WO2023117841A1

WO2023117841A1 - Aerosol deposition device

Info

Publication number: WO2023117841A1
Application number: PCT/EP2022/086551
Authority: WO
Inventors: Arno GUTLEB; Tommaso SERCHI; Elisa MOSCHINI; Servane CONTAL; Aline CHARY; Melissa SAIBENE; Tobias Krebs
Original assignee: Luxembourg Institute Of Science And Technology (List)
Priority date: 2021-12-21
Filing date: 2022-12-19
Publication date: 2023-06-29
Also published as: LU501054B1

Abstract

The invention is directed to a device (1) for applying an aerosol (22) on a biological system (10), preferably cells maintained in air-liquid-interface conditions, the device (1) comprising: a casing (18) delimiting an exposure chamber (20) filled with an aerosol (22); a plurality of cup-like inserts (6) with a microporous membrane receiving cells, the inserts (6) being arranged in wells (4.1) of a multi-well plate (4), the inserts (6) being partially immersed in liquid (5); and a cover (16) adapted to cover the peripheral gaps (8) between the inserts (6) and the well (4.1), the cover (16) being provided with openings (16.1) enabling the aerosol (22) to flow down onto the cells (10) lying on each respective membrane (6.1).

Description

AEROSOL DEPOSITION DEVICE

Technical field

[0001] The invention is directed to a device and a method for applying an aerosol to a biological system and in particular for applying an aerosol to cells maintained under air-liquid-interface conditions.

Background art

[0002] Prior art patent document WO 2015/027998 A1 discloses a device for sucking down an aerosol on cells. The cells are placed in a container and the container is positioned in a stainless-steel block. An aerosol is sucked down onto the cells by a vacuum source and the aerosol is thus impacted onto the cells. Such a device requires a complex set-up and complex postexposure operations, which prevent it from being used at a large or commercial scale. This device also results in an extremely low deposition efficiency as only between 0.1 and 1 % of the aerosol reaches the cells. This device requires a long exposure time and presents a high risk of damaging the cellular layer due to the continuous exposure to the air stream.

[0003] Another example of a device for aerosol deposition is given in WO 2014/147229 A1 . In this device, the inserts with the cells are inserted and taken out of the aerosol chamber one by one. This device entails many drawbacks such as a risk of chemical carry-over or of biological crosscontamination. The handling of the samples is cumbersome and this device does not propose a reliable way of deposing aerosol only on the apical side of the cells’ container.

Summary of invention

Technical Problem

[0004] The invention aims at providing a device for applying an aerosol on a biological system without the drawbacks of the known devices. In particular, the invention seeks to provide an efficient and reliable device, ensuring a faster handling of the biological system and of the parts of the device, and/or ensuring that any chemical/biological cross-contamination or carry over is prevented.

Technical solution

[0005] The invention is directed to a device for applying an aerosol on a biological system, the device comprising: a base provided with a housing; a casing, removably supported by the base and delimiting an exposure chamber therein, the casing being provided with an inlet for introducing an aerosol into the exposure chamber; a multi-well plate adapted to be removably introduced in the housing of the base and having a plurality of wells configured for receiving a liquid, the wells being delimited by respective well walls; a plurality of cup-like inserts, each having a peripheral wall and a bottom in the form of a microporous membrane for receiving a biological system, each insert being adapted to be positioned in a respective well so that the peripheral wall is partially immersed in the liquid, a peripheral gap being defined between the peripheral wall of the insert and the respective well wall; and a cover adapted to lie on the base and to cover the peripheral gaps, the cover being provided with openings enabling the aerosol to flow down onto the biological system lying on each respective membrane.

[0006] The biological system is preferably made of cells.

[0007] The casing enables to define an exposure chamber which can be occupied by an aerosol that will flow down onto the cells of several inserts at once.

[0008] The cells positioned in the inserts are exposed to the aerosol on the apical compartment of the insert and cells may be exposed to the liquid on the basolateral compartment of the insert. By covering the gap, the cover enables to avoid the basolateral side to be reached by the aerosol.

[0009] The preferred embodiments below further improve the efficiency and reliability of the process to various degrees.

