EP2384363A2

EP2384363A2 - Device for automatically cultivating cells in parallel

Info

Publication number: EP2384363A2
Application number: EP10721643A
Authority: EP
Inventors: Josef Seidl; Michael Wiechmann
Original assignee: Pan-Systech GmbH; PAN SYSTECH GmbH
Current assignee: Pan-Systech GmbH; PAN SYSTECH GmbH
Priority date: 2009-04-22
Filing date: 2010-04-22
Publication date: 2011-11-09
Also published as: JP2012524527A; WO2010121601A3; WO2010121601A4; WO2010121601A2; KR20110133589A; US20120034596A1

Abstract

The invention relates to a device for automatically cultivating cells in parallel in cell culture vessels, in particular microtiter plates (MT plates) (2), having a housing (23) in which are disposed an observation unit (3) comprising at least one microscope (7) and at least one camera (6), a receptacle device (4) for receiving the cell culture vessels, and a fluid distribution unit (5) for automatically filling and/or emptying the cell culture vessels, in particular the wells (17) of MT plates, with fluid, wherein the climatic conditions in the device, in particular the gas composition and temperature, can be regulated at least in the area of the cell culture vessels, wherein a climate chamber (21) is provided in the housing, in which a desired temperature and/or a particular gas composition can be adjusted automatically, and in which the receptacle device (4) having the cell culture vessels (2) is at least partially integrated.

Description

DESCRIPTION:

Device for the automated, parallelized cultivation of cells

The present invention relates to a device for the automated, parallelized cultivation of cells in cell culture vessels, in particular microtiter plates (MT plates).

The cultivation of isolated cells in vitro is a recognized valuable tool in biomedical research to identify reproducible data. However, cell cultivation is still predominantly carried out manually today. Since significant process variations are unavoidable in manual processes and sterility principles are frequently broken, the reproducibility and cell quality suffer, which in turn represents an immediate prerequisite for successful use, for example in drug development. Furthermore, there is an increased risk of incorrect or unstable handling by the human factor, especially when working with a large number of different cell lines. In addition to the quality, the throughput in manual cell cultivation is limited or scalable only by a corresponding staff. Cell cultures are widely used in the pharmaceutical, cosmetics and biotech industries, among others, in the search for new active principles and active ingredients in the pharmaceutical and plant protection sector and have become indispensable. Only standardized, automated cell culture methods with reliable monitoring of all cell culture parameters enable a comparison between the substances and their principles of action over the years, thus providing, among other things, an essential basis for the research and development activity in the biopharmaceutical industry in the areas of cell culture, media development and drug development.

The known from the prior art cell culture methods have been characterized by a high manual effort due to extensive serial testing with difficult documentation and reproducibility. Any desired change in the culture conditions currently leads to high overhead due to lack of flexibility and automation of cell culture and is associated with a high resource (staff, time) with corresponding cost implications for users.

It is an object of the present invention to provide a device for the automated, parallelized cultivation of cells in cell culture vessels, in particular microtiter plates, which overcomes the disadvantages of the prior art.

This object is achieved by a device having the features of claim 1.

The observation unit with a microscope and a camera enables constant observation and optical evaluation of cell growth and cell morphology.

Cell culture vessels can be, for example, cell culture bottles. Preferably, however, the cell culture vessels are MT plates. The MT plates are automatically filled by the fluid distribution unit or unneeded or used fluid is sucked off or replaced.

The modules of the system (fluid distribution unit, receiving device, climate chamber and fluid distribution unit) are arranged in a special housing, so that the device acts as a so-called sterile bench. With this sterile bench, an open fluid and media supply is possible because the risk of bacterial contamination through the housing is greatly minimized.

The entire device usually functions automatically, i.e., the essential steps in culturing the cells, such as e.g. Filling and aspiration of the wells (wells of the MT plates), microscopic observation and evaluation are carried out automatically. As a result, all the work steps can be performed in the closed system of the sterile bench and it must, for example, no MT plates are removed from the system. Due to the controllability of the climatic conditions in the device, the climate (temperature and / or gas mixture) can be adapted from the outside to the respective needs of the cell cultures.

