DE102022115737B3 - Device and method for automated cell cultivation - Google Patents

Abstract

The claimed incubator system comprises an incubator with a fixed internal volume; multiple flexible cell culture bags floating in a respective frame for each of the multiple cell cultures; several process locks connected to the respective cell culture bags, with inner and outer lock doors that can be actuated manually or automatically; an automatic agitation device associated with each of the flexible cell culture bags; an inner and an outer incubator axis system for automatically connecting multiple hardware components integrated into the incubator system to the multiple cell cultures; a cell counter mounted outside the incubator on the outer incubator axis system with a measuring cuvette provided separately for each cell culture bag, wherein the cell counter can be positioned in front of the process locks with the aid of the outer incubator axis system; an automatic weighing system with load cells connected to the inner incubator axis system; and a nutrient reservoir associated with each of the plurality of cell cultures, each cell culture bag being associated with its own nutrient reservoir.

Description

field of invention

The present invention generally relates to an incubator system for automated cell cultivation. In particular, the invention relates to such an incubator system for the simultaneous, automated cultivation of a plurality of cell cultures. The invention relates very specifically to an incubator system for cultivating a number of cell cultures which have identical, controllable environmental conditions.

Furthermore, the present invention also relates to a method for the simultaneous automated cell cultivation of a plurality of cell cultures using the incubator system according to the invention.

Background of the Invention/Prior Art

• - Temperatur Es ist nachgewiesen, dass bspw. bei Säuger-Zellen eine Temperaturabweichung von weniger als 1°C von der Solltemperatur einen Effekt auf die Zellkultivierung hat. Teilweise wird das bewusst dazu benutzt, ein Wachstum bspw. zu verlangsamen oder die Zellreaktion zu beeinflussen. In Inkubatoren wird dies bspw. mittels Peltier-Elementen, geregelter Warmluft oder einem geregelten Wassermantel eingestellt.
• - pH-Wert Zellen bzw. Zellkulturen sind pH-Wert-sensitiv. Es existieren verschiedene Möglichkeiten, diesen Wert zu regulieren. Sofern es notwendig ist, wird dem Zellkultur-Medium ein Puffer hinzugefügt, der in Reaktion mit der äußeren Atmosphäre die Regulierung einstellt (bspw. durch die Zugabe von Carbonat in Verbindung mit einer CO₂angereicherten Atmosphäre). Eingestellt wird dies systemspezifisch über der Suspension hinzugefügte Puffer und die äußere Atmosphäre, bspw. über den CO₂-Gehalt, der z.B. mittels Masseflussreglern eingestellt wird. Es kann aber auch dem Inkubatorvolumen eine vorgefertigte Gasmischung hinzugefügt werden.
• - Agitation Zellen neigen dazu, zu sedimentieren. Dadurch werden „überschichtete“ Zellen weniger gut mit Nährstoffen und Sauerstoff versorgt. Es existieren verschiedene Ansätze, die Zellen zu agitieren, bspw. die Zell-Suspension mit Gas zu durchspülen und dadurch die Suspension zu durchmischen, es können Rührer benutzt werden oder der ZellkulturBehälter kann geschüttelt oder gewippt werden. Die Art der Agitation hängt sowohl vom Zellkulturbehälter (Beutel, Tank, Hohlfasern, Flasche, etc.) als auch von der Zellkultur selbst ab. So besitzen Säugerzellen keine stabile Zellwand, sondern nur eine Zellmembran. Hierdurch sind sie empfindlich für Scherkräfte, eingebracht durch die Agitationsmethode, die die Zellen schädigen können. Bei einer Wippe (engl. wave reactor) stellen der Kippwinkel und die Frequenz die Freiheitsgrade dar. Andere Methoden werden bspw. über die Geometrie und die Drehzahl des Rührers definiert.
• - Sauerstoffsättigung Zellen benötigen zum Wachstum eine ausreichende Sauerstoffversorgung. Abgeschlossene Zellkulturbeutel bieten diverse Vorteile, da diese steril verpackt geliefert werden, und sowohl kostengünstig als auch in verschiedenen Volumina einfach verfügbar und nach Gebrauch einfach entsorgbar sind (was Kreuzkontamination verhindert). Der Austausch von Gasen, hier Sauerstoff und CO₂, erfolgt dabei mittels Diffusion durch die Beutelwand hindurch. Aus diesem Grund ist eine möglichst große bzw. definierte Oberfläche pro Volumen wichtig und ein entscheidendes Kriterium. Durch die Diffusion der Gasmoleküle durch die Wand des Zellkulturbeutels findet der Gasaustausch bei der Zellkultivierung statt. Ein großes Verhältnis von Oberfläche zu Volumen des Beutels ist daher grundlegend von Vorteil, da dann mehr Fläche für den besagten Austausch zur Verfügung steht. Sollen die Diffusionsprozesse zeitlich gesteuert werden, kann die Kontrolle des Verhältnisses Oberfläche zu Volumen ganz allgemein von Vorteil sein. Das Material des Zellkulturbeutels kann bspw. Polyethylen, PE; Polypropylen, PP; oder Polytetrafluorethylen, PTFE) sein.

