EP1664727A1

EP1664727A1 - Device and method for automatically carrying out laboratory procedure steps

Info

Publication number: EP1664727A1
Application number: EP04787010A
Authority: EP
Inventors: Peter RÖHNERT; Frank Striggow
Original assignee: KeyNeurotek AG
Current assignee: KeyNeurotek AG
Priority date: 2003-09-24
Filing date: 2004-09-24
Publication date: 2006-06-07
Also published as: DE10344284A1; WO2005031313A1; JP2007506419A; CN1856701A; US20070005169A1

Abstract

The invention relates to a device for automatically carrying out laboratory procedure steps on cell or tissue samples, comprising a number of modular stations, each of which carries out at least one procedure step, in a combined sequence of procedure steps and at least one support unit for the transport of sample containers to and between said stations. The device comprises at least one station from the automatic exchange of liquid media in the sample containers.

Description

Device and method for the automated execution of laboratory work steps

The present invention relates to a device for the automated implementation of laboratory work steps on line or tissue samples, which has a plurality of modular stations, each carrying out at least one work step in a total sequence of work steps, and at least one conveyor unit for conveying sample containers to and between the stations ,

The development of new active pharmaceutical ingredients is extremely time-consuming and costly. Within the conventional drug development process, the identification and validation of therapeutic targets, the discovery of potential drugs and the preclinical phase include steps that need to be optimized in order to reduce the time, costs and risk of product failures in later development phases. The bottlenecks in the preclinical phase are more recently due to advances in so-called "high-throughput screening" (HTS), combinatorial chemistry, computer-assisted biochemistry and virtual pharmacology, which provide a large number of potential candidates for a new drug, been tightened. Since the large number of possible drug candidates can practically no longer be evaluated or can only be evaluated with unreasonable effort in animal experiments, the preselection of promising drug candidates through in vitro tests on cells or cell tissue samples is becoming increasingly important. The in vitro pre-evaluation helps to sort out unsuitable drug candidates even before in vivo screening or to identify particularly suitable targets and drug candidates more quickly.

Even if corresponding in vitro methods for drug screening are relatively time-consuming and inexpensive compared to in vivo methods, the problem remains that cell tissue samples have to be cultured in a complex manner and over a longer period of time, so that due to the large number of samples to be processed simultaneously , each of which has to be subjected to a long sequence of complex, chemical or analytical processing steps, there is a need for automation of the laboratory work steps.

WO 02/49761 proposes an automated laboratory system, in particular for carrying out the PCR method. The device comprises a plurality of modular stations, each of which carries out at least one work step in a total sequence of work steps, and a modular station which passes material to another station without human intervention.

DE 198 53 184 A1 discloses a device for conveying a carrier element, in particular a titer plate, which has a plurality of sample wells to and between storage and / or processing stations, with a linear and in particular linearly connecting a loading and / or storage unit with at least one processing station Conveyor unit for transporting the carrier element, the processing station being equipped in a modular manner with at least one functional unit designed to carry out an automatable processing function on the carrier element or its test troughs, and for transferring a carrier element between the conveyor unit and functional unit, the processing station has a pivoting device which has at least one turntable or rotating arm , which is designed such that, in response to an electronic control signal, a support element held by the pivoting device by a predetermined angle by a rotation pivoted and can be transferred from the swivel device to an adjacent unit by moving. This strikes as functional units Document an incubator, a dispenser, a pipettor and a reading unit for a preferably optically readable identification provided on the carrier element.

The known laboratory automation devices are less suitable for handling line or tissue samples, since the cultivation and processing of line and tissue samples requires special conditions over a longer period of time. It is therefore an object of the present invention to provide a device for the automated execution of laboratory work steps on cell or tissue samples, with which these samples can also be cultivated over a longer period of time.

This object is achieved by a device for the automated execution of laboratory work steps on line or tissue samples, which has a plurality of modular stations, each carrying out at least one work step in a total sequence of work steps, and at least one conveyor unit for conveying samples to and between the stations and is characterized in that it has at least one station for automatically changing liquid media in the sample containers (media changing station).