[0010] According to a preferred embodiment, the cover contains generally circular protrusions protruding into the inserts and contacting the peripheral wall of the inserts, preferably at the inner side of the peripheral wall. According to a preferred embodiment, the contact between the protrusions and the inserts centres the inserts with respect to the openings. This facilitates the introduction of the inserts into the wells as they do not need to be manually centred. A manual centring would indeed increase the handling and thus the risk of biological contaminations. The inserts are indeed aligned all at once and without the need of any further manual handling when positioning the cover down on the inserts. This quickens the handling of the inserts and improves both the efficiency and reliability of the whole process of deposition, by facilitating the handling and ensuring a proper positioning of the cells. The cover and the inserts are neither permanently attached to one another nor stuck together, but they are merely in contact with each other through at least one point of contact at a given moment in time.

[0011] According to a preferred embodiment, the well walls have a respective top circular edge whereon the inserts lay or hang, preferably through a plurality of fingers. The contact between the inserts and the multi-well plate is therefore such that the inserts can be introduced and removed by hand or with a tweezer.

[0012] According to a preferred embodiment, the inserts are adapted to be loosely engaged in the respective wells. This again enables a simple and fast introduction of the inserts in the well. It also allows various sizes of inserts to be used with the same multi-well plate (simultaneously or sequentially).

[0013] According to a preferred embodiment, the cover is made of a biocompatible material. By providing such a disposable cover, the cross-contamination between two successive experiments is prevented and the need to clean the cover is waived, rendering the exposure process more reliable and more efficient.

[0014] According to a preferred embodiment, the cover is made of a material that is resistant to chemicals and autoclavable.

[0015] According to a preferred embodiment, the cover, the multi-well plate and the inserts are removable as a group from the base. This enables to leave the inserts in the wells and to carry them as a group to the post-exposure incubation, thereby preventing cross-contamination (as would be the case when removing each insert one after the other). Also, this enables to prepare an experiment by inserting the inserts in a multi-well plate and sealing the wells with the original cover, proper of the multi-well plate as individually provided, aside from the device, while another multi-well plate and other inserts are being processed in the chamber, improving thereby both reliability of the experiments and time efficiency. An additional lid (without holes) may be used in addition or in replacement with the cover, when handling the inserts and multi-well plate from and into the incubator. The additional lid covers the wells and inserts and prevents any contamination when handling the biological system.

[0016] According to a preferred embodiment, the base has a generally rectangular cross-section provided with two lateral extensions on the longest sides of the rectangular cross-section and the multi-well plate has a rectangular cross-section, thereby creating two recesses between the plate and the base enabling the removal by hand of the multi-well plate from the housing. According to a preferred embodiment, the base comprises external elastic tabs for a snap fit assembling of the casing on the base. Those aspects constitute further improvements for handling the device more efficiently.

[0017] According to a preferred embodiment, the base integrates heating means. As some applications require to maintain the cells at a pre-determined temperature (e.g., body temperature), integrating the heating means within the base enables a biologically and chemically non-intrusive heating and trustworthy temperature monitoring.

[0018] The invention also relates to a method for applying an aerosol on cells, optionally with a device in accordance with any of the embodiments discussed above, the method comprising the steps of: placing cells on the microporous membrane-like bottom of a plurality of inserts; placing the inserts in respective wells of a multi-well plate, thereby defining an apical compartment and a basolateral compartment, partially immersed in liquid; covering the inserts and the multi-well plate with a cover, the cover being provided with openings exposing the apical compartment of the inserts while sealing away the basolateral compartment; and flowing down an aerosol through the openings and onto the cells.

[0019] This method enables a reliable exposure of the cells. [0020] According to a preferred embodiment, after the step of covering and before the step of flowing, the multi-well plate, the inserts and the cover are introduced in a housing of a base of a cell cultivation device and a casing is positioned on the base to seal an exposure chamber. Processing time and reliability of the handling can thus be further improved.

