DE102020118369B3 - Exposure apparatus - Google Patents

Abstract

The invention relates to an exposure apparatus for carrying out in-vitro experiments with biological test systems which are perfused with an exposure medium while forming an exposure atmosphere.

Description

The invention relates to an exposure apparatus for carrying out in vitro experiments with technical replicas of at least one biological test system, which can be exposed to an exposure medium perfusively with the formation of an exposure atmosphere on or above the biological test system.

When performing in vitro experiments with biological test systems, in particular with cells, cell cultures or tissues, a substance is usually brought into contact with the biological test system, so-called exposure, regardless of the objective of the experiments or the investigations.

The biological test system or biological test system is exposed to an exposure atmosphere that is formed by an exposure medium. The exposure medium can contain the substance, which is also referred to as test substance or test substance, and / or form it. In some cases it may be desirable for the exposure medium to be in the form of a substance or test substance or test substance.

The biological test system can be a eukaryote culture, particularly preferably cell lines, primary cell isolates, tissue sections, reconstructed tissue such as cocultures, or genetically modified cells, or a prokaryote culture.

The exposure medium can be liquid or gaseous.

The gaseous exposure medium can be present as a pure gas or gas mixture, i.e. H. all substances contained therein, in particular atoms, molecules, etc., are in the gas phase, and / or it can also serve as a carrier for solid and / or liquid substances. In particular, aerosols, atomized liquids, small liquid droplets (e.g. pesticides as spray mist, etc.), suspended particles, solid particles (e.g. wood dust, etc.), gaseous suspensions, atomized suspensions or emulsions can be contained in the carrier gas as substances to be carried.

The in-vitro experiments mentioned can involve targeted manipulations using pharmacologically active substances. The in-vitro experiments mentioned can also be those that are intended to influence cell growth or differentiation, for example in the context of so-called tissue engineering techniques. The in-vitro experiments mentioned can also be toxicological investigations of a wide variety of substances or substances.

With regard to the way in which the substances are brought into contact with the biological test systems, a basic distinction can be made between two modes of operation, namely on the one hand static exposure and on the other hand dynamic exposure, which is also referred to as perfusion or perfusive exposure.

In the case of static exposure, the substance is added once or repeatedly, in particular additively, but not continuously, to the biological test system, on which it then remains for a certain period of time without being exchanged.

In the case of dynamic exposure, the substance is continuously added and discharged, for example in order to generate greater effectiveness or sensitivity in the investigation, or due to physical / chemical or other properties of the test substances, the biological test system or other boundary conditions of the investigation.

To carry out dynamic exposures or perfusion exposures with exposure media is in the WO 2015/027998 A1 discloses an advantageous exposure apparatus. In the WO 2015/027998 A1 there is a detailed overview of the state of the art relevant in this technical field.

• • Verwendung von in vitro kultivierten Zellen und Geweben
• • Verwendung von so genannten „Membrankulturen“, d.h. Zellen und Gewebe, die auf mikroporösen oder filterartigen Substraten kultiviert wurden
• • Verwendung einer möglichst großen Anzahl von Kulturen in einem einzigen experimentellen Ansatz (in verschiedenen „Wells“) als Voraussetzung zur Realisierung von Experimenten im höheren Durchsatz („Higher Throughput“, „HT“), resultierend in einer Verkleinerung der Fläche der einzelnen Kultur.
• • Oberseitige („apikale“) Heranführung eines Mediums (z.B. ein Aerosol in der Gasphase) an die Kultur („Expositionsmedium“)
• • Definierte Verteilung des Expositionsmediums über unterschiedliche Kulturen (als „technische Replikate“ verwendete, identisch behandelte Wells).
• • Versorgung der Kulturen mit Kulturmedium
• • Konstruktive Trennung der Bereiche, die der Heranführung des Expositionsmediums dienen, und der Bereiche, die der Kulturmediumversorgung dienen, um einen Kontakt und damit die physikalisch chemische Vermischung der beiden Medien auszuschließen
• • Thermostatisierung
• • Gezielte Wärmeverteilung, um Effekte wie „thermische Präzipitation“, auch „Thermophorese“ genannt, durch gezielt eingestellte Temperaturgradienten vorwiegend zwischen den Oberflächen der Kulturen und dem überströmenden Expositionsmedium zu ermöglichen
• • Beobachtung der Kulturen mittels fluoreszenzspektroskopischer Methoden in der Expositionssituation, z. B. durch Fluoreszenzmikroskopie oder ähnlichen Systemen, die als „Multiwell Platten Reader“ bekannt sind
• • Verwirklichung der technischen Lösung in Form eines möglichst kompakten Expositionsapparats

The Indian WO 2015/027998 A1 The exposure apparatus disclosed is constructed in such a way that, for example, the following requirements are met:

• • Use of cells and tissues cultivated in vitro
• • Use of so-called “membrane cultures”, ie cells and tissue that have been cultivated on microporous or filter-like substrates
• • Use of the largest possible number of cultures in a single experimental approach (in different "wells") as a prerequisite for carrying out experiments with higher throughput ("Higher Throughput", "HT"), resulting in a reduction in the area of the individual culture.
• • Upper ("apical") approach of a medium (eg an aerosol in the gas phase) to the culture ("exposure medium")
• • Defined distribution of the exposure medium over different cultures (identically treated wells used as "technical replicates").
• • Supply of cultures with culture medium
• • Constructive separation of the areas that serve to bring the exposure medium up and the areas that serve to supply the culture medium in order to exclude contact and thus the physico-chemical mixing of the two media
• • Thermostatic control
• • Targeted heat distribution in order to enable effects such as “thermal precipitation”, also known as “thermophoresis”, through specifically set temperature gradients mainly between the surfaces of the cultures and the overflowing exposure medium
• • Observation of the cultures using fluorescence spectroscopic methods in the exposure situation, e.g. B. by fluorescence microscopy or similar systems known as "multiwell plate readers"
• • Realization of the technical solution in the form of an exposure apparatus that is as compact as possible

The Indian WO 2015/027998 A1 The exposure apparatus disclosed mainly meets the requirements mentioned, in particular when using standard multiwell plates. The use of plate formats with higher throughput, e.g. 96-well plates, instead of the standard 12-well plates typically used, is associated with difficulties.

The use of corrugated sheets with a higher number of wells results in various fluidic, physical and constructional consequences, which are a pure scaling of the in the WO 2015/027998 A1 exposed exposure apparatus can no longer be the best solution.

Further exposure apparatus are from the DE 10 2009 039 868 A1 and the US 2010/0 112 690 A known.

On the basis of this prior art, the invention is based on the object of developing an exposure apparatus for carrying out in vitro experiments with technical replicates of at least one biological test system, which can be exposed to an exposure medium perfusively with the formation of an exposure atmosphere on or above the biological test system, to improve, in particular to improve it so that it is suitable for plate formats with higher throughput, e.g. for 96-well plates.

This object is achieved with an exposure apparatus which has the features of claim 1. Further developments and advantageous configurations of the invention emerge from the subclaims.

• • Eine Vervielfachung des Prinzips der individuellen Exposition einzelner Wells resultiert ebenso in der Notwendigkeit der entsprechenden Vervielfachung der Erzeugung von extern betriebenen Expositionsflüssen. Dies stellt einen erheblichen technischen Aufwand und eine erhöhte experimentelle Unsicherheit dar und führt zu einem massiven Raumbedarf der experimentellen Installation.
• • Die geometrischen Verhältnisse der einzelnen Wells in Plattensystemen mit einer höheren Anzahl von Wells, beispielsweise bei 96- Wellplatten, sind aus der Notwendigkeit des Beibehaltens der äußeren Plattenabmessungen, insbesondere aus Kompatibilitätsgründen, deutlich anders als in Multiwellplatten mit weniger Wells, beispielsweise bei 6- oder 12-Wellplatten. Mit zunehmender Anzahl von Wells pro Multiwellplatte wird daher das geometrische Verhältnis der Höhe des Wells zu seiner Bodenfläche immer größer; die Wells werden in ihrem Querschnitt immer schmaler und höher.

The invention is based on the knowledge that the pure scaling of the WO 2015/027998 A1 disclosed exposure apparatus to a higher number of wells - as well as any other system that is not based on standard multiwell plates - essentially has the following disadvantageous consequences:

• • A multiplication of the principle of the individual exposure of individual wells also results in the necessity of the corresponding multiplication of the generation of externally operated exposure flows. This represents a considerable technical effort and increased experimental uncertainty and leads to a massive space requirement for the experimental installation.
• • The geometrical relationships of the individual wells in plate systems with a higher number of wells, for example with 96-well plates, are clearly different than in multiwell plates with fewer wells, for example with 6 or 12-well sheets. As the number of wells per multiwell plate increases, the geometric ratio of the height of the well to its base area increases; the wells become ever narrower and taller in their cross-section.

Exposure apparatus, such as those in the WO 2015/027998 A1 are disclosed follow the principle of the so-called “stagnation flow”. The flow of the exposure medium is directed vertically and centrally onto the surface of the membrane cultures. The approach takes place through a so-called “inlet pipe”, which can be shaped according to its task according to the flow lines. If the geometry of a well is changed, this inlet tube must be lengthened and at the same time reduced in its diameter in order to have a similar distance to the surface of the cell culture to be exposed in order to achieve comparable exposure conditions. A reduction in the inner diameter in turn means a massive change in the geometric relationships between the inner surface and the open cross-section. If, for example, aerosols are used that contain very small particles, for example nanoparticles, the movement of which is predominantly diffusive, this means a considerable loss of particles in the aerosol due to adsorption effects on the inner wall of the inlet pipe. However, this also reduces the concentration of particles actually available for exposure. A significant one The result is a loss of effectiveness of the exposure process, for example when investigating the biological effects of inhalable aerosols.