The laboratory work steps can be part of complex and highly complex test procedures.

The device according to the invention has several modular stations, which can be known automatic pipetting, dosing or analysis machines, such as are described for example in WO 02/49761 or DE 198 53 184. The conveyor unit disclosed in DE 198 53 184, for example, is suitable as a conveyor unit for conveying sample containers to and between the stations.

The device according to the invention preferably comprises a

Sample injection station, at least one incubator station, one

Media change station, a data acquisition station and one

Probenausschleusungsstation.

The cell or tissue samples, for example juvenile cell tissue samples, in particular juvenile brain tissue samples (for example as hippocampus tissue sections), but also heart or liver tissue samples, such as cardiac atrial sections, are introduced into the device according to the invention in the sample introduction station. Are suitable for this Sample containers in the form of plates with one or more recesses, which can contain membrane inserts, the plates each having a cover plate. Corresponding sample containers are commercially available, for example, as so-called 6-hole plates (also multi-dishes) and are used as tissue culture plates. These plates consist of a base plate and a removable cover. The base plate contains six wells. A membrane insert can be inserted into each recess. These are bowls, the bottom of which is designed as a membrane, for example in the form of a glass frit. At the sample introduction station, the membrane inserts are loaded with tissue samples, especially manually. For example, the tissue samples can be hippocampal sections from juvenile rats or heart or liver sections. Both animal and human tissue samples can be used.

Usually, the device according to the invention provides a sample container at the sample introduction station on request by the operator, the recesses of which are already filled with a desired medium in a suitable amount. The sample container preferably contains water, in particular bidistilled water, between the wells in order to set a desired atmospheric humidity in the container. At the sample introduction station, the tissue sections are then placed in the membrane inserts, and the sample container is then automatically transferred to the conveyor unit of the device.

Previously, the device according to the invention had already taken an empty sample container from a storage container, for example, and filled the wells with the desired medium in a suitable amount and, if desired, the space between the wells with water in the media changing station. The multishell can then be equilibrated in an incubator station, in particular an oxygen incubator station, before being made available at the sample introduction station. This happens automatically for a predetermined period of time. Alternatively, the device can take over the empty sample container at the sample introduction station instead of from a storage container.

The sample container, which is preferably designed as a multishell, is then conveyed by the conveyor unit, for example, to an incubation station and stored there under controlled conditions (for example temperature and gas composition). In one possible embodiment, the device according to the invention comprises at least two different incubator stations, an oxygen incubator station and a nitrogen incubator station. The climatic conditions in the oxygen incubator can be, for example, 95% air and 5% CO ₂ at 36 ° C, the climatic conditions in the nitrogen incubator 95% N ₂ and 5% CO ₂ at 36 ° C.

For the successful cultivation of cell and tissue samples, the media used for storing the samples, in particular nutrient media, must be changed regularly. For this purpose, the sample containers are conveyed from the conveyor unit to the media changing station. The media changing station can have a unit for removing a liquid medium from the wells of the plate of a sample container and a unit for adding one or more liquid media into the wells of the plate of the sample container. Since the sample containers can be provided with a cover plate, the media changing station can also comprise a unit for removing and fitting such a cover plate.

In particular, when using multi-shells with membrane inserts as sample containers, there is also the problem that the liquid medium is located below the membrane insert in the recesses of the multi-shell. In order to make it easier to remove the liquid medium from the recesses in the multishell, the media change station can therefore additionally be equipped with a unit for removing and inserting the membrane inserts from the recesses in the plate of a sample container.

In particular, if several different liquid media are added to a recess in the plate of the sample container, it has also proven to be advantageous if the media changing station has a unit for mixing liquid media in the recesses of a plate of a sample container.

The media change station therefore preferably works as follows:

A sample container, preferably a multi-tray, is transferred from the conveyor unit of the device according to the invention to the media changing station. The media change station automatically recognizes whether the sample container has a cover plate. If the sample container has a cover plate, this is removed by the unit for removing the cover plate. This can be done, for example, by a robot gripper that can be used with a pair of pliers to grip the cover plate or, for example equipped with a suction device for suctioning the cover plate by means of vacuum.

