WO2007085621A1

WO2007085621A1 - Method and apparatus for exerting an effect on cells

Info

Publication number: WO2007085621A1
Application number: PCT/EP2007/050699
Authority: WO
Inventors: Ulrich Mohr; Josef Vogel; Ulrich Deschl
Original assignee: Boehringer Ingelheim International Gmbh; Boehringer Ingelheim Pharma Gmbh & Co. Kg
Priority date: 2006-01-27
Filing date: 2007-01-24
Publication date: 2007-08-02

Abstract

A method and an apparatus for investigating the biological effect of a test substance is described and is characterized in that (a) an aerosol generator and, where appropriate, a humidifier are supplied with pure air, (b) the substance to be tested is converted by the aerosol generator into a finely dispersed particle stream, (c) the streams from the aerosol generator and, where appropriate, the humidifier are mixed in a suitable manner to give an aerosol and passed to a distributor, and (d) defined volumetric streams of the aerosol are drawn off from the distributor and brought into direct contact with prokaryotic or eukaryotic in vitro cultures in a culturing/exposure apparatus.

Description

Method and device for charging cells

The present invention relates to a method for assaying the biological effect of a huable substance or composition on living cells and a device for carrying out such studies.

In vitro testing systems used in pharmaceutical research and development investigate potential drugs under conventional culture conditions. This also applies to substances that are best applied by inhalation. In these test systems, cells are submerged, i. cultured with medium coating and added or dissolved a potential active substance in the medium. A study of the biological effects of substances and combinations of substances that would need to be inhaled can not be tested in vitro in the form they are developed for the patient (for example, as an aerosol), but only in inhalation experiments on living animals.

DE 10014057 A1 and DE 1021 1324 describe culture devices which make it possible to expose the cells cultured therein to the damaging or therapeutic conditions of gaseous media, aerosols or particle streams. Se can be purchased commercially under the name CULTEX®.

The present invention is based on the idea of using such culture devices in a standardized test system with which the biological effect of inhalable substances or substance mixtures on living cells is systematically examined, whereby the substances to be investigated are brought into direct contact as aerosols with the prokaryotic or eukaryotic biological test cultures and then examine the effects of this contact on the biological systems. Such a system makes it possible to detect from a large number of substances those whose biological activity permits a statement about the therapeutic benefit and / or its toxicity, while at the same time the number of animal experiments and the required substance quantities are reduced. The present invention thus relates to an in vitro method for investigating the biological effect of a test substance, characterized in that

(a) an aerosol generator and possibly a humidifier are supplied with clean air,

(b) the test substance is transferred by the aerosol generator into a finely divided particle stream,

(c) the streams from the aerosol generator and optionally the humidifier are suitably mixed into an aerosol and fed to a distributor, and

(D) withdrawn from the manifold volume flows of the aerosol and brought into direct contact with prokaryotic or eukaryotic in vitro test cultures in a culture / exposure device.

It has proven to be particularly advantageous for carrying out the method to reduce or minimize the occurrence of condensation effects by means of thermal conditioning throughout the entire process. Particularly suitable is a conditioning to a fixed temperature value between 20 ° C and 45 ° C and preferably between 37 ° C and 42 ° C.

By moistening the air can be kept at a value of at least 60% relative humidity. Preference is given to saturated saturation of the clean air with water vapor, which then forms a carrier matrix for the test substance in the resulting aerosol.

Aerosols are understood as meaning gases which contain liquid or solid substances in extremely finely divided form. Typical particle sizes for inhalation purposes are from 0.1 μm to 10 μm, preferably from 1 μm to 5 μm aerodynamic diameter. Various possibilities of aerosol production are described in "Theory and Practice of Inhalation Therapy" by Dieter Köhler and Wolfgang Fleischer, Arcis Verlag, Munich, 2000, ISBN 3-89075-140-7 and summarized in Figure 22, page 33 of the book. For example, aerosols containing liquid particles (droplets) may be venturi-atomized (compressed air or steam), single-fluid nozzles (eg Respimat®), ultrasound (pressing the fluid through a vibrating perforated membrane, detaching droplets from surface waves) or vaporizing and subsequent condensation are made.