Advantages of the invention

[0021] The invention is particularly interesting in that it enables a separation between the apical compartment and the basolateral compartment so that the aerosol is prevented from reaching the basolateral compartment. Thus, only the cells in apical compartment are directly exposed to the aerosol. This is particularly relevant for experiments aiming at investigated substances whose mechanism of action does not imply direct exposure of the basolateral compartment (e.g., inhaled chemicals). This allows obtaining more reliable exposure and assessment conditions and thus preventing false positive and/or false negative results.

[0022] Various aspects of the invention participate in the reliability or the reduction of the risks of biological/chemical cross-contamination or chemical carry over, the need for post-exposure clean-up operations.

Brief description of the drawings

[0023] Figures 1 A and 1 B show respectively a cross-section and a partial top view of a schematic principle of a device of the invention;

[0024] Figure 2 shows an exploded view of an embodiment of the device of the invention;

[0025] Figure 3 shows an embodiment of the device in an assembled condition.

Description of an embodiment

[0026] The drawings show some features of the invention in a schematic way. The figures are not drawn to scale and some dimensions may be enlarged for better understanding of the concepts of the invention.

[0027] Figure 1 A shows a schematic view of part of a device for applying an aerosol sample on a biological system. [0028] The device 1 comprises a base 2 of a generally parallelepipedal shape and provided with a cavity or housing 2.1. At the bottom of the housing 2.1 , a series of protrusions 2.2 can be arranged. The base 2 can be made of stainless steel. It can integrate heating means (e.g., a resistor embedded into the base) to heat up and/or maintain the housing at a pre-determined temperature, such as for example a temperature between 20 and 60°C, and preferably 37°C for experiments on human cells. The base can also integrate control electronics for monitoring the temperature and an adapted interface (e.g., display) for displaying the current temperature and/or for setting a target temperature and/or a heating duration.

[0029] A multi-well plate 4 can be inserted in the housing 2.1 , for instance laying on the protrusions 2.2. The plate 4 may be a standard cell culture plate, containing a plurality of wells 4.1 (e.g., 4, 6, 12, 24, 48, 96, etc.). The wells 4.1 can be filled independently with liquid. The wells 4.1 are delimited by well walls 4.2 which can have a generally cylindrical shape. A generally circular edge 4.3 constitutes the top end of the well walls 4.2.

[0030] In each well, an insert 6 can be introduced. The insert 6 can have a generally cup-like shape. The insert 6 has a microporous membrane 6.1 as bottom (for example with pores comprised between 0.4 and 10 pm) and has a peripheral wall 6.2 which can be cylindrical or conical. At the top of the peripheral wall 6.2, a ring (not shown) or alternatively fingers 6.3 can be arranged, so that the insert 6 hangs in the well, supported by the circular edge 4.3.

[0031] The peripheral wall 6.2 and the well wall 4.2 can have any other regular or irregular geometric cross-section (e.g., polygon, etc.).

[0032] A gap 8 between the peripheral wall 6.2 and the well wall 4.2 facilitates the introduction and removal of the insert 6 into/from the well 4.1 (for instance with a tweezer). Various sizes of inserts 6 can be used with the same well plate 4.

[0033] Figure 1 B shows a partial top view of the device 1 , illustrating two exemplary inserts 6 in their respective wells 4.1 . Various numbers of fingers 6.3 can be foreseen. Between the well wall 4.2 and the inserts’ walls 6.2 lies the gap 8. [0034] Back in reference to figure 1A, cells 10 can be deposited on the membrane 6.1. An apical compartment 12 stands above the cells 10 and a basolateral compartment 14 lies below and around the insert 6. The basolateral compartment 14 is maintained immersed in the liquid 5, while the apical compartment 12 is not immersed.

[0035] To maintain the apical compartment 12 sealed away from the basolateral compartment 14, a cover 16 is provided. The cover 16 lays on the base 2 and/or on the multi-well plate 4 and/or on the inserts 6. The cover 16 has a generally plate-like shape with a plurality of openings 16.1. The cover 16 covers the gaps 8 and can be provided with protrusions 16.2 which extend downwards into contact with the inserts 6. The outline of the protrusions 16.2 matches the outline of the peripheral wall 6.2 of the inserts 6. The protrusions 16.2 help to seal the apical compartment 12 from the basolateral compartment 14. The protrusions 16.2 also help centring the inserts 6 with respect to the respective opening 16.1 .