The membrane inserts, if present, are then automatically removed from the recesses in the plate of the sample container. This can be done, for example, by robot grippers that can grip one or more of the membrane inserts simultaneously or one after the other and can remove them from the recesses. For example, the robot gripper can have a link that engages in the membrane inserts and spreads gripping elements apart there. The unit for removing the membrane inserts preferably has as many grippers as the sample containers used have membrane inserts so that the unit can grip and remove all membrane inserts of a sample container at the same time.

The unit for removing and inserting the membrane inserts should advantageously be able to selectively remove only one of the several membrane inserts from a sample container, so that, for example, samples identified as unusable can be sorted out during the experiment. This also enables samples from different origins, for example, to be mixed (pooled) in a sample container during an ongoing experiment.

Since the membrane inserts can contain extremely sensitive tissue samples, it has also proven to be advantageous if the media changing station also has a unit for the intermediate storage of membrane inserts under controlled conditions. This can be, for example, a multi-dish which contains suitable nutrient medium in the wells at a desired temperature. The unit for removing the membrane inserts can then insert the membrane inserts, which are removed from the sample container that has been transferred to the media changing station, into the recesses of the multi-dish, so that the tissue samples only have to remain separated from a nutrient medium for a short time.

In order to avoid carryover of media from a sample container into the unit for the intermediate storage of membrane inserts or from this unit into another sample container, the device according to the invention can furthermore have a unit for removing liquid medium from membrane inserts. This can be, for example, a filter paper, a fabric or a sponge on which the membrane inserts are set down and dried before being inserted into or after being removed from the unit for temporarily storing the membrane inserts.

After the membrane inserts have been removed from the plate of a sample container, the latter is conveyed in the media changing station to a unit for removing the liquid medium from the recesses of the plate of the sample container. This can be done, for example, using an automatic robot gripper or an assembly line. Alternatively, in the case of a stationary sample container, the unit for removing the membrane inserts can be exchanged for the unit for removing the liquid medium. The liquid medium can be removed from the wells of the plate, for example, by automatically tipping the liquid medium out of the wells or by suctioning off the liquid medium. It has proven to be advantageous if the plate is inclined, for example, by approximately 45 ° to remove the liquid medium, and the medium is then suctioned off. As a result, the medium can be removed from the depressions largely without residue.

The suction can take place, for example, by means of one or more pipes which automatically sink into the recesses from above and suck off the medium by means of a pump. The unit for removing the liquid medium preferably has as many tubes for suction as the sample container used has depressions so that the medium can be sucked out of all the depressions of the sample container used at the same time. It may also be desirable for the liquid medium removal unit to have several different suction tubes for each well of the sample container, which can optionally be used for suction, so that different media can be collected separately from one another, for example in different collecting containers. Alternatively, the medium changing station can have several units for removing the liquid medium, which are automatically selected depending on the liquid medium that is to be removed from the sample container in order to collect different liquid media in different collecting containers separately from one another.

After removing the liquid medium from the wells of the sample container, the latter is conveyed further to a unit for adding one or more liquid media into the wells of the sample container. This can be done either by automatically conveying the sample container, for example by a robot gripper, or by means of an assembly line. Alternatively, with stationary Sample container, the unit for removing the liquid medium can be exchanged for the unit for adding one or more liquid media.

The unit for adding one or more liquid media can be, for example, a pipetting unit with which a predetermined amount of one or more liquid media is introduced from one or more storage containers into a recess in the sample container. The unit can have one or more corresponding pipetting devices in order to be able to fill several different liquid media into the wells without a pipetting unit coming into contact with different media. The addition can take place simultaneously or in succession. The unit can also have as many pipetting units or combinations of pipetting units as the sample containers used have depressions in order to be able to fill all the depressions of a sample container at the same time. Alternatively, the unit can have fewer pipetting units or combinations of pipetting units than the sample container has depressions. In this case, the wells of a sample container are filled one after the other.