The device according to the invention has a climate chamber in which a desired climate, namely a desired temperature and / or a desired humidity and / or gas composition, is preferably automatically adjustable and in which the receiving device with the cell culture vessels, in particular the MT plates at least partially is integrated, wherein the climate chamber is arranged in the housing. The climate chamber makes it possible to create a specific climate only within a limited area within the enclosure. As a rule, all cell culture vessels used, in particular MT plates with the cell cultures located therein, are arranged inside the climate chamber. In the climate chamber, a specific, optimal for the respective cell cultures Climate set. The temperature is preferably set to values of 20 to 45 ° C. The CO ₂ concentration is usually set to 0 to 20%. The proportion of oxygen within the climate chamber is usually 10 to 40%. The control of the climate within the climate chamber is preferably carried out by appropriate sensors within the climatic chamber. Furthermore, it is achieved by the climate chamber that a further separation of the cell cultures from the outside world takes place and thus the danger of a bacterial contamination of the cell cultures in the cell culture vessels is further minimized.

In a particularly preferred embodiment of the device according to the invention separate gas containers are provided which contain different gases, preferably O ₂ , CO ₂ and N ₂ , wherein the gas streams are controllable from the gas cylinders and wherein certain amounts of gas are passed into a mixing chamber, from where the thus prepared gas mixture is passed into the climatic chamber. In this way, a precisely predetermined gas mixture can be prepared and initially tested for the desired composition. Only then is the gas mixture passed from the mixing chamber into the climate chamber. The adjustment of a particular gas composition is particularly important in the cultivation of cells. For example, tumor cells grow at significantly less oxygen than it does in the ambient air. On the other hand, one wants to investigate, for example, damaged neuronal cells at much higher O ₂ concentrations than those of the tumor cells mentioned. Advantageously, the gas mixture produced in the mixing chamber is brought to a certain temperature before it is passed into the climatic chamber. In this way optimal conditions for different cells to be cultivated can be produced. By way of example, MT plates of a different number and number of wells (eg 6, 12, 24, 48, 96, 384 wells per MTP) can be accommodated by the receiving device. In a preferred embodiment of the device according to the invention, the cell culture vessels, in particular MT plates, preferably in the region of the observation unit and / or in the area of the fluid distribution unit, are preferably transportable automatically from the climate chamber or into the climate chamber by a carriage or robotic arm. In this way, certain selected cell culture vessels, in particular MT plates for filling with fluid can be brought out of the climatic chamber for a short time. Also, certain cell culture vessels can be transported for observation in the area of the observation unit. These targeted, short-term transports from the climatic chamber have no negative effects on the climate within the chamber. The automatic transport of the cell culture vessels from the climate chamber or into the climate chamber replaces a manual movement of the MT plates within the system, which would be associated with a high risk of contamination.

Advantageously, the observation unit and / or the Fluidverteilungs- unit are arranged outside the climatic chamber. This has u.a. the advantage that these units are not exposed to the sometimes extreme climate within the climate chamber. For example, these units are protected from high levels of humidity that could damage these units. Furthermore, contamination of the cell cultures by said units are avoided.

Advantageously, the receiving device comprises at least two receiving units, preferably arranged one above the other, and preferably has holding devices for holding the cell culture vessels, in particular MT plates. The stacked receiving units a serious space savings is achieved, which also leads to a reduction in system costs. By the arrangement one above the other arranged receiving units much more cell culture vessels can be accommodated in the climatic chamber. The holding devices may be, for example, recesses within the receiving units whose shape is adapted to the shape of the cell culture vessels, in particular MT plates.

In a further embodiment of the device according to the invention, a cell culture vessel located in the region of the observation unit, in particular MT plate, can be moved, the movements preferably being controllable.

Advantageously, the optics (microscope incl. Camera) of the observation unit and / or the cell culture vessel to be observed are movable, wherein the movements of the camera or of the cell culture vessels are preferably controllable.

The microscope used in the optics of the device according to the invention can be a normal microscope (transmitted-light microscope) or else a phase-contrast microscope or any other detection device for the observation and / or analysis of cells. However, fluorescence microscopy can also be carried out with the device according to the invention, which is why it is often advantageous if the microscope is a fluorescence microscope.

The optics of the device according to the invention preferably has an automatic focusing. Furthermore, the optics usually has an automatic lens change.