Working without any inlet pipe, which would lead to a purely diffusive or sedimentation-driven separation of substances from the exposure medium, would also not be an advantageous option due to the lack of effectiveness and reproducibility that this creates.

• • mehrere Ableitungsrohre aufweist, die jeweils einem bestimmten, mit biologischem Testsystem versehenen Well der Multiwellplatte zugeordnet sind, wobei das Ableitungsrohr nach dem Aufsetzen des Expositionsaufsatzes auf die Multiwellplatte mit dem unteren Ende in den Well ragt,
• • zumindest einen Ableitungsraum aufweist, in den die Ableitungsrohre mit ihren oberen Enden münden,
• • zumindest einen Ableitungsanschluss aufweist, der mit zumindest einem Ableitungsraum strömungsverbunden ist und an dem eine Unterdruck erzeugende Vorrichtung anschließbar ist,
• • mehrere Zuleitungsrohre aufweist, die jeweils einem bestimmten, mit biologischem Testsystem versehenen Well der Multiwellplatte zugeordnet sind, wobei das Zuleitungsrohr nach dem Aufsetzen des Expositionsaufsatzes auf die Multiwellplatte mit seinem unteren Ende in den Well mündet,
• • zumindest einen Zuleitungsraum aufweist, in den die Zuleitungsrohre mit ihren oberen Enden münden,
• • zumindest einen Zuleitungsanschluss aufweist, der mit zumindest einem Zuleitungsraum strömungsverbunden ist und an dem eine Expositionsmediumquelle anschließbar ist,

wobei das untere Ende des Ableitungsrohres näher am Boden des Wells bzw. am biologischen Testsystem angeordnet ist als das untere Ende des Zuleitungsrohres,
wobei mittels der am Ableitungsanschluss angeschlossenen und einen Unterdruck erzeugenden Vorrichtung, ein Expositionsmedium aus der am Zuleitungsanschluss angeschlossenen Expositionsmediumquelle in den Zuleitungsraum, von dort in die mit dem Zuleitungsraum verbundenen Zuleitungsrohre, von dort zur Ausbildung der Expositionsatmosphäre in das jeweils einem Zuleitungsrohr zugeordnete Well bzw. über das darin angeordnete biologische Testsystem, von dort in das jeweils dem Well zugeordnete Ableitungsrohr, von dort in den mit den Ableitungsrohren strömungsverbundenen Ableitungsraum zum Ableitungsanschluss leitbar bzw. förderbar ist.The invention therefore provides an exposure apparatus for carrying out in-vitro experiments with technical replicates of at least one biological test system, which can be exposed to an exposure medium perfusively with the formation of an exposure atmosphere on or above the biological test system, which comprises an exposure attachment that is exposed to a multiwell plate can be attached, whereby the exposure apparatus

• • has several drainage tubes which are each assigned to a specific well of the multiwell plate provided with a biological test system, the drainage tube protruding into the well with the lower end after the exposure attachment has been placed on the multiwell plate,
• • has at least one drainage space into which the drainage pipes open with their upper ends,
• • has at least one discharge connection which is flow-connected to at least one discharge space and to which a device generating negative pressure can be connected,
• • has several feed pipes, each assigned to a specific well of the multiwell plate provided with a biological test system, the lower end of the feed pipe opening into the well after the exposure attachment has been placed on the multiwell plate,
• • has at least one supply space into which the supply pipes open with their upper ends,
• • has at least one supply line connection which is flow-connected to at least one supply line space and to which a source of exposure medium can be connected,

wherein the lower end of the drainage tube is arranged closer to the bottom of the well or to the biological test system than the lower end of the supply tube,
whereby, by means of the device connected to the discharge connection and generating a negative pressure, an exposure medium from the exposure medium source connected to the feed connection into the feed space, from there into the feed pipes connected to the feed space, from there to the formation of the exposure atmosphere into the well or via the respective feed pipe the biological test system arranged therein can be guided or conveyed from there into the drainage pipe assigned to the respective well, from there into the drainage space that is flow-connected to the drainage pipes to the drainage connection.

The Indian WO 2015/027998 A1 The exposure apparatus disclosed already discloses many advantageous features which, of course, can also be present in the exposure apparatus according to the invention. The features are, for example, the structural separation of the areas of exposure and culture medium, the structural anchoring of the possibility of working according to the thermophoresis principle through the implementation of thermal gradients (layer structure), and the implementation in a form that enables the observation of the Cells and tissue during exposure "from below" and which, in their construction, allow the use of standardized multiwell plates.