In particular if several different liquid media have been filled into a recess of a sample container, it has proven to be advantageous if the media changing station of the device according to the invention additionally has a unit for mixing the liquid media in the recesses of the sample container. This can be, for example, stirrers which engage in the depressions from above and mix the introduced liquid media by stirring. Alternatively, it can preferably be a vibrating table on which the sample containers are automatically placed. The various liquid media in the wells of the sample container are then mixed by shaking the container.

After adding the liquid medium or the liquid media into the wells of the sample container, the unit for inserting the membrane inserts reinserts them into the wells of the sample container. If necessary, the membrane inserts are removed from the intermediate storage unit for this purpose and, if desired, cleaned from the membrane inserts by the liquid medium removal unit. When inserting the membrane inserts into the wells of the sample container, it is important that the device according to the invention inserts the membrane inserts in such a way that no air bubbles form between the surface of the filled liquid medium and the underside of the membrane insert, since these form the Exchange of substances between the medium and, for example, the tissue sections on the surface of the membrane could hinder.

After inserting the membrane inserts into the wells of the sample container, the cover plate is possibly replaced and the multiplate is returned from the media change station to the conveyor unit, which automatically conveys the sample container to the next modular station, for example an incubator station.

In the course of the overall sequence of work steps, the cell or tissue samples can, for example, be exposed to disease-simulating conditions in order to then examine the effects of these conditions on the samples and / or the influence of test substances on the samples under these conditions. For this, e.g. in the media changing station, a nutrient medium is exchanged for another medium that does not contain the nutrients required by the tissue, in order to simulate a nutrient deficiency. The samples can also be incubated in a nitrogen incubator, for example, instead of in an oxygen incubator, in order to simulate an oxygen deficiency.

Simultaneously or alternatively, in a station for adding reagents to the sample containers, one or more test substances, the effect of which on the cell tissue samples is to be tested, can be added to the liquid media in the wells of the sample containers. The station for adding reagents is preferably coupled to the media changing station in order to avoid unnecessary work steps. At the same time or independently of the media change, the desired substance can then be introduced in a suitable quantity into the wells of the sample container, for example by means of an automatic pipette.

The substances added can, for example, also be specific substances for inducing or simulating different disease-related events (for example lipopolysaccharides (LPS) and / or specific cytokines for inducing inflammatory events). In addition, molecular biological tools (e.g. small interference RNA (siRNA) or antisense sequences) or molecular biological constructs (e.g. viral vectors) can be added to manipulate tissue-specific gene expression with the aim of either disease-relevant situations (e.g. Alzheimer's disease). similar pathophysiology) or to identify or validate drug-relevant target structures (targets).

As an alternative or in addition to the test substance, the effect of which on the cell tissue samples is to be tested, a substance which supports the recording of measurement data of the cell tissue samples can optionally be added from the station for adding reagents to the wells of the sample containers. Such a substance can be, for example, propidium iodide (PI), which is suitable for staining nucleic acids. Appropriately stained samples can then be examined in the data acquisition station using fluorescence microscopy. As additional substances to support the acquisition of measurement data (markers / readouts), for example, specific apoptosis markers, proliferation markers, cellular differentiation markers and fluorescence-labeled antibodies are suitable for the visualization of distinct proteins.

In order to be able to determine the influence of the incubation conditions (e.g. composition of the liquid or gaseous medium) and / or of test substances on the tissue samples, the device according to the invention preferably also includes a data acquisition station in which, for example, a microscope unit (e.g. a confocal, non-confocal or Infrared microscopy unit) relevant data of the samples can be recorded at the beginning, during or at the end of the overall sequence of work steps. For example, transmission images and / or fluorescence images of corresponding tissue sections can be taken under the microscope and / or after staining with PI. A comparison of the different recordings before, during and after the corresponding treatment steps then enables the operator to determine whether and, if appropriate, to what extent the test substances used and / or changes in the incubation conditions have effects on the tissue cultures.