The observation unit is preferably capable of online evaluation of cell morphology, cell physiology, cell behavior and cell growth. The evaluation of the data of the cell cultivations is usually carried out by a PC with a specifically created software and user interface. In a preferred embodiment of the device according to the invention, the fluid distribution unit comprises a plurality of containers for storing different fluids, such as cell solutions, nutrients and dyes, and a microdosing for targeted, controlled addition of the fluids in the cell culture vessels, in particular in the wells of the MT plates and preferably also for removing the fluids from the cell culture vessels, in particular from the wells of the MT plates, wherein the movements and actions of the microdosing unit are preferably controllable. By carrying out the fluid distribution unit with a plurality of containers for storing different fluids, it is possible, for example, to add a plurality of different fluids to specific wells of MT plates. If, for example, fluorescence microscopy is to be used, dyes for fluorescence microscopy can also be introduced into some wells in addition to cell solutions and nutrients. If some wells are again to be freed from fluids, this can also be done by the microdosing unit of the fluid distribution unit. The containers can be positioned around the microdosing unit. In such an embodiment, the microdosing unit is preferably rotatable and / or raised and lowered, so that the microdosing unit can approach the individual containers by means of rotational and / or lifting and lowering movements.

Advantageously, the cell culture vessels, in particular MT plates and preferably also the wells of the MT plates are indicated. In this case, as a rule, each plate and preferably each well is assigned an assignment number (index), which is recognized by the system, in particular by the software described below.

For a particularly precise control of the individual process steps and precise assignment of the cell culture substances (for example cells, fluids) to the individual culture vessels and the culture vessels to the individual process stations (fluid station, incubation station, optical station), preferably a 4-stage indexing takes place. In the first At the level of indexing, each culture vessel is indexed for unique assignment and identification and provided with an internal index / number (for example MTP 1 to MTP 24). In the second stage of indexing, each sub-vessel of a culture vessel (typically each well of an MT plate) is indexed. In the third stage of indexing, each pitch is indexed in the cradle (especially in the climatic chamber) for unambiguous assignment and identification (for example, pitch 1 to 24). In the fourth stage of indexing, each process space of the overall system is indexed for unique assignment, identification and control (eg fluid station = 1, pickup station = 2, optical station = 3).

Particularly preferably, the cell culture vessels are transparent. As a result, all types of microscopy, especially fluorescence microscopy can be performed. Used MT plates are preferably formed very thin, whereby the lenses of the microscope can be brought as close as possible to a cell culture to be observed. The MT plates may, for example, have thin film bottoms. The MT plates may be made of any translucent materials, such as e.g. Glass, plastic, etc. be made.

As already explained above, other cell culture vessels, such as cell culture bottles such as T25 or T75 or closed autoflasks can be used as an alternative to MT plates. In a preferred embodiment of the device according to the invention, said different culture vessels can be used both alternatively and side by side. For this purpose, for example, the receiving device for receiving the cell culture vessels to different adapters, through which an adaptation to different shapes and sizes of culture vessels is possible.

In a particularly preferred embodiment of the device according to the invention, these sensors have the control of all relevant parameters, in particular temperature, gas concentration, in particular CO ₂ concentration, O ₂ concentration, N ₂ concentration, humidity and / or supply of fluids into the cell culture vessels, in particular in the wells of MT plates on. These sensors are the basis for fully automatic control and regulation of the climatic conditions as well as the supply of fluids into the cell culture vessels.

A particularly preferred embodiment of the device according to the invention is characterized by a computer-controlled control and control system for automatic control and control of at least one, preferably all listed below parameters or steps: a) temperature, in particular in the climatic chamber; b) O ₂ content in the gas, in particular in the climatic chamber; c) CO ₂ content in the gas, in particular in the climate chamber; d) N ₂ content in the gas, in particular in the climatic chamber; e) filling and / or draining selected cell culture vessels, in particular selected wells, of selected MT plates with selected amounts of selected fluids; f) microscopic observation and evaluation of cell cultures in selected cell culture vessels, in particular selected wells of selected MT plates at selected times with selected technique, such as fluorescence or phase contrast microscopy.

Preferably, all parameters or work steps are fed into the system. From this point on, for example, the climatic conditions are automatically regulated by the system. Should one or more parameters deviate from the entered values in the course of the cell cultivation cycles (eg as a consequence of a short-term opening of the climatic chamber for transporting an MT plate out or in), this will be immediately readjusted to the specified value. For specific filling of selected wells of selected MT Plates with selected amounts of selected fluids are indicated as the MT plates, and preferably also the wells of the MT plates. As a result, the wells of the MT plates can be entered in a particularly simple manner in the system and recognized by this. With the help of the optics are preferably continuously made of the cultured cells, the recordings are preferably stored by the system. The system also preferably recognizes certain changes in the cell cultures and can thereby react to certain changes in the cell cultures by readjusting the above-mentioned parameters or work steps.