The exposure apparatus according to the invention is distinguished from that in FIG WO 2015/027998 A1 disclosed exposure apparatus in that the flow path of the exposure medium is reversed. The reversal has the effect that the geometry of the supply pipe in the area of the approach of the exposure medium to the cultures can be constructed much more favorably, i.e. with a larger volume / surface ratio. The disadvantageous route of the exposure medium through a central and - in the case of a multiwell plate with a higher throughput necessarily - narrow and long supply pipe is bypassed.

A dense approach of the exposure medium to the biological test system which can be designed is nevertheless possible through the drainage pipe, which can advantageously be made narrow as desired, since losses due to an unfavorable cross-section / length ratio are no longer relevant when the exposure medium is transported away from the biological test system.

It can be advantageous if the temperature of the multiwell plate can be controlled, wherein preferably at least one electrical heating device, in particular a heating plate, or a thermostating device, which is preferably arranged below the multiwell plate, can be used. The heating device or thermostating device is advantageously designed and arranged in such a way that it controls the temperature of one or more wells or of a growth medium to optimize the growth conditions of the biological test system, whereby on a separate one Incubator or incubator can be dispensed with and, in particular, operation of the exposure apparatus away from the laboratory is possible. The heating device or thermostating device is advantageously designed and arranged in such a way that a thermal gradient is formed within the exposure apparatus, which can be used in particular for the targeted separation of substances from an aerosol flow by means of thermophoresis. To form the thermal gradient, it can be advantageous if a heating device or thermostating device is also arranged at at least one other point of the exposure apparatus not provided below the multiwell plate, for example on the exposure attachment.

It can be advantageous if the lower end of the discharge pipe is arranged in the lower third, preferably in the lower quarter, particularly preferably in the lower fifth, very particularly preferably in the lower sixth of the well.

It can be advantageous if the discharge pipe is exchangeable, preferably by a discharge pipe with a different dimension, for example with a different inner diameter and / or a different length, and / or with a different material. Through the choice of material, the properties of the discharge pipe can be specifically influenced in the desired manner. The choice of material can thus have an influence on the surface properties of the inner wall of the discharge pipe, as a result of which an effect is achieved with regard to particle adsorption on the inner wall. In addition, the electrostatics, heat conduction and heat capacity of the discharge pipe can be influenced in the desired manner through the choice of material.

In addition, it can be advantageous if the length with which the drainage tube protrudes into the well is adjustable, preferably by moving the drainage tube within the exposure attachment.

The supply pipe, which can also be designed as a very short simple opening within a wall or as a sleeve, thanks to its design in length and shape, enables the flow of the exposure medium to the surface of the biological test system, in particular the cell surface, to be influenced .

It can be advantageous if the lower end of the supply pipe is arranged in the upper third, preferably in the upper quarter, particularly preferably in the upper fifth, very particularly preferably in the upper sixth of the well.

It can be advantageous if the feed pipe is exchangeable, preferably by a feed pipe with a different dimension, for example with a different inner diameter and / or a different length, and / or with a different material. The properties of the supply pipe can be influenced in a targeted manner by the choice of material. The choice of material can thus have an influence on the surface properties of the inner wall of the feed pipe, whereby an effect is achieved with regard to particle adsorption on the inner wall. In addition, the electrostatics, heat conduction and heat capacity of the supply pipe can be influenced in the desired manner through the choice of material.

It can be advantageous if the length with which the supply pipe protrudes into the well is adjustable, preferably by moving the supply pipe within the exposure attachment.

By providing at least one discharge space, into which several discharge pipes open, and at least one feed space, from which several feed pipes branch off, and the flow direction of the exposure medium provided according to the invention, a considerable reduction in the technical effort for the necessary control and regulation tasks is achieved.

Without the provision of such inlet and outlet spaces and the associated combination of inlet and outlet spaces, for example, 96 individual supplies with exposure flows and 96 feeds of the exposure medium would have to be implemented in a standard 96-well plate. That would correspond to 192 hose connections on a base area of the multiwell plate in a format of 13 x 8.5 cm and would therefore no longer be manageable.

It can be advantageous if the wells of the multiwell plate can be subdivided into different groups, with different groups of wells being connected to different inlet and / or outlet spaces via correspondingly assigned inlet and outlet pipes, so that each group of wells individually with the same or different exposure volume flows of an exposure medium with or without test substance is flowed through.

The connection to a group leads to a factor of 8 when merging columns of a 12 x 8 multiwell plate in 96-well format or by a factor of 12 when merging rows of a 12 x 8 multiwell plate in 96-well format.

It can be advantageous if the division can be carried out along the rows and / or columns of the multiwell plate. There can also be several Rows and / or several columns of wells belong to a group of wells.

It can be advantageous if no inlet and outlet pipes are assigned to a group of wells of the multiwell plate, this group of wells being able to be carried along as a so-called control sample in the in vitro experiment.

It can be advantageous if a group of wells is connected to an inlet and outlet space via appropriately assigned inlet and outlet pipes through which the exposure medium can be fed to the group of wells without test substance and carried along as a so-called blank sample in the in vitro experiment is.