The transmission microscope unit can take transmission pictures fully automatically. For this purpose, a picture of the complete membrane insert is first made and the position of the individual tissue sections in a membrane insert is automatically recognized by suitable software. The individual tissue sections are then individually enlarged to record the transmission images. The same can be done for the acquisition of the fluorescence images. Devices for flow cytometry (FACS analysis), laser-based microdissection, mass spectrometry (e.g. MALDI / TOF analysis), spectroscopy (e.g. IR spectroscopy, fluorescence correlation spectroscopy) and electrophysiology (e.g.

Multi-electrode arrays). Suitable data acquisition stations can be selected by the person skilled in the art depending on the experiment carried out and the markers used.

After the end of the experiment, the sample containers are preferably conveyed by the conveyor unit to a sample ejection station and collected there, for example, in order to later be able to be either disposed of or recycled. The device according to the invention can also have several, for example two, sample ejection stations for sample containers, for example with different liquid media, if these media are to be collected and disposed of separately from one another.

In one possible embodiment, the device according to the invention has a combination of the following stations: a sample introduction station in which cell tissue samples are introduced manually into membrane dishes which are located in depressions of a multi-dish, the depressions containing a nutrient medium for the cell tissue samples, an oxygen incubator station in which the Cell tissue samples are cultured, a media change station in which the nutrient medium in the multi-dishes is changed after predetermined time intervals, a reagent addition station in which one or more test substances, the effects of which on the cell tissue samples are to be tested and / or, if appropriate, one or more substances which increase the uptake Support of measurement data of the cell tissue samples, into which wells of the multi-dishes are added, optionally a nitrogen incubator station, in which the cell tissue samples are additionally incubated as needed in the course of the experiment t, a data acquisition station in which measurement data of the cell tissue samples are recorded, for example before and after addition of a test substance and / or cultivation in the nitrogen incubator, in order to determine their influence on the cell tissue samples, a sample ejection station in the multishells after completion of the examinations from the device are discharged, and a conveyor unit for conveying the multishells from the sample injection station to and between the stations up to the sample discharge station. The device according to the invention has the advantage that, for example, tissue sections can be cultivated fully automatically and the effects of test substances and disease-simulating conditions on these tissue sections can be examined. The device according to the invention thus enables functional, tissue-based screening, in particular on the basis of disease-related effects. This overcomes a bottleneck between the very fast HTS processes, which deliver a large number of possible drug candidates, and the comparatively slow and, in particular, costly in vivo tests. The present invention thus also relates to the use of the device according to the invention for carrying out investigations of chemical, biological and / or physical influences on physiological or pathophysiological processes on samples such as cell tissue samples. In particular, the device can be used to test the effect of substances on samples, preferably on cell tissue samples. Corresponding uses are described, for example, with reference to vital mammary heart tissue cells kept ex vivo in DE 103 22 986.8 from KeyNeurotek AG.

In addition, a method for testing the effect of substances on samples, preferably on cell tissue samples, is provided, which comprises introducing samples into a device according to the invention and evaluating measurement data recorded in the data acquisition station. This method has the particular advantage that only the samples have to be prepared manually and the measurement data have to be evaluated manually. The laborious, time-consuming and cost-intensive intermediate steps, even of the most complex test sequences, are carried out fully automatically by the device according to the invention. A large number of different samples and test substances can therefore be examined simultaneously.

Another advantage of the device according to the invention is its modular structure, which makes it possible, depending on the course of the desired experiment, to exchange individual modular stations for others, or the device, for example to increase capacity with additional incubators or e.g. can also be provided with an additional media change station. In principle, the device according to the invention is not subject to any capacity limits, since its capacity can be increased at any time by adding further modular stations.

The present invention will now be explained in more detail on the basis of a specific exemplary embodiment, but without restricting it to this. The attached FIG. 1 shows schematically a top view of a possible embodiment of the device according to the invention.

Figure 2 shows the perspective view of another possible embodiment of the device according to the invention.

1 and 2 show a sample injection station 1, two oxygen incubation stations 2 and 2 ", a nitrogen incubator station 3, a media change station 4, a data acquisition station 5, a sample container supply station 6 and (only in FIG. 1) two sample discharge stations 7 and 7 'and a conveyor unit 8 for conveying the sample containers to and between the stations.