By spraying with propellants (CFCs, HFCs), solid or liquid particles may form, depending on whether it is a suspension of solid particles in the propellant or a solution of the active ingredient and any excipients in the propellant gas. Solid particle aerosols can be generated by dispersing the powder with a suitable gas, especially air. In this case, the powder can be metered in accordance with an inhalation with individual strokes (for example HandiHaler®) or in large quantities as a heap. Solid particles aerosols can also be removed by one or more sharp edges of a compact of active ingredient and excipients. Evaporation and subsequent condensation can also produce aerosols with solid particles.

The technology of aerosol generation, i. the selection of the aerosol generator takes place depending on the agent to be characterized (liquid aerosol, solid / powder aerosol) but is of minor importance for the claimed method. Typical examples of aerosol generators which can be used in the present invention are known by the name of Respimat® for the production of droplets and Handihaler® for the production of solid particles. Preferred aerosol generators can produce a defined aerosol concentration of the substance in the range of 1 μg / L to 20 mg / L. The size of the particles produced should be in the range of 0.1 μm to 10 μm, preferably 1 μm to 5 μm.

The test atmosphere generated by mixing the streams from the aerosol generator and possibly the humidifier in the distributor should be kept at a constant temperature. Depending on the applied cell type, the temperature should be maintained between 20 ° C and 45 ° C and preferably between 37 ° C and 42 ° C. - A -

In order to be able to remove different samples reproducibly from the distributor, the withdrawn volume flow should be able to be varied in the range from 1 ml / min to 200 ml / min, preferably from 5 ml / min to 10 ml / min.

Cell cultures which are cultivated in so-called CULTEX® modules are particularly suitable for charging cells with the test atmosphere generated in the distributor by mixing the streams from the aerosol generator and optionally the humidifier. Part of the test atmosphere in the distributor is preferably via a flow guide with an inlet for introducing the

Test atmosphere i n the flow guide and an opening above the cell culture output opening in the Kutur / exposure device forcibly guided and comes into direct contact with the cells in order to develop a possibly biological effect can. In a particular embodiment, the flow guidance is characterized in that the outlet orifice of the flow guide opens in the direction of flow corresponding to the bell of a trumpet (hereinafter "trumpet-shaped"), thereby ensuring an intensive loading of the cells with the test atmospheres opening starting orifice opposite a circular cylindrical inlet pipe is that in particular difficult to control

Ablösezonen be avoided at the mouth edge, for example, lead to self-contained vortices and thus dead zones with no or undefined exposure above the culture surface. This specially shaped exit port ensures that the flow of the test atmosphere is smoothly passed through the cells, free of charge, and contained in the flow

Substances, in particular particles etc., are distributed as evenly as possible over the culture surface. The flow guide can be designed in such a way that the flow flow of the test atmosphere is generated over the surface of the cell culture and through an annular gap existing between the flow guide and the culture inner wall, for example by means of a vacuum pump arranged downstream of said annular gap. She leaves the admission area, for example, via holes in a circle around the Inlet nozzles of the flow guide are arranged and the output side are bundled in an output port. The flow guide may be connected on the input side to an intake manifold, which is connected to the generation source of the test atmosphere. The exposure attachment includes one or more, for example, three separate flow guides that allow separate loading of the cells in separate cell culture vessels of the exposure device.

For the flow guidance various materials can be used, which are based on the properties of the test atmosphere, e.g. Teflon, metallic materials, polished stainless steel and specially non-stick or glass-coated trumpet-shaped flow guides, which allow application of cultured cells, such as cells of the respiratory tract, with ultrafine particles.

