I. Introduction:
Continued industrial development and new technological improvements (eg, nanotechnology) have led to an increase in air pollution, which has led to a significant increase in lung disease over the past few decades. New products (eg, nanoparticle sprays) have been widely used in the electrical industry, and everyday consumer and medical applications, especially in spray or powder form applications, are considered to be particularly harmful to human health. The increased exposure to harmful substances, such as shoe care, detergents, antibacterial sprays, or technical processing of products such as plastics, using nanospray can significantly increase lung exposure. However, little is known about the toxicity and basic pathological mechanisms of inhalable substances. Since 2006, the EU Chemical Regulations (Registration, Evaluation, Authorization and Restriction of Chemicals, EC 1907 / 2006) require toxicity assessments of existing and newly introduced chemicals, including identification of their potential inhalation Toxic hazard. Toxicological data in the human body is rare because human poisoning is usually accidental, unpredictable, and uncontrollable. It is very difficult to have the same or similar experimental specimen data. In addition, the study of lung toxicity in the human body is also not feasible due to social moral reasons. Therefore, toxicological data is usually produced in animal experiments. However, animal models also have their limitations, such as interspecies differences. In addition, legislation needs to consider the “3R†principle (reduction, optimization, and replacement) and its development, that is, the need to establish and validate alternative methods for current animal testing. Therefore, an in vitro exposure-based exposure technique based on in vitro experiments is necessary for lung toxicity evaluation studies.
Second, the cell exposure technology
1. Immersion in vitro exposure
Various in vitro culture models have been used to perform acute toxicity assessment and research of aerosols in the air. Traditional immersion in vitro exposure technology is the simplest and most effective method for in vitro exposure to the drug. So far, many laboratories are still in use. The method firstly immerses the cells in an immersion culture, and then the aerosol material to be exposed needs to be dissolved in the culture solution to reach the cell surface, and the virus is subjected to an interaction experiment by means of interaction between the cells and the culture solution (as shown in FIG. 1). .
Figure 1 Immersion-type in vitro exposure pattern
However, for intrapulmonary epithelial cells, this approach is different from the real gas-liquid interface exposure environment: 1. Exposure to immersion conditions, resulting in potential changes in aerosol properties; 2. For water-insoluble solid particles The uniformity of distribution in the culture solution is low; 3. Compared with the gas, the movement speed of the particles in the liquid is lowered, and the efficiency of contact with the cells is lowered.
2. Gas-liquid interface in vitro exposure
Because of the innate shortcomings of immersion in vitro exposure, scientists have begun to explore a new in vitro exposure technology that can more realistically mimic the biological effects in vivo. As described in 1975, Voisin et al. presented a description of the in vitro exposure of gas-liquid interface (ALI), which provides a basis for the gas-liquid interface exposure (ALI) of cultured lung cells in vitro. In this theory, cells can obtain the medium through the basement membrane, ensuring the in vitro survival rate of the cells, and direct exposure to the top and gas (including the aerosol of the test substance).
Figure 2: In vitro exposure pattern of gas-liquid interface
2.1 The first generation of gas-liquid interface cell exposure device CULTEX: In 1999, according to Voisin et al. and Tarkington et al. on the concept of dynamic exposure, Dr. Ulrich Mohr, Dr. Michaela, Professor of Toxicology, Hannover Medical School, Germany Aufderheide et al. successfully developed the first generation of in vitro cell culture and exposed devices, namely the linear flow-based in vitro exposure module Cultex CG (Figure 3).
Figure 3 CULTEX CG (Linear Flow External Exposure System)
The first generation of gas-liquid interface in vitro exposure to the system is characterized by a linear flow distribution of the gas path. The main structure has two modules, the two modules form a tight closed system. The lower module is a cell culture part, the laboratory cell culture chamber is placed therein, the bottom of the chamber is provided with a 37 ° C water bath and a flowable medium supply; the upper part is a linear gas inlet and outlet module corresponding to the culture chamber, which is connected through the back end. The air pump realizes the negative pressure in the closed chamber, and the exposed substances are not discharged into the closed system to complete the exposure and the poison is discharged from the rear end. A good cell culture environment is formed in the cell culture chamber, and the cells can be directly contacted with the gas coming in from the top to achieve direct gas-liquid interface exposure.