The use of the device according to the invention for screening possible active substances against cerebral ischemia is described by way of example below. Normal brain functions depend largely on the constant supply of glucose and oxygen. An interruption in cerebrospinal blood flow due to a stroke, brief heart failure or as a side effect of cardiac surgery quickly leads to ischemic conditions in the brain and then neuronal cell death. To determine the effect of test substances on cerebral ischemia, hippocampal sections can first be cultivated according to known protocols and then exposed to ischemic conditions through oxygen and glucose deprivation. Damage to neuronal cells from these conditions can then e.g. can be visualized by the intracellular fluorescence of added propidium iodide (Pl), which is only incorporated into damaged cells. A possible positive effect of substances to be tested on the ability of neuronal cells to survive ischemic conditions is determined in a comparative experiment under the same conditions with simultaneous addition of the test substance to the sewing medium.

The laboratory work steps required for this can be carried out by the device according to the invention as follows, for example. First, empty multishells with membrane inserts inserted into the depressions are introduced into the device via the sample introduction station 1. The conveyor unit 8 conveys the multishells to the media changing station 4, in which bidistilled water between the wells and glucose medium is filled into all the wells of the multishells. Then be the multishells are conveyed by the conveyor unit 8 into an oxygen incubator 2 or 2 ', where they remain between 20 minutes and 24 hours for equilibration. The multishells are then conveyed from the oxygen incubator 2 or 2 'to the sample introduction station 1. There, the membrane inserts of the multi-shells are loaded by the operator with hippocampal sections that were previously prepared manually. The conveyor unit 8 then conveys the loaded multi-dishes back into an oxygen incubator 2 or 2 '.

In order that the samples can be clearly sorted at any time, the multishells are preferably marked, for example with a barcode, which can then be read at any time by the conveyor unit and also by the various stations of the device according to the invention and compared with the database.

The hippocampal sections are cultivated in an oxygen incubator 2 or 2 'for a period between six hours and 13 days or longer. During this dwell time, cyclic media changes are carried out (e.g. changing the glucose medium). For this purpose, the multi-dishes are conveyed to the media changing station 4 at intervals of 60 to 72 hours and back into the incubator after the media has been changed. Cultures are cultivated until the operator requests them for inspection, usually after six to 13 days.

In the next step, the samples are pre-checked. For this purpose, a multishell is conveyed from an oxygen incubator 2 or 2 'to the data acquisition station 5. There, transmission images of the complete membrane inserts and the individual sections of a membrane insert are made in a microscope unit. The multishell is then conveyed back into an oxygen incubator 2 or 2 '.

The individual tissue sections are then evaluated on the basis of the transmission recordings and an experiment specific is assigned to each individual membrane insert of the multishells and communicated to the device according to the invention, for example, via a computer terminal.

In accordance with the assignment concerned, the membrane inserts of the multi-dishes are either removed if they have been identified as unsuitable, or they are put together again in multi-dishes, such membrane inserts which are to be subjected to the same cultivation conditions being combined in one multi-dish. This "pooling" can take place, for example, in the media changing station by means of the unit for removing and inserting membrane inserts from multi-shells and the intermediate storage of multi-shells in the unit for the intermediate storage of membrane inserts under controlled conditions. At the same time, the database of the device is updated accordingly, so that the individual samples in the multishells can be clearly assigned even after the membrane inserts have been pooled.

To carry out the experiments, each membrane insert is assigned, for example, one of three possible conditions via the experiment specifics:

Condition 1 OGD without addition of substance (s), condition 2 OGD with addition of substance (s), condition 3 addition of substance (s) without OGD.

As used herein, "OGD" means that the cultures have ischemic conditions, i.e. be subjected to an oxygen and glucose deprivation (OGD).

The individual conditions can be further specified, e.g. by specifying the type and amount of the test substance to be used, the duration of the OGD, etc.