Prokaryotic test systems are usually cultured on media solidified with agar or chelatine, while eukaryotic cells are preferentially cultured on membranes for direct loading. These are basally supplied with culture medium via the membrane, so that the cells grow without medium (apical).

Examples of eukaryotic cells are cells of the animal or human respiratory tract; macrophages; endothelial cells; or fibroblasts. As cells of the respiratory tract, for example, nasal, bronchial or alveolar epithelial cells come into question for the investigations. The cells grow apically and come into direct contact with the test atmosphere at the air / liquid boundary layer. They can be cultured on microporous membranes (see DE 198 01 763 A1). Examples of suitable microporous membranes are coated membranes, for example the companies Becton Dickinson (FALCON) and Corning (Costar), which ensure appropriate cell growth on account of their membrane properties. A special culture guide not only allows the study of the drugs with the above cell types, but also allows their combination with other cells, such as macrophages, endothelial cells and fibroblasts, which may play a role in the development of the effect of the respective substance. This takes place in so-called co-cultures, wherein the cells are grown on both sides of a membrane. Such a culture device in combination with the method, after direct exposure of the apical cell type to the test atmosphere, allows an analysis of the interactions between the various cell types present on the membrane in order to obtain further information on the effect of the test substance.

Vessels which receive cells grown on microporous membranes and cultured to be exposed to the test atmosphere (e.g., in so-called transwell inserts) are preferably in a separate subset, with each vial being individually supplied with nutrients. The supply of nutrients takes place via a feed line which is connected via a suitable connection, e.g. a hose connection is connected to the nutrient reservoir, and via an inlet nozzle allows a supply of nutrients in the relevant vessel of the pedestal. The vessel can be filled to a certain level with medium to supply the cells over the time of exposure. Another nozzle opens up the possibility of a constant medium flow in which the inflowing volume flows off or is removed in an analogous manner via the outlet nozzle. The medium flow may e.g. controlled by a peristaltic pump. Through these measures, a continuous supply of medium over long admission times is achieved.

The test method described makes it possible, for example, to apply different target cells of the respiratory tract or suitable bacterial test systems to potentially therapeutically active substances which are taken up by inhalation and act directly. This is not limited to gaseous and liquid aerosols, but also includes agents that are administered as solid aerosol particles. The procedure ensures an immediate Loading the cultured cells or bacteria with a test atmosphere and allows a precise analysis of the resulting cellular reaction pattern to describe the expected therapeutic and possibly unwanted toxic effect. The bioavailability and efficacy of substances applied in solid particulate form can be studied depending on particle size and support material, an aspect that can not be realized under conventional conditions.

Another advantage of the method is the ability to test not only individual substances, but also combinations thereof in different mixing ratios of cells to select therapeutically relevant agents or combinations thereof.

The treatment of cultured cells with therapeutic aerosols by direct application contains no limitation with regard to the determinable biological endpoints or effects. Typically certain parameters are e.g. Viable cell count, metabolic activity, membrane integrity, energy status, or oxidative stress. A comprehensive characterization of the treated cells, for example with respect to the cytotoxic and genotoxic activity of the test atmosphere, their inflammatory potential or molecular biology aspects (Genomics, Metabono mics) is fully possible, as well as receptor studies, the determination of mediators or other biomarkers, the analysis of signal transduction pathways and bioavailability conditions of the test atmosphere.

The investigations preferably carried out in the context of the method relate to the biological effect of aerosols / test atmosphere on cells cultured in vitro. They are typically aimed at

The effectiveness of active substances or combinations of active substances of particulate aerosols, depending on their particle size and optionally the carrier material used; The bioavailability of active substances or combinations of active substances of particulate aerosols as a function of their particle size and optionally the carrier material used;

The cytotoxic and genotoxic effects of gases, aerosols or mixtures on cell cultures;

The inflammatory potential of gases, aerosols or mixtures for cell cultures;

• the cellular response of cell cultures to direct contact with the gases, aerosols or their mixtures; and • molecular biology aspects of the direct contact of the cells with the gases, aerosols or mixtures thereof.