It can be advantageous if a group of wells is connected to an inlet and outlet space via appropriately assigned inlet and outlet pipes, through which the exposure medium with test substance can be fed to the group of wells and carried along as the actual sample in the in vitro experiment .

It can be advantageous if the discharge pipe is provided with an element which determines the flow and preferably narrows the internal cross-section of the discharge pipe, which element is preferably arranged exchangeably at the upper end of the discharge pipe.

The narrowing of the cross-section increases the flow resistance to generate a pressure drop. This means that different exposure conditions and different exposure doses can be set.

Differently designed elements can also be used advantageously to define different groups or different subgroups within groups. Such groups or subgroups can also be defined using different biological test systems.

The organization in rows or columns carried out by the drainage and supply spaces and the associated drainage and supply pipes therefore does not restrict the possibilities that are desired due to the high number of wells due to the formation of subgroups.

There are different possibilities for the technical realization of defined, vacuum-driven exposure medium flows, for example mechanical needle valves or electrical mass flow controllers. In addition to the fluidic functionality, ease of use and price are also decisive for sensible technical use. In the exposure apparatus according to the invention, a high number of exposure medium flows should be realized reliably. At the same time, the necessary measures for the user should be kept as low as possible, since the focus of the application of the exposure apparatus is on the examinations with the biological test systems, for example cell cultures, which require considerable attention from the user.

Flow-determining elements, in particular also so-called “critical nozzles”, which generate defined volume flows through a pressure drop with a given negative pre-pressure, are preferred according to the invention. The structural merging with the drainage pipe also enables an extremely compact design.

It can be advantageous if the exposure attachment can be placed on and connected to the multiwell plate in such a way that the exposure atmosphere that forms within each specific well during the in vitro experiment is tightly sealed, with exposure medium only via the supply pipe assigned to the respective well can be fed to the well and exposure medium can only be discharged from the well via the respective discharge tube assigned to the well.

It can be advantageous if one or more than one sealant is provided between the exposure attachment and the multiwell plate to seal off the exposure atmosphere.

It can be advantageous if the sealing means is a seal surrounding the respective well or the opening of the respective well and / or a seal surrounding the respective supply and / or discharge pipe.

It can be advantageous if the sealant is a sealing mat that can be or is attached to the multiwell plate and / or attached or attached to the side of the exposure attachment facing the multiwell plate with corresponding recesses for the specific wells or openings of the specific wells and / or with corresponding Recesses for the supply and / or drainage pipes assigned to the specific wells.

It can be advantageous if the discharge pipe is arranged at least partially inside the supply pipe and protrudes therefrom in the direction of the well. In this respect, the supply pipe and the discharge pipe can be at least partially concentric. Advantageously, according to the pipe-in-pipe principle, the flow can thus be carried out at least partially in a correspondingly coaxial countercurrent. It can be advantageous if the drainage pipe is in its biological test system facing end area widens, in particular conically or by non-linear change of its inner diameter with the length, for example hyperbolic

It can be advantageous if the exposure medium that can be conducted through the line elements is liquid or gaseous, the exposure medium being able to be provided with or without a test substance.

According to the invention, individual wells, which are also referred to as depressions or cavities, for receiving the biological test systems are arranged essentially in the form of a matrix on a multiwell plate. The wells can be receptacles that are already integrated in the multiwell plate or those that are received as separate culture vessels, so-called inserts, in existing integrated wells or in correspondingly arranged recesses in the multiwell plate. It can be advantageous if the bottom of at least one well of the multiwell plate is designed as a permeable carrier or, if necessary, also has such a carrier, on which the biological test system is arranged. Preferably the permeable carrier can be a microporous membrane. To accommodate the biological test system, the well can also be designed as a commercially available so-called cell culture insert that can be introduced into the multiwell plate, the permeable carrier forming the bottom membrane of the well.

It can be advantageous if the device generating a negative pressure is a peristaltic pump for liquid or a membrane pump for gaseous exposure media.

It can be advantageous if a maintenance medium can be supplied to or assigned to the multiwell plate which, for supplying the biological test system, is in direct contact with the underside of the well or the permeable carrier on which the biological test system is arranged. A maintenance medium within the meaning of the invention is understood here and below to mean a culture medium, also referred to as nutrient medium, with or without additives or a salt solution.

It can be advantageous if the maintenance medium can be supplied perfusively, i.e. in continuous flow, in order to enable, for example, the use for so-called “organ-on-a-chip” approaches in which different organ cultures are cultivated together, or something like that to enable said kinetic transport studies with regard to the mass transfer via the exposed cells, or at the same time to be able to supply the exposed cells, for example, pharmacologically active substances through the culture medium.

• 1 schematisch im Querschnitt einen Teilausschnitt aus einem erfindungsgemäßen Expositionsapparat.

The invention is explained below using an exemplary embodiment which is shown in the drawing. In this shows

• 1 schematically, in cross section, a partial section from an exposure apparatus according to the invention.

