Field of invention This invention relates to an exposure device using thermophoresis, a method for testing aerosols and/or gases and a method for toxicological testing of aerosols/gases.

Background

Exposure of viable cells to particles and gases in aerosols can be examined using air-liquid interface (ALI) technique accompanied with electrostatic, thermophoretic or other deposition mechanism of the particles. Thermophoresis is based on a temperature gradient between two opposing surfaces resulting in migration of aerosol particles towards the colder surface.

Examples of prior art methods are based on droplet formation with aerosol nebu- lizer and sedimentation of the particles or droplets onto the cell culture by gravitation and an exposure device, based on thermophoresis (prit-systems.de) where the aerosol sample is directed towards the cell culture, which serves as the deposition surface. There are also some exposure devices available without any deposition improvement techniques, e.g. original Vitrocell and Cultex. They are based simply on gravitation and natural diffusion of the particles.

Comouth et al. (2013) describes an electrostatic method and device for particle deposition for cell exposure at air-liquid interphase. The aerosol flow is directed vertically to the cell surface in cell culture inserts.

ΒΓθββΙΙ et al. (2013) describes a thermophoretic device for testing of airborne and inhalable substances in cells at the air-liquid interface. Said device comprises two parallel metal plates forming a longitudal channel for gas flow. The temperature is regulated using semiconductive elements. The top plate serving as a cold plate is equipped with two cavities for culture inserts, front insert for receiving the particles and rear insert for assaying the effect particle free gas. Prior art ALI devices and methods have at least some of the drawbacks relating to deposition efficiency, uniformity of deposition, measurement of the exposure dosage and possibilities to select parameters without compromising the well-being and viability of the target cells. Thus, there is a need for improved devices and method that at least reduce these problems.

Summary of the invention

An object of this invention is to provide novel device and method for use in in vitro analyses of aerosols and/or gases, and especially for particles in aerosols.

These and other objects are achieved by the present invention as described and claimed below.

The first aspect of the invention is an exposure device using thermophoresis as a deposition mechanism for in vitro aerosol exposure. Characteristic features of said device are given in claim 1 .

The second aspect of the invention is a method for in vitro aerosol exposure. Characteristic features of said device are given in claim 6.

The third aspect of the invention is a method for testing of aerosol in vitro. Characteristic features of said device are given in claim 9. Brief description of the drawings

Figure 1 shows one embodiment of the device according to this invention. The device comprises a base module (1 ) having at least one duct for aerosol flow (4); a middle module (2) having at least one cavity (5) arranged to receive a culture insert (6); and a top module (3). Figure 2 shows another embodiment of the device according to this invention.

Figure 3 shows a SEM image (magnitude 5000 X) before and after deposition on foil.

Figure 4 is a graphical view showing a feed Ag particles; Concentration~1 e5 number of particles/cm3; Diameter -50-400 nm Ag particles, Number Size Distri- bution of the test aerosol Ag-particles. Measured with SMPS-Device.

Figure 5 shows a SEM image (magnitude 2000 X) before and after deposition on foil replacing the cell culture.

Figure 6 Comparison of observed (SEM) number size distribution and predicted by calculation from SMPS measurement results number concentration -70 % of predicted (SMPS) number concentration were found from the foil. Detailed description of the invention

The inventors have surprisingly found that thermophoretic deposition methods for living cell cultures at air-liquid interphase can be improved by optimizing the prior art devices and especially by controlling the temperature of the device including the insert carrying the target cells. Requirements of the deposition are dependent on the aerosol to be tested. Deposition of representative sample of particles requires sufficient aerosol flow, exposure time and temperature gradient. These typically cause remarkable stress to the target cells and thus may compromise the sensitivity and reliability of the test. Prior art methods have compromised means for controlling the temperature and humidity conditions inside the device as well as the stress caused by airflow. Optimal relative humidity for target cells is close to 100%. However, no condensation of water should issue. The device and method according to the present invention optimizes both the particle deposition and the viability of the target cells. The device using thermophoresis according to the present invention is suitable for in vitro aerosol and/or gas exposure. The device comprises

- a base module having at least one duct for aerosol/gas flow extending along the longitudal axis and means for controlling the temperature of said module and optionally an insulation layer facing the middle module; and - a middle module having at least one cavity arranged to receive a culture insert positioned so that the bottom of said insert is in parallel position with the duct for aerosol/gas flow and means for controlling the temperature of said module; and

- a top module for closing the culture inserts, wherein the top module is equipped with means for controlling the temperature of said culture insert when positioned into said insert.