Examples of molecular biology studies include receptor studies, determination of mediators or other biomarkers, or analysis of signal transduction pathways.

Another object of the invention relates to a device based on the method just described. Such a device is used to study the biological effect of an inhaled substance comprises

• a humidifier;

• an aerosol generator for solid or liquid substances, which uses humidified clean air of the humidifier as carrier gas;

• a distributor, which is fed with the generated aerosol; and • one or more cell culture units connected to the manifold via one or more

Sampling points; and is characterized in that the generated aerosol stream is passed from a sampling point to the connected cell culture unit where it is brought into direct contact with the cultured cells. In a particular embodiment, the device described additionally comprises on-line analyzers for measuring aerosol parameters, which are connected to the distributor via sampling points and, like the cell culture units, are supplied with samples from the distributor.

For example, cold traps or activated carbon filters for gaseous components, particle filters for aerosol particles or impactors for defined particle sizes can be connected as sampling devices.

For example, devices for determining the particle concentration, devices for determining the particle size distribution or devices for determining the concentration of certain gaseous constituents can be connected as online analysis devices.

Typical examples of aerosol generators which can be used in the context of the present invention are known under the names Respimat® and Handihaler®. A suitable culture and exposure unit are those available as CULTEX® modules.

It proves to be advantageous to heat-condition the entire device in order to reduce or minimize the occurrence of condensation effects. Particularly suitable is a conditioning to a fixed temperature value between 20 ° C and 45 ° C and preferably between 37 ° C and 42 ° C.

The essential method steps or elements of the present invention are explained in greater detail in the exemplary flow diagrams of FIGS. 1 and 6. It shows the aerosol exposure structure. The complete construction consists of at least two such constructions, which are used for the simultaneous exposure of A549 cultures on microporous membranes to a control substance (aerosolized PBS) and a test substance (eg aerosolized tertiary butyl hydroperoxide). Examples:

Example 1: Apparatus for the Generation and Exposure of Droplet Aerosols on a Cell Culture Scale A modular construction in parallel arrangement allows the simultaneous operation of several nebulizers. Thus, the simultaneous, parallel exposure of cells to microporous membranes is e.g. to a test aerosol and a control aerosol (e.g., PBS as the vehicle or solvent of the test substance) in an experiment. To produce the droplet aerosol from liquids, commercially available nebulizers of the "Miniheart Low Flow Nebulizer" brand (Westmed Inc., Tucson, USA) are used, which are continuously pumped into the nebulizer storage vessel by means of a perfusor pump for constant levels in the nebulizer 37 ° C, 2 l / min) The aerosols are sampled for cell exposures immediately above the outlet In the further flow path of the test aerosol is the sampling position for an online aerosol analysis before the test aerosol is passed through a cold trap and a combined activated carbon / particulate filter.If necessary, the parallel operated position de s Kontrollaerosols be extended by such a cleaning unit and online aerosol analysis. The entire flow path of the aerosols above the Nebulizer is heated by an electric winding heater to approx. 37 ° C constantly thermostated.

Example 2: cell culture

From a continuously passaged A549 culture (DSMZ, Braunschweig) cells are harvested during the routine culture split and seeded on membranes (BD Falcon). After a 72 h attachment and growth phase under standard culture conditions, the cells on the membranes are microscopically controlled and provided with fresh medium. Eighteen hours before the actual exposure, the adherently-growing cells become submerged in culture spent in an air / lifted cultural tour. The cells conditioned in this way are used for exposure to the test aerosol or the control aerosol or as incubator control. Following the exposure period, the cells are worked up or post-cultured under submerged conditions in the incubator for further analysis.

Example 3: Analysis of cellular effects

Immediately after exposure, the membranes are removed from the exposure equipment, fitted with fresh medium, and first analyzed for tetrazolium-malt salt by WST-1 assay. WST-1 assay

WST-1 dye reagent (Boehringer, Mannheim) is added to the culture medium supernatant. The cells are reincubated under cell-type-specific conditions in the incubator for 30 min. The measurement of the absorption in the microtiter plate reader at the wavelengths 450 nm / 630 nm is then carried out from the medium.