A preferred embodiment of the device according to the invention is characterized by a loading and Entdeckelungsvorrichtung for, preferably automatic loading and Entdeckein the cell culture vessels (see also figure description).

In a preferred embodiment of the device according to the invention, a controlled selective use of disposable and multiple pipettes is possible in the fluid distribution unit. This can prevent cross-contamination between individual culture vessels and wells. In particular, two types of pipettes are used in the device according to the invention. So come on the one hand so-called working pipettes used, which are used several times during a cultivation, which brings the need for a sterile recording and sterile filing. The working pipettes are stored in sterile boxes within the device according to the invention. In the area of the fluid distribution unit, a robot arm generally takes up the working pipettes in a sterile manner from the receiving boxes. Thereafter, the desired Fluidaktionen be performed on the cell culture containers. Subsequently, the robot arm of the fluid distribution unit rests the working pipettes again sterile in the receiving box. This work cycle can take place several times during a cell cultivation. In addition to the working pipettes come in the inventive device usually also disposable pipettes used. These disposable pipettes are used only once during cell culture, with the need for one-time sterile ingestion and safe disposal after a one-time procedure. The disposable pipettes are stored in sterile disposable boxes in the area of the fluid station. In use, the fluid robot arm takes a disposable pipette sterile from a receiving box. Subsequently, the desired fluid action is performed on a cell culture container, in particular an MT plate. After the action, the robotic arm drops the disposable pipette into a waste box. This working cycle takes place only once with a disposable pipette during a cell cultivation.

The present invention further relates to a method for the automated, parallelized cultivation of cells in cell culture vessels, in particular microtiter plates, which are in a cell culture system, in particular in a device according to one of claims 1 to 13, comprising the following steps: a) filling the system with cell culture vessels ; b) filling the system with containers containing fluids to be used, such as cell culture medium, cell suspension, test substance, dye; c) loading the system with containers containing gases to be used, such as O ₂ , CO ₂ , N ₂ ; d) Programming the automatic tuning system for at least one of the following parameters:

Temperature O ₂ content in the gas CO ₂ content in the gas N ₂ content in the gas; e) programming the automatic filling system and / or

Emptying of cell culture vessels, in particular selected wells of selected MT plates with selected amounts of selected fluids, at selected times;

f) programming the system for microscopic observation and evaluation of cell cultures in selected cell culture vessels, in particular in selected wells of selected MT plates at selected times with selected technique, e.g. Fluorescence or phase contrast microscopy or any other method of detection and / or analysis of cells.

In the apparatus according to the invention or in the method according to the invention, the system automatically regulates the climatic conditions in the sense of a control loop for the constant maintenance of the climatic conditions. Furthermore, the system carries out the microscopic evaluation according to the programmed values for the microscopy.

For the optical evaluation, multiple freely selectable observation points (points of interest = POI) in the culture vessel or in the subunits (for example POI 1 to 10 in the well 2 of the culture vessel 4) can be defined in each culture vessel. Here, a specific control software with a user interface can freely choose which POI is to be evaluated in which culture vessel at which time and which optical method (for example transmitted light, phase contrast, fluorescence). Particularly preferably, each POI can be approached by the traversing unit (cross table) in three axes x-y-z (ie horizontal and vertical), focused by the system, recorded, evaluated and stored. Owing to the possibility of this three-dimensional recording and storing of POIs, different levels can be focused in a culture vessel or in a subunit of a culture vessel.

Each POI can be approached and evaluated very accurately over time with a precision of <1 μm. However, in addition to individual images and individual image evaluations, you can also use time-lapse photography and dynamic evaluations of each POI are made possible by composition of the individual images. Furthermore, automatic analysis of the optical image information by means of a specific software and user interface is usually possible (for example cell number, cell movement, cell shape, color information of fluorescently labeled cell components, number of stained cells).

Furthermore, the system can remove used media and feed new media.

Filling the system with cell culture vessels and filling the system with containers containing fluids to be used is usually done manually. This also applies to the loading of the system with containers containing gases to be used. For this purpose, the device according to the invention may have a further housing, which may contain the said gas cylinders.