When compared to diffusion based or other methods thermophoresis based deposition for particles smaller than 1000 nm is less dependent on size and density of the particles to be deposited. Thermophoresis allows easier calculation of the de- posited dose from the number size distribution results measured (e.g. by SMPS) prior entering the device. The device and the method of the present invention is suitable for deposition of particles having average diameter of 1200 nm or less, preferably 1000 nm or less, more preferably 800 nm or less, still more preferably 500 nm or less. The device and the method are especially suitable for deposition particles of nano size having an average diameter of 100 nm or less, down to 1 nm and even smaller.

Said means for controlling the temperature of said culture insert are useful in avoiding temperature differences within the insert and the cell culture. Any 'cold spots' in device and especially in the cell culture may result in water condensation being harmful to living cells. In addition, any changes in optimal relative humidity of the gas or aerosol in the device or optimal temperature of the cell layer have negative effect to living cells and cause noise into the toxicological analysis. Liquid circulation is one preferred method for controlling the temperature as the e.g. volume of the circulating liquid makes it possible to maintain accurate temperature over the desired period of time.

Said cavity arranged to receive a culture insert positioned so that the bottom of said insert is in parallel position with the duct for aerosol makes is possible to use parallel airflow in relation to the surface of the cell culture. Thus, airflow in the sen- sitive surface of the cell culture is close to zero in the case of laminar flow and does not stress the cells.

The temperature of the top module and middle module should be suitable for the target cells. Temperature can be chosen to be optimal for the target cells and chosen toxicological method; e.g. for human cells a temperature of 36 to 38 °C, pref- erably about 37 °C is suitable. For the bottom module, a higher, controllable temperature can be chosen of about 45 to 65 °C, preferably 50 to 60 °C in order to establish optimal temperature gradient for deposition by thermophoresis.

In one embodiment, the means for controlling the temperature are arranged by liquid circulation embedded within the base module or middle module or top mod- ule or in any combinations thereof. This allows maintaining any targeted, stable temperature over the exposure time and on overall surfaces of the duct for aerosol flow and cavities for receiving the culture insert. This reduces the risk of water condensation. Controlled and optimized temperature of the target cells and relative humidity of aerosol or gas enhances the well-being and viability of the cells and allows variations in temperature gradient, exposure time and flow rate without compromising the viability of the cells.

In one embodiment of the invention the duct for aerosol flow is formed by a thermal insulating layer (e.g. teflon) positioned on the base module on the side facing the middle module. This allows easily changing the dimensions (width, depth, length, inlet angle etc.) and number of the aerosol ducts without need for dressing the metal surface. This increases flexibility of the system allowing variations in e.g. aerosol flow and temperature gradient without compromising the viability of the cells. The duct(s) must be gas tight and thus usually it is necessary to use some locating means such as locating pins or screws, especially when formed using the insulation layer.

In one embodiment the device comprises two, three, four, five or more parallel ducts allowing parallel exposures by the same aerosol using similar or different target cells.

General interaction and effectiveness of various forces acting on particles and their deposition in said device follow the rules outstandingly explained in example in Optimisation of a thermophoretic personal sampler for nanoparticle exposure studies by Azong-Wara et al. J. Nanopart Res. (2009) 1 1 :161 1 -1624.

In practice, the airflow on the outer bottom surface of the culture insert due to laminar flow is minimal and very gentle to the living cells thereby maintaining the via- bility of cells.

The means for controlling the temperature of the culture insert can be a plug adapted to the size of the cavity in said culture insert and equipped with a liquid circulation. Cavities for liquid circulation are connected to a temperature controlling unit. A plug as means for controlling the temperature allows maintaining an optimal temperature also in inserts with different sizes.

In one embodiment the means of controlling temperature (e.g. in form of a plug) is submerged in the culture medium in the insert This further enhances maintenance of the optimal temperature and well-being of the target cells, which minimizes the stress to the cells caused by the temperature and reduces risk of condensation of water on cells. The material for the means of controlling the temperature should be selected as heat conducting material as possible and non-reactive with the chosen cell culture media. A person skilled in the art is able select the heat conducting material so that it complies with the medium used. For example stainless steel has been found compatible with culture medium for human lung cells. Submerged means for controlling the temperature can be equipped with means for circulation or renewal of culture medium. The means for circulation can be tubes forming a liquid connection between the medium in the plug and a medium reservoir. Preferably, said reservoir is heat regulated. This further improves possibilities of providing the cell culture with fresh medium having an optimal temperature thereby allowing longer exposure time, preventing lack of nutrients and/or changing pH

The device can also comprise or be combined with various means for controlling the aerosol flow, valves for aerosol inlet and/or outlet before/after each duct for aerosol flow, means for controlling the temperature of the aerosol feed, means for controlling the humidity of the aerosol feed etc. Said means are known within the art and a person skilled in the art is able to combine them with the device according to the invention.

In one embodiment the device comprises also means for taking samples from the gas or aerosol feed located before the duct for aerosol flow.