Approximately one hour before OGD, the multishells with membrane inserts, which are intended for condition 2 or condition 3, are conveyed from the conveyor unit 8 to the media changing station 4. There, the glucose medium is exchanged for a glucose / test substance medium, and the multishells are then conveyed back into an oxygen incubator 2 or 2 '.

Before OGD, empty multishells without membrane inserts are also introduced into the device either via the sample container supply station 6 or via the sample introduction station 1 and conveyed from the conveyor unit 8 to the media changing station 4. Exactly the number of new multi-shells is introduced which corresponds to the number of multi-shells which contain membrane inserts for the upcoming experiment, which are assigned to condition 1 or condition 2. In addition, a system-internal complementary assignment of the new multi-shells takes place, with exactly one newly introduced multi-shell being uniquely assigned to one multi-shell, which is for the upcoming one Experiment contains membrane inserts that are intended for condition 1 or condition 2.

In the media changing station 4, double-distilled water is filled between the wells of the newly introduced multi-dishes. In addition, a mannitol medium is placed in those wells of the newly introduced multishells in which there are membrane inserts on the complementary multishell intended for condition 1, and a mannitol / test substance medium in those wells of the newly introduced multishells in which there are complementary multi-shell membrane inserts, which are intended for condition 2, filled. The newly introduced multishells are then conveyed by the conveyor unit 8 into a nitrogen incubator 3, where they are equilibrated between 20 minutes and 24 hours.

Approximately one to five minutes before OGD, those multishells containing the membrane inserts with the tissue sections intended for condition 1 or condition 2 and the respective complementary multishells with the mannitol medium are conveyed from the conveyor unit 8 to the media change station 4. There, the membrane inserts, which are intended for condition 1 or condition 2, are converted from the multishells with the glucose medium into the complementary multishells with the mannitol medium. The multishells with the mannitol medium are conveyed to the OGD in the nitrogen incubator 3 and the multishells with the glucose medium in an oxygen incubator 2 or 2 '.

For OGD, the multishells with the mannitol medium remain in the nitrogen incubator 3, for example, for five to 90 minutes or longer, depending on the experiment specificity. Immediately after OGD, the multishells with mannitol medium from the nitrogen incubator 3 and the respective complementary multishells with the glucose medium from an oxygen incubator 2 or 2 'promoted to the media change station 4. There, the membrane inserts, which are intended for condition 1 or condition 2, are converted from the multishells with the mannitol medium into the complementary wells of the complementary multishells with the glucose medium, and the multishells with the glucose medium are transferred from the delivery unit 8 into an oxygen incubator 2 or 2 'promoted. The empty multi-dishes with the mannitol medium are discharged via the sample ejection station 7 or 7 '. Multishells with membrane inserts which are intended for condition 2 or condition 3 are conveyed from conveyor unit 8 to incubator station 4 approximately one hour after OGD and in those wells of the multishell which contain membrane inserts for condition 2 or condition 3, a media change with glucose medium is carried out. The multi-dishes are then conveyed into an oxygen incubator 2 or 2 'by incubating for a period of between two and 72 hours.

If, according to the experiment specification, the OGD is to be carried out several times for some multishells, the steps of the experiment cycle described above are repeated accordingly.

For the evaluation of the experiment, the multishell is conveyed from the conveying unit 8 from an oxygen incubator 2 or 2 'to the media changing station 4 approximately two hours before the data acquisition, and propidium iodide (PI) is added to the wells of the multishell in a predetermined concentration. The multi-dishes are returned to an oxygen incubator 2 or 2 'and remain there for about two hours. Immediately prior to the data acquisition, Pl can again be added in the media changing station 4 in the same or a different concentration to the recesses of the multishell.

The multishells are then conveyed to the data acquisition station and automatically transferred to the microscope unit there. There, transmission and fluorescence images of the complete membrane insert and the individual sections of a membrane insert are made under the microscope. The multishells are then taken over from the data acquisition station 5 by the conveyor unit 8 and, depending on the medium, are discharged via a sample ejection station 7 or 7 '.

The experiment can be evaluated manually by the operator using the transmission and fluorescence images.