• • mehrere Ableitungsrohre 20 aufweist, die jeweils einem bestimmten, vorzugsweise mit biologischem Testsystem 10 versehenen Well 22 der Multiwellplatte 18 zugeordnet sind, wobei das Ableitungsrohr 20 nach dem Aufsetzen des Expositionsaufsatzes 16 auf die Multiwellplatte 18 mit dem unteren Ende 24 in das Well 22 ragt,
• • zumindest einen Ableitungsraum 26 aufweist, in den die Ableitungsrohre 20 mit ihren oberen Enden 28 münden,
• • zumindest einen hier nicht dargestellten Ableitungsanschluss aufweist, der mit zumindest einem Ableitungsraum 26 strömungsverbunden ist und an dem eine hier nicht dargestellte Unterdruck erzeugende Vorrichtung anschließbar ist,
• • mehrere Zuleitungsrohre 30 aufweist, die jeweils einem bestimmten, vorzugsweise mit biologischem Testsystem 10 versehenen Well 22 der Multiwellplatte 18 zugeordnet sind, wobei das Zuleitungsrohr 30 nach dem Aufsetzen des Expositionsaufsatzes 16 auf die Multiwellplatte 18 mit seinem unteren Ende 32 in das Well 22 mündet,
• • zumindest einen Zuleitungsraum 34 aufweist, in den die Zuleitungsrohre 30 mit ihren oberen Enden 36 münden,
• • zumindest einen hier nicht dargestellten Zuleitungsanschluss aufweist, der mit zumindest einem Zuleitungsraum 34 strömungsverbunden ist und an dem eine hier nicht dargestellte Expositionsmediumquelle anschließbar ist,

wobei das untere Ende 24 des Ableitungsrohres 20 in unmittelbarer Nähe des biologischen Testsystems und damit näher am Boden 38 des Wells 22 bzw. am biologischen Testsystem 10 angeordnet ist als das untere Ende 32 des Zuleitungsrohres 30,
wobei mittels der am Ableitungsanschluss angeschlossenen und einen Unterdruck erzeugenden Vorrichtung, ein Expositionsmedium 14 aus der am Zuleitungsanschluss angeschlossenen Expositionsmediumquelle in den Zuleitungsraum 34, von dort in die mit dem Zuleitungsraum 34 verbundenen Zuleitungsrohre 30, von dort zur Ausbildung der Expositionsatmosphäre 12 in das jeweils einem Zuleitungsrohr 30 zugeordnete Well 22 bzw. über das darin angeordnete biologische Testsystem 10, von dort in das jeweils dem Well 22 zugeordnete Ableitungsrohr 20, von dort in den mit den Ableitungsrohren 20 strömungsverbundenen Ableitungsraum 26 zum Ableitungsanschluss leitbar bzw. förderbar ist.The exposure apparatus according to the invention for carrying out in vitro experiments with technical replicates of at least one biological test system 10 that are perfusive with the formation of an exposure atmosphere 12th on or above the biological test system 10 with an exposure medium 14th can be acted upon, includes an exposure attachment 16 on a multiwell plate 18th can be put on, with the exposure apparatus

• • several drainage pipes 20th has, each with a specific, preferably with a biological test system 10 provided well 22nd the multiwell plate 18th are assigned, the drainage pipe 20th after putting on the exposure attachment 16 onto the multiwell plate 18th with the lower end 24 in the well 22nd protrudes
• • at least one drainage room 26th has, in which the drainage pipes 20th with their upper ends 28 flow out,
• • has at least one discharge connection, not shown here, which has at least one discharge space 26th is flow-connected and to which a device that generates negative pressure, not shown here, can be connected,
• • several supply pipes 30th has, each with a specific, preferably with a biological test system 10 provided well 22nd the multiwell plate 18th are assigned, the feed pipe 30th after putting on the exposure attachment 16 onto the multiwell plate 18th with its lower end 32 in the well 22nd flows into
• • at least one supply room 34 has, in which the supply pipes 30th with their upper ends 36 flow out,
• • has at least one lead connection, not shown here, which has at least one lead space 34 is flow-connected and to which an exposure medium source (not shown here) can be connected,

being the lower end 24 of the discharge pipe 20th in the immediate vicinity of the biological test system and thus closer to the ground 38 of the well 22nd or on the biological test system 10 is arranged as the lower end 32 of the supply pipe 30th ,
wherein by means of the device connected to the discharge connection and generating a negative pressure, an exposure medium 14th from the connected to the supply connection Exposure medium source in the supply room 34 , from there into the one with the supply room 34 connected supply pipes 30th , from there to the formation of the exposure atmosphere 12th in each of the supply pipes 30th assigned well 22nd or via the biological test system arranged therein 10 , from there into the respective well 22nd associated drainage pipe 20th , from there into the one with the drainage pipes 20th flow-connected discharge space 26th can be conducted or conveyed to the discharge connection.