Measurement of the living cell count

After removal of the medium from the inserts, a trypsin / EDTA solution is added and the cells are reincubated at 37 ° C for 5 min in the incubator. Subsequently, the trypsin effect is stopped by addition of trypsin inhibitor and the cells are carefully suspended. To determine the viable cell count by means of an electronic cell analyzer (CASY, Fa. Sharp), an aliquot of cell trypsinate is suspended in a weak electrolyte and sucked at a constant flow rate through a capillary of defined geometry. Each cell suspension is subjected to a triple measurement.

Example 4: Exposure Experimental groups

Incubator controls are incubated in the incubator for the period of exposure under air-liquid culture conditions.

Exposure controls are cultures that are exposed simultaneously and simultaneously to the aerosol exposure under the same conditions as the cells in aerosol exposure. The only difference with aerosol exposure is that this exposure occurs exclusively against aerosolized PBS. In aerosol exposure, the cultures are exposed for 30 minutes to various concentrations of a freshly generated droplet aerosol of a model substance.

exposure doses

Various concentrations of a model substance are used. The cells are exposed to the generated aerosols at an exposure flux of 5 ml / min / cm ² for 30 min each. To determine the actual exposure dose, for each test substance in cell-free experiments, the deposition of the aerosol is quantified using Na fluorescein (10 μg / ml in the Nebulizer filling) on the microporous membranes. Under conditions of cell exposure, they are overflowed with exposure fluxes of 5 ml / min for periods of 10 to 30 minutes. Thereafter, the membranes are removed, cut out of the holders, extracted with ultrapure water and quantified the fluorescence of the extract at the wavelengths 435 nm (ex.) 510 nm (em.) Against reference solutions of Na-fluorescein in ultrapure water. By cutting out the membranes, only the growth areas are included in the measurement, on which the adherent cell cultures will be located.

For example, the volumes of PBS deposited during the exposure time can be determined by staining the Nebulizer filling with sodium fluorescein and detecting in cell-free experiments. The maximum deposited volume is within 30 minutes at 1 ul / cm ² .

exposure situation

In addition to the modulation of common exposure parameters such as time, concentration or temperature, the medium level ("medium level") below the membrane can also be an influence on the exposure situation of apically adherently cultivated cells when using microporous membranes. Therefore, cell-grown membranes are exposed to the aerosolized model substances or the corresponding PBS control for the duration of exposure as a function of the medium level in up to three different variations: (1) under the conditions of a "high" medium level, and a resulting maximum hydrostatic level Pressure on the microporous membrane (2) minimal "contact" between the membrane and the basal medium, and (3) no membrane and medium contact, a so-called "low" medium level (Figure 2-2).

exposure controls

Exposure controls are used concurrently with each aerosol exposure in parallel construction and are the reference for the aerosol exposure results as they are subject to all exposure factors, except for the effect of the test substance. The cell-grown originating from the respective control situations

Membranes are processed in parallel with the membranes derived from the aerosol exposures. In the following, the results for the incubator controls and the exposure controls depending on the respective exposure day are shown separately after endpoints. The results of the tetrazolium salt conversion (WST-1) after exposure of the A549 cells to a PBS droplet aerosol as a control situation for the aerosol exposures carried out with model substances are shown in FIG. 2 for two exposure days (Di and Mi) as a function of the exposure situation (mean values with standard deviation for medium high, medium contact and medium deep). The data are related to the respective incubator controls currently carried out during each exposure.

The values are in the fluctuation range of the incubator controls and document that the exposure situation has no influence on the tetrazolium salt conversion of the cells. The results of the living cell count determination, which was recorded as a further vitality parameter following the exposure, are compiled in FIG. Here, too, the values on both days of exposure coincide on average Exposure controls in all exposure situations with incubator control values.