[0036] The cover 16 may be made of a biocompatible material so as to be disposable (e.g., biocompatible polymer such as polyethylene, ABS, polycarbonate, etc.). The cover 16 may be chemically resistant and autoclavable. The cover may be manufactured by injection moulding to limit the costs involved and facilitate mass production.

[0037] The cover 16, the multi-well plate 4 and the inserts 6 are removable as a group from the base.

[0038] Above the base 2, a 5-sides box-like casing 18 can be arranged. The casing 18 can be made of any material, as for example, polycarbonate, plexiglass, glass, acrylic or polypropylene. The casing 18 delimits a cavity therein, i.e., the exposure chamber 20. An aerosol 22 can be formed in the exposure chamber 20. The aerosol 22 may be of any nature. For example, a substance that is to be tested on the cells 10 can be diluted in water solution with salt. A nebulizer (not shown), e.g., a high-voltage membrane arranged horizontally, can nebulize the solution to form a thick cloud that distributes evenly in the exposure chamber 20, before settling down onto the cells 10. Various technologies for the generation of the aerosol can be envisaged, such as those well-detailed in WO 2014/147229 A1 . [0039] The openings 16.1 allow the aerosol 22 to flow down onto the cells 10, while the design of the cover 16, of the inserts 6 and of the multi-well plate 4 prevent the aerosol 22 from reaching the basolateral compartment 14.

[0040] Figure 2 illustrates an exploded view of the various parts of an embodiment of the device 1. The same numbers as in figure 1 are used to depict the same parts.

[0041 ] One can see that the base 2 comprises two lateral extensions 2.3 which can form a recess between the plate 4 and the base 2. These recesses enable an easy insertion and/or removal of the plate 4 by hand. The base 2 can furthermore have ribs or chamfers to adequately position the plate 4 laterally (in the horizontal plane). A user interface 2.4 (display, buttons, alarm, etc.) can be provided, preferably on one of the extensions 2.3. This interface 2.4 is connected to electronics controlling the temperature of the base 2 through sensors and heating means.

[0042] Figure 2 also shows the cover 16 with the protrusions 16.2 (upside down view on the right-hand side) and a peripheral lip 16.3 which can enhance the sealing of the aerosol away from the multi-well plate 4, or the positioning of the cover 16: the plate 4 can be in contact with the inner side of the lip 16.3 and/or the base 2 can be in contact with the outer side of the lip 16.3.

[0043] The casing 18 exhibits an opening 18.1 , preferably at the top of the casing 18, serving as an inlet for the aerosol.

[0044] Figure 3 shows the device in an assembled condition. The base 2 is adapted for a snap-fit engagement with the casing 18, potentially with elastic tabs (not shown). Other assembling design can also be provided (flanges, screws, quick-lock, etc.).

[0045] The present invention can be used in an in vitro model of a human lung for assessing the irritation potential, the sensitization or toxicity of inhalable products such as particles or molecules on the alveolar barrier of lungs (see e.g., WO 2018/122219 A1 ). For that purpose, epithelial cells and macrophages can be present at the apical side of the membrane, and endothelial cells and dendritic-like cells are present at the basal side of the membrane, immersed in a co-culture medium. [0046] The device and method can however be used for any other application where an aerosol is to be deposited reliably on a biological system.

[0047] In an example that is not illustrated, a discharging device lets a substance fall onto the biological system. For example, needle printing can be used instead of a chamber discharging an aerosol.