The device can also comprise or be combined with various known means for measuring humidity, temperature or flow of the aerosol and adjusting volume of the airflow.

In one embodiment the device is connected to a device for adjusting the relative humidity of the aerosol before it is introduced into the device according to the invention. In one embodiment, the gas or aerosol is humidified until essentially 100% relative humidity.

In one embodiment, the device is connected with a control device and/or a data collection unit connected to a data processing unit. In one embodiment the top module is equipped with means for control closing the cavity (cavities) arranged to receive a culture insert. This protects the microporous membrane on the bottom of the insert and the target cells cultured on it.

The culture insert to be positioned to cavities of the middle module may be a commercial insert having a container for a culture medium and a microporous bot- torn allowing culturing the cells on the outer surface of the bottom. Culture medium located inside the insert provides nutrients to the cell culture and forms a thin film covering the culture. Examples of suitable commercial product are e.g. Corning Falcon® inserts, Millipore Millicell®, Thermo Nunc™ inserts, Greiner ThinCert™ inserts and Corning Transwell® inserts. It is possible to use fitting rings between the cavity and the insert. This allows easy exposure using an air-liquid interface without contact of the aerosol and the culture medium. In addition, the volume of the culture medium is relatively large allowing temperature control by the top module equipped with means for controlling the temperature of said culture insert when positioned into said insert. Medium separate from the target cells can also be circulated.

This method (as all ALI methods) is especially suitable for the cell types that attach to the membrane inserts, i.e., e.g. epithelial cells, endothelial cells and differentiated macrophages or other attaching immunological cells.

Also a method for testing of aerosol in vitro using thermophoresis is within the scope of this invention. The method comprises the steps of a. providing a duct for aerosol flow (4) extending along the longitudal axis of a base module (1 ) and a middle module (2);

b. creating a temperature gradient between said base module and said middle module;

c. positioning a culture insert (6) carrying living cells to a cavity (5) of the middle module so that the bottom of said insert is in parallel position with the duct for aerosol flow; and

d. maintaining optimal temperature for the cell culture medium by regulation the temperature of said culture insert to equal the temperature of said middle module; and

e. exposing said culture inserts to a lateral aerosol flow in duct for aerosol flow formed in step (a).

Temperature gradient results in deposition of particles of the aerosol (or gas) to the culture on insert bottom surface.

Also a method for testing of aerosol in vitro is within the scope of this invention. The method comprises the steps of a. providing a duct for aerosol flow (4) extending along the longitudal axis of a base module (1 ) and a middle module (2);

b. creating a temperature gradient between said base module and said middle module;

c. positioning a culture insert (6) carrying living cells to a cavity (5) of the middle module so that the bottom of said insert is in parallel position with the duct for aerosol flow; and

d. maintaining optimal temperature for the cell culture medium by regulation the temperature of said culture insert to equal the temperature of said middle module; and e. exposing said culture inserts to a lateral aerosol flow in duct for aerosol flow formed in step (a); and

f. after exposure of step (e) subjecting the cell culture to toxicological test to analyze the effect of aerosol to viability of the cell culture.

Temperature gradient results in deposition of particles of the aerosol to the culture on insert bottom surface. Toxicological analysis can be performed thereafter. As evident to a skilled person, a result of the exposure should be estimated in light of suitable controls; e.g. exposure with clean air in similar conditions.

Reduced viability or other markers indicating reduced well-being of the cell culture after the exposure, when compared to a suitable control exposure e.g. using clean air, indicates that the aerosol comprises particles and gases, which are harmful or toxic to said cell type. The device and the methods of the present invention pro- vides good living conditions to the cell culture during the exposure thereby reducing the noise in toxicological tests after the exposure. E.g. for human lung epithelial cell cultures a temperature about 37 °C and relative humidity close to 100 % is suitable. In one embodiment more than one culture insert is used and the temperature gradient and the aerosol/gas flow can be set so that particles are deposited before the last (from the aerosol/gas inlet) insert. In this embodiment said last insert serves as a particle fee control culture in exactly same exposure conditions as the culture^) wherein the particles are deposited as explained in detail by Brossell et al. 2013. In another embodiment the particle deposition is distributed equally within the culture inserts. A person skilled in the art is able to select suitable parameters.

In one embodiment the methods of the invention further comprise a step of adjusting the relative humidity of the aerosol/gas before step (e). This enhances mainte- nance of even humidity during the exposure.

In one embodiment the aerosol/gas flow is chosen so that flow is laminar meaning that the well-known Reynolds number of sample aerosol/gas in the said duct is <1000, more preferably below 800 and most preferably below 500.

The method is suitable for testing particles having an average diameter below 1200 nm, preferably below 1000 nm, more preferably below 500 nm and most preferably below 100 nm. Respectively the method is suitable for particles having average diameter above 1 nm ore even smaller particles.