In biotechnology, there are several systems or processes to multiply cell cultures, ie to cultivate them. The aim here is to enable cells of an initial cell culture to divide in order to obtain a desired cell culture size (ie a specific volume) and/or a desired cell density (e.g. cells/ml). Depending on the type, cells as biological systems make very different demands on the environment or the medium surrounding them, e.g. with regard to temperature, humidity, pH value, volume-surface ratio, agitation, etc. The influences are sometimes different :

• - Temperature It has been proven that, for example in mammalian cells, a temperature deviation of less than 1°C from the target temperature has an effect on cell cultivation. This is sometimes used deliberately, for example to slow down growth or to influence the cell reaction. In incubators, this is set, for example, by means of Peltier elements, controlled warm air or a controlled water jacket.
• - pH value Cells or cell cultures are pH value sensitive. There are various ways to regulate this value. If necessary, a buffer is added to the cell culture medium, which stops regulating in reaction to the external atmosphere (e.g. by adding carbonate in connection with a CO ₂ -enriched atmosphere). This is set system-specifically via the buffers added to the suspension and the external atmosphere, for example via the CO ₂ content, which is set, for example, by means of mass flow controllers. However, a ready-made gas mixture can also be added to the incubator volume.
• - Agitation Cells tend to sediment. As a result, "overlayered" cells are less well supplied with nutrients and oxygen. There are various approaches to agitating the cells, for example flushing the cell suspension with gas and thus mixing the suspension, stirrers can be used or the cell culture container can be shaken or rocked. The type of agitation depends both on the cell culture container (bag, tank, hollow fibers, flask, etc.) and on the cell culture itself. Mammalian cells do not have a stable cell wall, only a cell membrane. This makes them sensitive to shear forces introduced by the agitation method, which can damage the cells. In the case of a wave reactor, the tilt angle and the frequency represent the degrees of freedom. Other methods are defined, for example, via the geometry and the speed of the stirrer.
• - Oxygen saturation Cells need an adequate supply of oxygen to grow. Sealed cell culture bags offer several advantages as they are supplied in sterile packaging, are inexpensive, are readily available in various volumes and are easily disposable after use (which prevents cross-contamination). The exchange of gases, in this case oxygen and CO ₂ , takes place by means of diffusion through the bag wall. For this reason, the largest possible or defined surface per volume is important and a decisive criterion. The gas exchange during cell cultivation takes place through the diffusion of the gas molecules through the wall of the cell culture bag. A large surface area to volume ratio of the bag is therefore fundamentally advantageous, since more surface area is then available for said exchange. In general, controlling the surface-to-volume ratio can be beneficial if the diffusion processes are to be time controlled. The material of the cell culture bag can be, for example, polyethylene, PE; Polypropylene, PP; or polytetrafluoroethylene, PTFE).

If these cell-specific conditions are not present or not sufficiently met, a reduced rate of cell division or cell death occurs.