The present invention is not limited to the above embodiment. For example, the device can be supplemented with additional stations or individual stations can be exchanged for others in order to adapt them to a corresponding test sequence. There is also the possibility within a respective station given to adapt it flexibly to a desired test procedure due to the low set-up or changeover effort.

The device according to the invention enables a large number of test substances to be tested sequentially or in parallel with little manual effort for their effect on a tissue sample under normal conditions or under disease-simulating conditions in vitro. In this way, for example, the preclinical development of pharmaceuticals or the preclinical development of potential pharmaceuticals is significantly accelerated, so that they can be carried out more cost-effectively for a large number of substances. In addition, constant test conditions are ensured by the automated work steps, so that the measurement data obtained are highly reproducible and meaningful.

Claims

claims:

1. Device for the automated implementation of laboratory work steps on cell or tissue samples, which has a plurality of modular stations, each carrying out at least one work step in a total sequence of work steps, and at least one conveyor unit for conveying sample containers to and between the stations, characterized in that the device has at least one station for automatically changing liquid media in the sample containers (media changing station).

2. The apparatus of claim 1, comprising a sample injection station, at least one incubator station, a media change station, a data acquisition station and a sample discharge station.

3. Apparatus according to claim 2, which has an oxygen incubator station and optionally a nitrogen incubator station.

4. Device according to one of the preceding claims, which has a station for adding reagents to the sample containers.

5. Device according to one of the preceding claims, the sample container in the form of plates with one or more recesses, which may contain membrane inserts, wherein the plates can each have a cover plate.

6. Device according to one of the preceding claims, wherein the media changing station optionally a unit for removing and fitting a cover plate of a sample container, optionally a unit for removing and inserting membrane inserts from wells of a plate of a sample container, a unit for removing a liquid medium from wells of a Plate of a sample container, a unit for adding one or more liquid media in the wells of a plate of a sample container and optionally a unit for mixing liquid media in the wells of a plate of a sample container.

7. The device according to claim 6, wherein the media changing station further comprises a unit for the intermediate storage of membrane inserts under controlled conditions.

8. The device according to claim 6 or 7, wherein the media changing station further comprises a unit for removing liquid medium from membrane inserts.

9. Device according to one of claims 2 to 8, wherein the data acquisition station has a microscopy unit, a flow cytometer, a mass spectrometer, a spectroscope and / or devices for laser-based microdissection and / or electrophysiology.

10. Device according to one of the preceding claims, comprising: a sample introduction station, in which cell tissue samples are introduced manually into membrane dishes which are located in depressions of a multi-dish, the depressions containing a nutrient medium for the cell tissue samples, an oxygen incubator station in which the cell tissue samples are cultivated a media change station, in which the nutrient medium in the multi-dishes is changed after predetermined time intervals, a reagent addition station, in which one or more test substances, their effect on the cell tissue samples are to be tested and / or, if applicable, one or more substances which are used to record measurement data of the cell tissue samples, into which wells of the multishells are added, optionally a nitrogen incubator station, in which the cell tissue samples can also be incubated as needed in the course of the experiment, a data acquisition station, in the measurement data of the cell tissue samples, for example before and after addition of a test substance and / or cultivation, are recorded in the nitrogen incubator in order to determine their influence on the cell tissue samples, a sample ejection station in which multi-dishes are discharged from the device after completion of the tests, and a conveyor unit for Conveying the multishells from the sample injection station to and between the stations to the sample injection station.

1 1. Use of a device according to one of claims 1 to 10 for carrying out studies of chemical, biological and / or physical influences on physiological or pathophysiological processes on samples, preferably cell tissue samples.

12. Use according to claim 11 for testing the effect of substances on samples, preferably on cell tissue samples.

13. Use according to claim 11 for the identification and validation of therapeutic targets.

14. A method for testing the effect of substances on samples, preferably on cell tissue samples, which comprises introducing samples into a device according to one of claims 1 to 10 and evaluating measurement data recorded in the data acquisition station.

15. The method of claim 14, which is drug screening.