Preferably, the device described here is used for the methods. Advantages dis- cusses in connection of said device apply also to the method here described.

Description of an embodiment with references to drawings

Reference is now made to Figures, which show various embodiments of the device. Figures 1 and 2 show two graphical views of embodiments of the invention. Both figures show a base module (1 ) having at least one duct for aerosol/gas flow (4); a middle module (2) having at least one cavity (5) arranged to receive a culture insert (6), a top module (3) and the means for controlling the temperature of said culture insert (7). Preferred means for controlling the temperature is means for circulating heated liquid. Figure 2 shows an upper view of the means for con- trolling the temperature of said culture insert; in this embodiment the means are arranged as a liquid circulation and the ducts for liquid are shown as feature (7b). In embodiments shown, the means for controlling the temperature of the modules are arranged using a liquid circulation; ducts (connecting the heating device and forming the liquid circulation) for said liquid are embedded inside the modules but the inlet and outlet connections for liquid are shown (8).

The gas or aerosol/gas to be tested is introduced to, and removed from the duct for aerosol/gas flow (4) via aerosol/gas inlet and outlet connections (9).

One optional embodiment of the invention comprises a gas connection (10) for feeding the aerosol/gas to the duct. Said connection allows taking samples of the aerosol/gas as it is while introduced to the duct for aerosol/gas flow (4). Sampling ports are shown as (13). Optional insulating layer (12) is shown in Figure 1 . It makes it easier to create desired temperature gradient and reduces temperature changes and especially possible cold spots in the modules. Another optional embodiment of the invention shown in Figure 2 comprises a means to control closing the cavity for receiving insert(s) (1 1 ).

It is to be understood that the terminology employed herein is for the purpose of description and should not be regarded as limiting.

The features of the invention described here as separate embodiments may also be provided in combination in a single embodiment. Also various features of the invention described here in the context of a single embodiment, may also be pro- vided separately or in any suitable sub-combination. It should be understood, that the embodiments given in the description above are for illustrative purposes only, and that various changes and modifications are possible within the scope of the disclosure. The invention is illustrated by the following non-limiting examples. It should be understood that the embodiments given in the description above and the examples are for illustrative purposes only, and that various changes and modifications are possible within the scope of the invention.

Examples

Example 1 : Deposition test on foil

Silver particles for particle deposition tests were produced with well-known method, described e.g. Lehtinen et al. Nuclear Engineering and Design 213 (2002) 67- 77. Aluminum foil was placed on the (outer surface of) the bottom of the insert. Produced test aerosol number size distribution was measured with SMPS (Scanning Mobility Particle Sizer) before entering aerosol conditioning for humidification. Measured aerosol number size distribution was used to calculate predicted particle deposition at the insert surface equipped with aluminum foil for later SEM (Scan- ning Electron Microscopy) analysis. Observed number size distribution from SEM analysis was compared to that of calculated from the SMPS measurement. Detected particle number size deposition (SEM) agreed more than 70% of predicted by calculations from SMPS results. Outcome is good confirming wanted operation of the device. Particle losses at the humidification were not taken to account in prediction calculations. The detection of particle number size distribution measured by SMPS is based on electrical mobility of particles hence causing error when compared to the results observed by SEM. Results of the test are shown in Figures 3 to 6.

Example 2: Preparation of cell cultures and cell insert Epithelial cells (A549) are maintained in flasks in cell line specific medium (DMEM), supplemented with L-Glutamine, FBS and antibiotics. The maintenance culture of the cells was performed usually on Mondays and Fridays, with a medium chance on Wednesday.

On day 1 of the membrane culture of the cells for exposures, the microporous membrane inserts (BD Falcon ^® ) were inverted and 1 ml of the cell suspension was added on the membrane. After attachment period (1 h) of the cells, the inserts were turned around, placed on the support plates and the compartments were filled with cell culture medium (DMEM). Thus the cells grow on outer surface of the inserts, facing downwards. On day 4, the cell culture medium was changed on the upper compartment and the medium was removed from the lower compartment to build an air-liquid-interface. On day 5 to 7 the cells were used in the exposures to Ag nanoparticles, described in the example 1 . The cell density used in the preparation of the inserts, depended on the day of the exposure. After exposing the cells e.g. 1 h in the exposure device, the culture inserts were moved on support plates and new medium was changed in the inserts and the cells were allowed to recover until the next day. The toxicological parameters were measured 24 h after ending the exposures. The method is suitable for analysis of various cell viability parameters as well as for measuring production of inflammatory mediators.

The experiments with clean air showed that cell viability remained at an excellent level -95% compared to incubator control. In the experiments with Ag nanoparti- cles a reduction in cell viability was observed. The exposure device has also been tested with emission aerosols from car engine and wood combustion. The exposure device has shown to be suitable for such experiments.