains alcohol for 3 times, each time for 10 min; the waste liquid is discharged through a tee 11. Adding PBS solution into the inner tank of the co-culture/co-incubation device, fully filling, and opening a circulating pump 7 in the filling process to ensure that the whole pipeline has PBS for 5 times, 5min each time;

4) the cells were co-cultured or co-incubated using a co-culture/co-incubation device with a circulation pump flow rate of 1 ml/min.

Respectively exposing 0 dose of smoke and 1/2 doses of smoke in the small chambers of the co-incubated Beas-2b, HUVEC, human lung macrophage and human liver microsome inactivated Beas-2b, and respectively marking as a liver microsome-free control group and a liver microsome-free smoke group; during this period, the circulation pump provides power to accelerate the exchange of liquid in the co-culture/incubation system at a flow rate of 1 mL/min.

The chambers of co-incubated Beas-2b, HUVEC, human lung macrophages and human liver microsome Beas-2b were exposed to 0 dose of smoke and 1/2 doses of smoke, respectively, as indicated in the liver microsome control and liver microsome smoke groups, during which time the circulation pump was at a flow rate of 1 mL/min. After the exposure is finished, after incubation and culture are carried out for 24h, the cell survival rate of each cell is detected by using CCK-8.

TABLE 2 survival rates of individual cells in each group

Grouping	Beas-2b(％)	HUVEC(％)	Human lung macrophage (%)
				Inactivated liver microsome control group	100	100	100
Liver microsome smoke inactivation group	38	89	95
				Liver microsome control group	100	100	100
Liver microsome smoke group	82	95	123

Note: inactivation conditions: boiling at 100 deg.C for 5min

As can be seen from Table 2, after the smoke is contaminated from the Beas-2b cell under the co-incubation of Beas-2b without liver microsome, HUVEC, human lung macrophage and inactivated human liver microsome, the smoke can diffuse to the environment where the HUVEC and human lung macrophage are located through a Transwell membrane, so as to cause the damage of the HUVEC and human lung macrophage, which suggests that the process can simulate the process of inhalable substance through the lung-blood circulation.

Under the co-incubation of the Beas-2b with active liver microsomes, the HUVEC, the human lung macrophages and the human liver microsomes, after the smoke is infected from the Beas-2b chamber, the cell survival rates of the Beas-2b, the HUVEC and the human lung macrophages are obviously higher than those of three cells in an inactivated liver microsome smoke group, and the existence of the active liver microsomes is prompted to carry out certain metabolic activation on the smoke. The Beas-2b and HUVEC in the co-cultured cell culture medium provided by the invention are prompted to well co-incubate human lung macrophages and human liver microsomes to carry out in-vitro metabolism of a test object, so that the survival rate of cells is influenced.