In summary, the following advantages of the device according to the invention and of the method according to the invention can be mentioned, inter alia:

Automated, parallelized cultivation of cells in cell culture vessels, especially in microtiter plates in a complete system with automatic control of all cell culture parameters

(eg temperature, CO ₂ concentration, O ₂ concentration, N ₂ concentration, air humidity, fluid support, active substances);

- low costs;

- open system platform solution for the biopharmaceutical industry to both develop new improved cell lines and to screen and develop new drugs efficiently and highly reproducibly;

low space requirement and uniform, intuitive operation with ringer training time for the operating personnel;

economic use through multiple use of expensive components;

simple, ergonomic, modular design, durable components with high biocompatibility;

allows parallel, simultaneous cultivation of different cells under identical conditions;

Possibility of continuous microscopic observation and documentation and storage of all relevant data for later retrieval and evaluation;

direct measurement of the effects when changing the culture conditions and possibility of evaluation depending on the composition of the medium;

Immediate recognition of morphological changes and improvement of developmental quality through meaningful results;

Possibility of setting optimal concentrations of individual substances and stepless change of culture conditions;

Reduction of development times by a factor of 2 to 4 through parallelization (for example reduction of the time for media optimization from 12 months to 3 to 6 months);

Reduction of development costs through automation by a factor of 2 to 3 (for example reduction of media optimization costs from formerly 75,000 EUR to 25,000 EUR to 40,000 EUR);

automated performance of complex cell cultures e.g. for toxicity studies to reduce the need and the number of

Animal experiments. The following fields of application are possible with the device according to the invention or the method according to the invention (by way of example):

Development of serum-free systems;

- nutrient media optimization;

- stem cell research;

Klonentwicklung;

- drug discovery;

Cell cycle studies;

- cell interactions;

- organ simulation;

Cytotoxicity assays;

Cell processing;

Development of therapeutic approaches on a cellular basis;

Automated execution of complex cell cultures to reduce animal testing.

Further features of the invention will become apparent from the following description of preferred embodiments of the invention in conjunction with the drawings and the dependent claims. In this case, the individual features can be implemented individually or in combination with each other. In the drawings show:

1 shows a side view of a device according to the invention (without housing and climatic chamber);

FIG. 2 is a perspective view of the device of FIG. 1; FIG.

Fig. 3 is a plan view of the device of Fig. 1;

FIG. 4 shows a perspective view of the device of FIG. 1 with climate chamber; FIG.

FIG. 5 shows the device of FIG. 4 with housing; FIG.

Fig. 6: control and regulation scheme for a device according to the invention.

7 shows a perspective illustration of a de-capping device in a device according to the invention

FIG. 8 is a plan view of the de-capping device of FIG. 7. FIG.

FIG. 1 shows a side view of a device 1 according to the invention for automated, parallelized cultivation of cells in microtiter plates 2 with an observation unit 3, a recording device 4 for receiving the microtiter plates 2 and a fluid distribution unit 5. The observation unit 3 comprises a CCD camera 6 , and a microscope 7. The camera 6 and the microscope 7 are arranged on a holder 8. The observation unit 3 further comprises a receiving table 9 for receiving the MT plates 2.

The receiving device 4 comprises four superimposed receiving plates 10. The receiving plates 10 have (not shown here) recesses into which the MT plates are fitted. The receiving device 4 can be raised or lowered. Within the receiving plates 10 of the receiving device 4, the MT Plates 2 are moved. In the present illustration, it is shown how an MT plate 2a is transported by the receiving device 4 into the region of the observation unit 3 with the aid of a carriage 11. By the lifting and lowering movements of the receiving device 4 and by the transportability of the MT plates 2 within a plane MT plates, for example, the observation unit 3 or the fluid distribution device 5 can be selectively supplied.

The fluid distribution unit 5 comprises a microdosing device 12 with a movable, in particular rotational movements and / or lifting and lowering movements exporting arm 13, which is provided with a fixed or exchangeable pipette 14. The fluid distribution unit further comprises a plurality of containers 15, in which different fluids, such as cell suspensions, nutrients, dyes, etc., are included. The containers 15 are arranged in a certain arrangement around the microdosing device 12, wherein the pipette 14 by rotational movements and / or lifting and lowering movements of the Verteilarms 13 and / or by movement of the pipette along the upper portion 16 of the Verteilarms 13 to the individual containers can be brought. To fill the wells 17 of the MT plates 2, the MT plates 2 can be automatically transported by the receiving device 4 into the region of the fluid distribution unit 5 by means of a carriage. In the present representation, an MT plate 2b is arranged in the region of the fluid distribution unit 5 and is filled there with the aid of the microdosing device 12 with specific fluids.