Wells are here 22nd the multiwell plate 18th shown, which are divided into three groups I, II, III. Further wells, not shown here, belonging to each group I, II, III are located behind and in front of the plane of the paper. Any group of wells 22nd stands over appropriately assigned inlet and outlet pipes 30th , 20th with one inlet and one outlet room each 34 , 26th in connection so that each group I, II, III of Wells 22nd individually with the same or different exposure volume flows of an exposure medium 14th is flowed through with or without test substance.

The exposure essay 16 is like this on the multiwell plate 18th placed on and connected to this, that the during the in-vitro experiment within each specific well 22nd educational exposure atmosphere 12th is tightly sealed, with just over each of the wells 22nd associated supply pipe 30th Exposure medium 14th the well 22nd can be fed and only via that in each case to the well 22nd associated drainage pipe 20th Exposure medium 14th from the well 22nd can be derived.

For sealing off the exposure atmosphere 12th is a sealant 42 between exposure attachment 16 and multiwell plate 18th intended. In the present case, the seal leads to a structural separation between the exposure atmosphere 12th and the room 46 for the preservation medium not shown here.

According to the present invention, each discharge pipe is 20th with a flow-determining, preferably the inner cross-section of the discharge pipe 20th narrowing element 40 provided, which is interchangeable at the top 28 of the discharge pipe 20th is arranged. The constriction leads to a desired pressure drop, which has the desired effects on the volume flow of the exposure medium.

In 1 is still the upper degree 44 of the exposure attachment 16 shown. This can for example have an electrical heating device, in particular a heating foil, or a thermostating device including a Peltier element. Below the multiwell plate 18th is a degree 54 shown, which has an electrical heating device, in particular a heating plate, or a thermostating device including a Peltier element. The heating device or thermostating device of the lower closure 54 is advantageously designed and arranged in such a way that it is the temperature of one or more wells 22nd or a growth medium for optimizing the growth conditions of the biological test system 10 controls, which means that a separate incubator or incubator can be dispensed with and, in particular, it is also possible to operate the exposure apparatus away from the laboratory.

The heating device or thermostating device ensures that a thermal gradient is formed within the exposure apparatus, which can be used in particular for the targeted separation of substances from an aerosol flow by means of thermophoresis.

The drainage pipe 20th and the flow-determining element 40 consist of highly thermally conductive material to stabilize the thermal gradient between the exposure medium 14th and the surface of the biological test system 10 , especially the cell surface.

To form the thermal gradient, the exposure apparatus is constructed in layers and includes an upper end 44 , preferably with an electrical heating device or a thermostating device, an upper part 48 made of a thermally conductive material and in a sealing demarcation 50 plus a middle part 52 made of a material with low thermal conductivity and underneath the multiwell plate 18th a lower degree 54 , which has an electrical heating device, in particular a heating plate, or a thermostating device.

List of reference symbols

10

biological test system

12th

Exposure atmosphere

14th

Exposure medium

16

Exposure essay

18th

Standard multiwell plate

20th

Drainage pipe

22nd

Well

24

lower end

26th

Drainage room

28

top end

30th

Supply pipe

32

lower end

34

Supply room

36

top end

38

floor

40

element

42

sealant

44

upper degree

46

Space for maintenance medium

48

Upper part of the exposure apparatus

50

Demarcation (seal)

52

Middle part of the exposure apparatus

54

lower end (with heating plate)

Claims (24)