Claims

1 . A device (1 ) for applying an aerosol (22) on a biological system (10), the device (1 ) comprising:

- a base (2) provided with a housing (2.1 );

- a casing (18), removably supported by the base (2) and delimiting an exposure chamber (20) therein, the casing (18) being provided with an inlet (18.1 ) for introducing an aerosol (22) into the exposure chamber (20);

- a multi-well plate (4) adapted to be removably introduced in the housing (2.1 ) of the base (2) and having a plurality of wells (4.1 ) configured for receiving a liquid (5), the wells (4.1 ) being delimited by respective well walls (4.2);

- a plurality of cup-like inserts (6), each having a peripheral wall (6.2) and a bottom (6.1 ) in the form of a microporous membrane for receiving a biological system (10), each insert (6) being adapted to be positioned in a respective well

(4.1 ) so that the peripheral wall (6.2) is partially immersed in the liquid (5), a peripheral gap (8) being defined between the peripheral wall (6.2) of the insert (6) and the respective well wall (4.2); and

- a cover (16) adapted to lie on the base (2) and to cover the peripheral gaps (8), the cover (16) being provided with openings (16.1 ) enabling the aerosol (22) to flow down onto the biological system (10) lying on each respective membrane

(6.1 ).

2. Device (1 ) according to claim 1 , characterized in that the cover (16) contains generally circular protrusions (16.2) protruding into the inserts (6) and contacting the peripheral wall (6.2) of the inserts (6), preferably at the inner side (12) of the peripheral wall (6.2).

3. Device (1 ) according to claim 2, characterized in that the contact between the protrusions (16.2) and the inserts (6) centres the inserts (6) with respect to the openings (16.1 ).

4. Device (1 ) according to any of the preceding claims, characterized in that the well walls (4.2) have a respective top circular edge (4.3) whereon the inserts (6) lay or hang, preferably through a plurality of fingers (6.3). Device (1) according to any of the preceding claims, characterized in that the inserts (6) are adapted to be loosely engaged in the respective well (4.1 ). Device (1) according to any of the preceding claims, characterized in that the cover (16) is made of a biocompatible material. Device (1) according to any of the preceding claims, characterized in that the cover (16) is made of a material that is resistant to chemicals and autoclavable. Device (1) according to any of the preceding claims, characterized in that the cover (16), the multi-well plate (4) and the inserts (6) are removable as a group from the base (2), potentially with an additional lid for covering the openings

(16.1 ) of the cover (16). Device (1) according to any of the preceding claims, characterized in that the base (2) has a generally rectangular cross-section provided with two lateral extensions (2.3) on the longest sides of the rectangular cross-section and the multi-well plate (4) has a rectangular cross-section, thereby creating two recesses between the plate (4) and the base (2) enabling the removal by hand of the multi-well plate (4) from the housing (2.1 ). Device (1) according to any of the preceding claims, characterized in that the base (2) comprises external elastic tabs for a snap fit assembling of the casing (18) on the base (2). Device (1) according to any of the preceding claims, characterized in that the base (2) integrates heating means. A device (1) for applying an aerosol (22) on a biological system (10), the device (1 ) comprising:

- a base (2) provided with a housing (2.1 );

- a cover (16) adapted to lie on the base (2) and to cover the peripheral gaps (8), the cover (16) being provided with openings (16.1 );

- a discharging device configured for discharging a substance over the cover (16) such that the openings (16.1 ) of the cover enable a deposition of the substance onto the biological system (10) lying on each respective membrane (6.1 ). Method for applying a substance or an aerosol (22) on cells (10), optionally with a device (1 ) in accordance with any of claims 1 to 12, the method comprising the steps of:

- placing cells (10) on the microporous membrane-like bottom (6.1 ) of a plurality of inserts (6);

- placing the inserts (6) in respective wells (4.1 ) of a multi-well plate (4), thereby defining an apical compartment (12) and a basolateral compartment (14), partially immersed in liquid (5);

- covering the inserts (6) and the multi-well plate (4) with a cover (16), the cover (16) being provided with openings (16.1 ) exposing the apical compartment (12) of the inserts (6) while sealing away the basolateral compartment (14); and

- flowing down a substance or an aerosol (22) through the openings (16.1 ) and onto the cells (10). Method according to claim 13, characterized in that after the step of covering and before the step of flowing, the multi-well plate (4), the inserts (6) and the cover (16) are introduced in a housing (2.1 ) of a base (2) of a cell cultivation device (1 ) and a casing (18) is positioned on the base (2) to seal an exposure chamber (20).