Figure 2 shows a perspective view of the device of Figure 1. Good to see in this illustration, the superimposed receiving plates 10 of the receiving device 4. The individual receiving plates 10 have recesses 18 which are dimensioned so that the MT plates 2 received therein and can be kept. FIG. 3 shows a plan view of the device 1 of FIG. 1 and FIG. 2. In addition to the actual working space, which is located below the housing (not shown here), a receiving chamber 19 for receiving gas cylinders 20 is arranged.

FIG. 4 shows the device 1, wherein the receiving device 4 is arranged in a climatic chamber 21. The housing 21 a of the climate chamber may be transparent or opaque. The housing 21a encloses the receiving device 4 substantially airtight and thus contributes to avoiding contamination of the MT plates. Within the climate chamber 21, a desired climate can be adjusted. For example, the temperature, the O ₂ concentration, the CO ₂ concentration, N ₂ concentration and the air humidity can be set. In this way, the climatic conditions can be adjusted to the particular needs of the cells to be cultivated. The observation unit 3 and the fluid distribution unit 5 are arranged outside the climate chamber 21 and are thus not exposed to the climatic conditions which are necessary for optimum cell cultivation. Furthermore, impurities (for example lubricating greases) are prevented from entering the MT plates 2 from the observation unit 3 or the fluid distribution unit 5. In the housing 21 a two openings 22 are arranged, which allow the transporting of MT plates from the climatic chamber 21 out or in the climatic chamber 21 inside. The openings 22 are arranged in the region of the observation unit 3 as well as in the area of the fluid distribution unit 5 and allow transport of MT plates 2 there. The openings 22 can be closed after the MT plates have been transported.

Figure 5 shows the device 1 with attached housing 23. The housing 23 may be completely transparent or completely opaque. The housing 23 can also-as in the present exemplary embodiment-have a transparent region 24 in the form of a glass pane. The glass pane 24 may be formed as an open window, so that, if necessary, can be passed through the open window 24 into the interior of the housing 23. The entire device 1 is designed as a so-called "sterile bench." The housing 23 closes the cultivation space substantially airtight and prevents penetration of dirt, bacteria, etc. from the outside into the interior.

All climatic conditions inside the climate chamber 21 and in the housing 23 are controlled by a computer-controlled control and control system. Sensors inside the housing 23 or inside the climate chamber 21 constantly provide information about the current conditions in the respective rooms. Through the control and control software, the climatic conditions are always kept at a predetermined level. Also, the filling of wells 17 of certain MT plates with certain amounts of particular fluids is controlled by the computer controlled control system. For this purpose, the MT plates (2) and the individual wells 17 of the MT plates 2 are indexed and recognized by the system. The computer controlled control system may be programmed to fill certain wells (17) of selected MT plates (2) with certain amounts of particular fluids at specific times.

Likewise, the computerized control system for microscopically observing and evaluating certain cell cultures in particular wells (17) of certain MT plates (2) at certain times with certain technique, such as e.g. Programmed fluorescence or phase contrast microscopy and / or any other detection method for observation and / or analysis of cells. Furthermore, the system can be programmed to take pictures of cell cultures at specific times, with recordings usually being stored and continually evaluated.

Figure 6 shows a schematic representation of the control and regulation of the climatic conditions in the interior of the climate chamber 21 and in Inside the housing 23. In the climate chamber 21 gas sensors 25 are arranged, namely a CO ₂ sensor 26, an N ₂ sensor 27 and an O ₂ sensor, not shown here. Furthermore, sensors for other gases may be present. The concentration values determined by the gas sensors are fed to a climate chamber controller. An air humidity sensor 29 constantly detects the humidity in the interior of the climate chamber 21 or in the interior of the housing 23. Furthermore, a temperature sensor 30 continuously measures the temperature. The humidity sensor 29 and the temperature sensor 30 also transmit their values to the climate chamber controller 28. Another temperature sensor 31 constantly measures the temperature inside the housing 23. A so-called laminar hot air heater 32 sets a temperature in the housing, preferably the temperature in the climatic chamber corresponds, a. This prevents condensation processes in the climate chamber. The climate chamber controller 28 regulates the humidity in the interior of the climatic chamber 21 via a humidifier 33. Between the humidifier 33 and the climatic chamber 21, a filter element 34 is interposed. Furthermore, fresh air 35 can be introduced into the interior of the housing 23 or the climatic chamber 21. About the climate chamber controller 28, the gas flows from the gas cylinders 20 are controlled by gas regulators. In this case, certain amounts of gas are passed into a mixing chamber 36. Via a gas circulation pump 37, which is also regulated via the climate chamber controller 28, the desired gas mixture is passed into the climatic chamber 21. The desired gas mixture is brought to the desired temperature by a gas mixture line heater 38.