Exposure apparatus for performing in vitro experiments with technical replicas of at least one biological test system (10), which can be exposed to an exposure medium (14) perfusively with the formation of an exposure atmosphere (12) on or above the biological test system (10), comprising an exposure attachment (16), which can be placed on a multiwell plate (18), the exposure apparatus • has several discharge tubes (20) which are each assigned to a specific well (22) of the multiwell plate (18) provided with a biological test system (10), the discharge tube (20) after the exposure attachment (16) has been placed on the multiwell plate ( 18) protrudes with the lower end (24) into the well (22), • has at least one discharge space (26) into which the discharge pipes (20) open with their upper ends (28), • has at least one discharge connection which is flow-connected to at least one discharge space (26) and to which a device generating negative pressure can be connected, • has several supply pipes (30) which are each assigned to a specific well (22) of the multiwell plate (18) provided with a biological test system (10), the supply pipe (30) after the exposure attachment (16) has been placed on the multiwell plate ( 18) opens with its lower end (32) into the well (22), • has at least one supply space (34) into which the supply pipes (30) open with their upper ends (36), • has at least one supply line connection which is flow-connected to at least one supply line space (34) and to which a source of exposure medium can be connected, wherein the lower end (24) of the drainage tube (20) is arranged closer to the bottom (38) of the well (22) or to the biological test system (10) than the lower end (32) of the supply tube (30), whereby, by means of the device connected to the discharge connection and generating a negative pressure, an exposure medium (14) from the exposure medium source connected to the feed connection into the feed space (34), from there into the feed pipes (30) connected to the feed space (34), from there for training the exposure atmosphere (12) into the well (22) assigned to a supply pipe (30) or via the biological test system (10) located therein, from there into the drainage tube (20) assigned to the respective well (22), from there into the discharge space (26) which is flow-connected to the discharge pipes (20) can be guided or conveyed to the discharge connection. Exposure apparatus according to Claim 1 , characterized in that the temperature of the multiwell plate (18) can be controlled. Exposure apparatus according to Claim 1 or 2 , characterized in that the lower end (24) of the drainage tube (20) is arranged in the lower third of the well. Exposure apparatus according to one of the preceding claims, characterized in that the lower end (32) of the feed pipe (30) is arranged in the upper third of the well (22). Exposure apparatus according to one of the preceding claims, characterized in that the feed pipe (30) is exchangeable. Exposure apparatus according to Claim 5 , characterized in that the feed pipe (30) can be replaced by a feed pipe (30) with a different dimension and / or with a different material. Exposure apparatus according to one of the preceding claims, characterized in that the discharge pipe (20) is exchangeable. Exposure apparatus according to Claim 7 , characterized in that the discharge pipe (20) can be replaced by a discharge pipe (20) with a different dimension and / or with a different material. Exposure apparatus according to one of the preceding claims, characterized in that the length with which the feed pipe (30) and / or the discharge pipe (20) protrudes into the well (22) is adjustable. Exposure apparatus according to Claim 9 , characterized in that the length can be adjusted by moving the supply pipe (30) and / or the discharge pipe (20) within the exposure attachment (16). Exposure apparatus according to one of the preceding claims, characterized in that the discharge pipe (20) is provided with a flow-determining element (40). Exposure apparatus according to Claim 11 , characterized in that the element (40) narrows the inner cross-section of the discharge pipe (20). Exposure apparatus according to Claim 11 or 12th , characterized in that the element (40) is arranged exchangeably at the upper end (28) of the discharge pipe (20). Exposure apparatus according to one of the preceding claims, characterized in that the wells (22) of the multiwell plate (18) can be subdivided into different groups, with different groups of wells (22) having different inlet and outlet pipes (30, 20) assigned to them - and / or drainage spaces (34, 26) are connected, so that each group of wells (22) is flowed through individually with the same or different exposure volume flows of an exposure medium (14) with or without a test substance. Exposure apparatus according to one of the preceding claims, characterized in that the division can be carried out along the rows and / or columns of the multiwell plate (18). Exposure apparatus according to one of the preceding claims, characterized in that no inlet and outlet pipes (30, 20) are assigned to a group of wells (22) of the multiwell plate (18), this group of wells (22) being used as a so-called control sample in the in -vitro experiment can be carried out. Exposure apparatus according to one of the preceding claims, characterized in that a group of wells (22) is connected to an inlet and outlet space (34, 26) through correspondingly assigned inlet and outlet pipes (30, 20) through which the exposure medium ( 14) can be fed to the group of wells (22) without test substance and can be carried along as a so-called blank sample in the in vitro experiment. Exposure apparatus according to one of the preceding claims, characterized in that a group of wells (22) is connected to an inlet and outlet space (34, 26) through correspondingly assigned inlet and outlet pipes (30, 20) through which the exposure medium ( 14) can be fed with test substance to the group of wells (22) and can be carried along as the actual sample in the in vitro experiment. Exposure apparatus according to one of the preceding claims, characterized in that the exposure attachment (16) can be placed on and connected to the multiwell plate (18) in such a way that the exposure atmosphere ( 12) is tightly sealed, with exposure medium (14) being able to be fed to the well (22) only via the supply pipe (30) assigned to each well (22) and exposure medium (14) only via the respective drainage tube (30) assigned to the well (22) ) can be derived from the well (22). Exposure apparatus according to one of the preceding claims, characterized in that one or more than one sealing means (42) is provided between the exposure attachment (16) and the multiwell plate (18) to seal off the exposure atmosphere (12). Exposure apparatus according to one of the preceding claims, characterized in that the sealing means (42) encircling the respective well (22) or the opening of the respective well (22) and / or the respective supply and / or discharge pipe (30, 20) Poetry is. Exposure apparatus according to one of the preceding claims, characterized in that the bottom (38) of a well (22) of the multiwell plate (18) is designed as a permeable carrier or has such a carrier, on which the biological test system (10) is arranged. Exposure apparatus according to one of the preceding claims, characterized in that the device generating a negative pressure is a peristaltic pump for liquid or a membrane pump for gaseous exposure media. Exposure apparatus according to one of the preceding claims, characterized in that the multiwell plate (18) can be supplied or assigned a maintenance medium which is in direct contact with the underside of the well (22) or the permeable carrier to supply the biological test system (10) in which the biological test system (10) is arranged.

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