With regard to the vitality parameters examined, no effects whatsoever can be observed for the A549 cells in the described exposure control situations even with variation of the medium level. The vitality measurements on control-exposed cells even lead to approximately congruent results as determined for cells under air / liquid conditions in the incubator.

exposures

To create dose-response relationships is tert as an example. Butyl hydroperoxide (tBuOOH) in various concentrations of 0.02M to 1 M in the construction nebulized and its effect on the cells by determining the metabolic activity (WST-1 assay) and living cell count investigated. The medium level "contact" is used.

Example 5: Evaluation

FIG. 4 shows the results of this series of experiments. The results from the study of the tetrazolium salt conversion at the median level "contact" against the dose determined by the deposition study are shown: Measurement points taken into account for the regression are marked with "x", not taken into account with "o." The plotted curves correspond to the regression line with The corresponding confidence limits (95%) A dose-response relationship is calculated which describes the complete vitality range of 100% to 0% within the investigated doses compared to the PBS-exposed control.

The corresponding representation of the results for determining the cell number is plotted in FIG. From these dose-response curves, characteristic values, for example ED ₅₀ values (value at which the measured signal of an end point, eg metabolic activity in the WST-1 assay, is reduced by 50% compared to the control culture) can be calculated allow a direct comparison of effects of different substances. For tBuOOH, this value, based on the measurement of the metabolic activity (WST-1 assay) under the conditions mentioned, is 0.05 μmol / cm ² or 0.08 μmol / cm ² for the cell number.

Claims

Claims:

1 . In vitro method for investigating the biological effect of a test substance, characterized in that (a) an aerosol generator and optionally a humidifier are supplied with clean air,

(d) withdrawn from the manifold volume flows of the aerosol and brought into direct contact with prokaryotic or eukaryotic in vitro cultures in a culture / exposure device.

2. The method according to claim 1, characterized in that the cell cultures are cultured in a CULTEX® module and brought into contact with the aerosol.

3. The method according to claim 1, characterized in that defined by the aerosol generator aerosol concentrations of the substance can be generated.

4. The method according to claim 1, characterized in that the finely distributed by the aerosol generator substances are solid or liquid.

5. The method according to claim 4, characterized in that as aerosol generator, a Respimat® or Handihaler® is used.

6. The method according to claim 1, characterized in that the volume flow for the sampling can be varied.

7. The method according to claim 1, characterized in that the investigations are directed to

The effectiveness of active substances or combinations of active substances of particulate aerosols, depending on their particle size and optionally the carrier material used;

The bioavailability of active substances or combinations of active substances of particulate aerosols as a function of their particle size and optionally the carrier material used;

The inflammatory potential of gases, aerosols or mixtures for cell cultures;

The cellular reaction pattern of the cell cultures after direct contact with the gases, aerosols or their mixtures; • molecular biology aspects of the direct contact of the cells with the

Gases, aerosols or mixtures thereof.

8. The method according to claim 7, characterized in that the molecular biological aspects by receptor studies, the determination of mediators or other biomarkers or by analysis of

Signal transduction pathways are investigated.

9. An arrangement for examining the biological effect of an inhaled substance comprising (a) a humidifier;

(b) an aerosol generator for solid or liquid substances which uses humidified clean air of the humidifier as a carrier gas;

(c) a manifold which is charged with the generated aerosol; and

(d) one or more cell culture units associated with the manifold via one or more sampling points; characterized in that the generated aerosol stream is passed from a sampling point to the connected cell culture unit where it is brought into direct contact with the cultured cells.

10. Arrangement according to claim 9, characterized in that in addition one or more online analyzers for measuring aerosol parameters are connected via sampling points to the distributor and as well as the cell culture units are loaded with samples from the distributor

1 1. Arrangement according to claim 9, characterized in that in order to avoid condensation effects, a thermal conditioning of all components is realized.