It is understood that a device according to the invention can also be designed differently than is shown in the figures. Thus, the receiving device for receiving the MT plates can also be constructed of superposed, independently rotatable turntables. Also, the individual receiving elements of the receiving device may be formed in the manner of assembly lines, through which a linear arrangement of the MT plates and a linear trans- the same port is reached.

Furthermore, it is conceivable that the climate chamber is transparent at least in the region of the observation unit and can be microscoped or observed through the wall of the climate chamber, so that the MT plates do not have to be moved out of the climate chamber. For this purpose, the climatic chamber can also have a bulge into which an MT plate to be observed can be brought in for observation.

Regardless of the embodiment, the positions of the MT plates are defined to be the same for each observation, i. with repeated passage, the observation position should be approached reproducibly with high accuracy. The accuracy of the system is dictated by the accuracy of the outside dimensions of the MT plates. There are different variants with regard to the equipment of the plates. The MT plates can be covered with a foil at the bottom and thus optically defined. However, certain deviations occur in the Z direction. Another embodiment of the MT plates is that the plates are equipped with a glass sheet and optically defined. These MT plates have hardly any deviations in the Z direction.

Repeated presentation of the MT plates to the optics, there is a need to align the cell positions in the MT plates with the deviations in the system. This too is done by the computerized control system.

Figure 7 shows a perspective view of a Entdeckelungsvor- direction (40) of a preferred embodiment of a device according to the invention. The de-capping device comprises a robot arm (41) and a lid holding device (42). Figure 8 shows the lid holding device (40) in a plan view. The Entdeckelungsvor- direction (40) is used for loading and uncovering MT plates (2). In As a rule, the MT plates are stored in the climate chamber for incubation with the lid closed. In the fluid or optical station, however, the MT plates are to be processed without a lid. Therefore, the MT plates must always be entdeckelt or what is possible with the Entdeckelungsvorrichtung (40). Of course, the term decapping device also includes that the device (40) is also suitable for covering. If, for example, an MT plate is to be moved from the incubator to the optics station, the robot arm (41) retrieves the corresponding MT plate from the selected place in the climate chamber ("shelf space"). The lid holding device (42) has horizontally displaceable retaining clips (43) which are movable by means of a spring or mechanically movable pin (45) When the MT plate (2) is raised onto the lid holding device (42) Finally, the lid (44) of the MT plate (2) is inserted into the holding device (42) and held by the retaining clips (43), and then the robot arm (41) moves down with the MT plate (2) the lid (44) remains in the lid holding device (42) .The decapping device (40) is positioned in the climatic chamber (21).

Subsequently, the robot arm (41) moves the opened MT plate (2) out of the climatic chamber into the observation unit (optical station). While the MT plate remains in the observation unit for a certain time, the lid (44) is still held in the lid holding device.

When the actions at the optics station have been completed, the robot arm (41) moves the open MT plate back from the optics station back into the climate chamber. There, the robot arm (41) moves the open MT plate (2) upwards in the direction of the cover holding device (42), wherein ultimately the open MT plate is accurately connected to the cover (44). Now let the retaining clips (43) the lid (44), causing the MT plate (2) is finally covered again. Now the robot arm (41) moves the MT plate down and spends it to the desired "parking space".

Claims

CLAIMS:

1. Apparatus for automated, parallelized cultivation of cells in cell culture vessels, in particular microtiter plates (MT plates) (2) with a

Housing (23) in which an observation unit (3), comprising at least one microscope (7) and at least one camera (6), a Aufhahmevorrichtung (4) for receiving the cell culture vessels and a fluid distribution unit (5) for automatic Befallen and / or emptying the cell culture vessels, in particular the wells (17) of MT plates are arranged with fluids, wherein the climatic conditions in the device, in particular the gas composition and the temperature are adjustable at least in the region of the cell culture vessels, wherein in the housing a climatic chamber (21) is provided, in which a desired temperature and / or a specific gas composition is automatically adjustable and in which the Aumahmevorrichtung (4) with the cell culture vessels (2) is at least partially integrated.