2.2 Second-generation gas-liquid interface cell exposure device CULTEX RFS:
When the test object is a gas, whether the gas path design is linear or not is not important because the gas distribution is uniform. However, when the test object is a particulate aerosol, its linear direction transport will have a very large influence on the deposition of particulate matter due to its complex particle size distribution and physicochemical properties. (1) The concentration of particles and the distribution of particle size have an effect on the stability of the dilution system; (2) the concentration and particle size of the particles deposited on different cell compartments will change, resulting in the deposition of particles in different cell compartments. Parallelism and repeatability are poor. Obviously, the dilution of the intake air of the compressed air is unable to break the laminar flow distribution of the test substance, which will not change the deposition effect as described above.
In view of these shortcomings, the cell culture chamber and aerosol inlet distribution need to be fundamentally changed. In order to avoid the unevenness of the exposed particles, Cultex introduced a new concept of the second generation of exposed poisoning module Cultex RFS (Figure 4, Figure 5).
Figure 4 CULTEX RFS appearance Figure 5 CULTEX RFS internal gas diagram
Here, the aerosol will enter through a central inlet with a nozzle (Mohr, 2013) where the aerosol will be evenly dispersed into the channels of the three central radiation distributions, through which the cells enter the radiation-distributing cell culture chamber. After such a process, the uniformity and repetition of aerosol deposition in the three cell compartments will be greatly enhanced. The pictures and data results after deposition of different particles in this experiment showed very good results, and the particle deposition deviation and standard deviation between the three cell compartments were very satisfactory. Compared with the first-generation linear flow system, we found that the second-generation radiation flow exposed the poison module lost some of the particles, that is, the total mass of the deposited particles in each cell was reduced. This is due to the fact that we have added large particle (coagulated particles) pre-separation technology in the Cultex RFS module, which reduces the possibility of deposition of large or agglomerated particles on the cell surface, ie provides uniformity of particle size distribution, allowing experiments The test substance is more stable, which not only reduces the influence of coarse particles on the experiment, but also further improves the experimental repeatability and stability.
2.3 Third-generation gas-liquid interface cell exposure device CULTEX RFS COMPACT:
Figure 6 COMPACT structure diagram
Gas-liquid interface cell exposure exposure to the second generation technology CULTEX RFS, has a very high experimental repeatability and stability, but there are still some problems in practical applications, such as fewer channels, only 3 exposure channels, And all channels exposed the same aerosol material, lacking the control experimental group. To this end, CULTEX Labs Technologies of Germany introduced the third generation of gas-liquid interface cell exposure device CULTEX RFS COMPACT (see Figure 6). COMPACT inherits the main advantages of CULTEX RFS radiation flow design and increases cell exposure. The number of chambers reached 6 and was divided into two groups for comparative experiments. At the same time, the optimization reduces the amount of cell culture medium added to each chamber, and the overall use of electric heating thermostat system is more flexible and convenient, and is easy to conduct outdoor atmospheric test.
This is the development of the gas-liquid interface cell exposure technology. In the early stage, mainly cell lines (such as A549) and primary cells (such as macrophages) were used in the dose-response relationship in the field of acute inhalation toxicology. set up. Over time, in vitro exposure to the system has become more and more complex, and can now be used for in vitro exposure to 3D cells differentiated from different parts of the respiratory tract, especially the recent Insert Cup device introduced by CULTEX. Cell co-culture models can be well established in vitro. The development of CULTEX's complex in vitro exposure system has also opened up new avenues for toxicology testing strategies. In addition to the establishment of stable verification methods in the field of acute inhalation toxicology, repeated repeated in vitro exposure concepts, especially non-toxic dose experiments, have been carried out in the field of subacute trials.
At present, the main interest of in vitro exposure technology is to simulate the biological effects in human body in vitro and to carry out in vitro exposure and exposure experiments of various specified experimental substances.
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