2. Apparatus according to claim 1, characterized in that separate gas containers are provided, which contain different gases, preferably O ₂ , CO ₂ and N ₂ , wherein the gas streams from the gas cylinders are controllable and wherein certain amounts of gas are passed into a mixing chamber of where the gas mixture thus produced is passed into the climate chamber, wherein the gas mixture is preferably brought to a desired temperature before being introduced into the climatic chamber.

3. Device according to one of claims 1 or 2, characterized in that cell culture vessels (2) preferably in the region of the observation unit (3) and / or in the region of the fluid distribution unit (5), preferably automatically by a carriage (11) or robotic arm from the climatic chamber (21) or in the climate chamber are transportable.

4. Device according to one of the preceding claims, characterized marked, that the observation unit (3) and / or the fluid distribution unit (5) outside the climate chamber (21) are arranged.

5. Device according to one of the preceding claims, characterized in that the Aufhahmevorrichtung (4) at least two, preferably arranged one above the other receiving units (10) and preferably holding means (18) for holding the cell culture vessels (2).

6. Device according to one of the preceding claims, characterized in that the optics (microscope (7) incl. Camera (6)) of the observation unit (3) is movable, wherein the movements are preferably controllable.

7. Device according to one of the preceding claims, characterized in that in the region of the observation unit (3) located cell culture vessel (2a) is movable, wherein the movements are preferably controllable.

8. Device according to one of the preceding claims, characterized in that the microscope (7) is a transmitted light and / or phase contrast and / or fluorescence microscope.

9. Device according to one of the preceding claims, characterized in that the fluid distribution unit (5) a plurality of containers for storing different fluids, such as cell solutions, nutrients and dyes, and a microdosing (12) for targeted, controlled addition of fluids into the cell culture vessels, in particular in the wells (17) of MT plates (2) and preferably also for removing the fluids from the cell culture vessels, in particular from the wells of the MT plates, wherein the movements and actions of the microdosing unit are preferably controllable.

10. Device according to one of the preceding claims, characterized in that the cell culture vessels, in particular the MT plates (2) and preferably also the wells (17) of the MT plates are indexed.

1 1. Device according to one of the preceding claims, characterized in that the cell culture vessels (2) are transparent.

12. Device according to one of the preceding claims, characterized by sensors (25, 26, 27, 29, 30, 31) for controlling all relevant parameters, in particular temperature, CO ₂ concentration, O ₂ concentration, N ₂ - concentration, humidity and / or supplying fluids to the cell culture vessels, in particular into the wells (17) of the MT plates (2).

13. Device according to one of the preceding claims, characterized by a computer-controlled control and control system for the automatic control and control of at least one, preferably all listed below parameters or steps: a) temperature, in particular in the climatic chamber (21); b) O ₂ content in the gas, in particular in the climate chamber; c) CO ₂ content in the gas, in particular in the climate chamber; d) N ₂ content in the gas, in particular in the climatic chamber; e) filling and / or emptying selected cell culture vessels, in particular selected wells (17) of selected MT plates (2) with selected quantities of selected fluids, at selected times; f) microscopic observation and evaluation of cell cultures in selected cell culture vessels, especially in selected wells of selected MT plates at selected times with selected technique, such as fluorescence, transmitted light or phase contrast microscopy.

14. Device according to one of the preceding claims, characterized by a loading and Entdeckelungsvorrichtung for preferably automatic loading and Entdeckein the cell culture vessels.

15. Device according to one of the preceding claims, characterized in that in the fluid distribution unit, a controlled selective use of disposable and multiple pipettes is possible.

16. A method for automated, parallelized cultivation of cells in cell culture vessels, in particular microtiter plates (2), which are in a cell culture system, in particular in a device (1) according to any one of claims 1 to 15, comprising the following steps: a) filling the system with cell culture vessels (2); b) filling the system with containers (15) containing fluids to be used, such as cell culture medium, cell suspension, test substance, dye; c) loading the system with containers (20) containing gases to be used, such as O ₂ , CO ₂ , N ₂ ; d) programming the system to automatically set at least one of the following parameters: - temperature

O ₂ content in the gas

- CO ₂ content in the gas

- N ₂ content in the gas; e) programming the system for automatic filling and / or emptying of cell culture vessels, in particular selected wells (17) of selected MT plates (2) with selected quantities of selected fluids, at selected times; f) programming the system for microscopic observation and evaluation of cell cultures in selected cell culture vessels, in particular in selected wells of selected MT plates at selected times with selected technique, such as fluorescence, transmitted light or phase contrast microscopy.