Two well-known and effectively used processes for cultivating cells are the batch and fed-batch processes. in batch In this method, the starting cell culture is placed in a specific volume with a specific amount of nutrients. If the environmental conditions are suitable, the cells multiply until the nutrient medium is used up. In the fed-batch process, the singular batch process is run through several times by adding further nutrients to the cell culture at suitable times in order to run through another cultivation cycle. This process usually ends when the desired culture size and/or cell density is reached. In order to determine an optimal addition of nutrients, a sample is usually taken from the current cell culture, which allows conclusions to be drawn about the current cell density and cell vitality using certain methods. Apparatuses are used to determine the number of cells and thus the cell density (number in relation to a defined test volume). These are mostly based on optical measuring methods (e.g. recording a microscopic image using a digital camera with subsequent automatic image evaluation). By recording consecutive images, conclusions can also be drawn about the cell dynamics or vitality. Many systems can be cultivated more effectively by a fed batch process than by a single batch process.

The cultivation cycles of a fed-batch process depend very much on the intended use or the cell culture itself. This means that for effective cultivation, the respective cycle must be carried out regardless of the time of day or day of the week. Since the culture volume also increases in the course of cell cultivation, the fulfillment of the condition requires a constant or set volume-to-surface ratio (here it is about the gas exchange between the cell culture in the bag and the environment, which is urgently required for cell cultivation - via the most precise possible By adjusting the ratio of the surface of the bag to the volume of the bag, the gas exchange and thus one of the essential parameters of the cell cultivation itself can be controlled - cf. above), usually the decanting of the cell suspension into a larger volume. To do this, the sterile environment must be broken and new sterile connections must be made. This regular work cannot currently be carried out automatically, or only with a very high level of technical effort, especially if several cell cultures are to be cultivated at the same time. Furthermore, cell cultivation is heavily dependent on the starting cell culture, which is why a prepared, standardized, sterile connection is difficult to reproduce economically. As a result, such manual work is associated with a great deal of effort and a regularly recurring risk of contamination.

Systems that have been described in the prior art or are commercially available offer both automated cell cultivation and expandable cell culture bags.

For example, the WO 2015/162211 A1 an automated method for generating genetically modified T-cells, T-cell subsets and/or T-cell precursors with various steps, all steps being carried out in a closed and sterile cell culture system.

In the WO 2009/072003 A1 a centrifugation chamber for separation methods, which is suitable for culturing cells, is described with the help of which the method from FIG WO 2015/162211 A1 can be carried out.

In the WO 2015/009153 A1 describes a bioreactor system with a variable volume bioreactor container for culturing cells, which includes a flexible cell culture bag and a squeezing roller, by means of which the volume of the cell culture bag can be changed. Furthermore, the bioreactor system has means for gently agitating the cell culture bag.

From the US 2013/0157353 A1 a system for multiplying stem cells from a rough biopsy is known, comprising a cell culture bag whose working volume can be continuously varied by means of a roller system.

Finally revealed the WO 2016/161174 A1 an automated cell culture incubator for the autologous production of mammalian cell cultures. The incubator has an external door that can be opened to an inner chamber. Disposed within the interior chamber are a plurality of cell culture containers, each container having a channel which allows materials to be aseptically introduced into and removed from the container. Each cell culture container can contain cell samples from different subjects. A constant temperature prevails in the incubator and there are no automatically expanding cell culture bags, only dimensionally stable containers with permeable membranes for introducing or removing material. The system has only a low degree of automation.

The main disadvantage of the systems of the prior art is that they are predominantly limited to a single cell culture at the same time or, when using several cell culture containers, they are not located separately from each other in closed systems, so that cross-contamination occurs can. In addition, it is not possible to maintain the sterile barrier during the entire cultivation process (even over several days). The systems of the prior art also have a low degree of automation and require a high level of worker intervention. In addition, they allow only little control over the cultivation process (recording of the actual state, readjustment of the nutrient medium, control of the gas exchange, etc.).

Summary of the Invention

The invention is therefore based on the object of further developing an incubator system for the simultaneous automated cultivation of a plurality of cell cultures in such a way that the disadvantages of the prior art described are avoided.

These and other objects are solved by the incubator system according to claim 1 and the method according to claim 17. Further advantageous configurations are listed in the dependent claims.

character list

• 1 eine schematische Darstellung des erfindungsgemäßen Inkubator-Systems;
• 2 eine schematische Darstellung des erfindungsgemäßen in einem Rahmen gefassten Zellkulturbeutels mit einem Zellzähler-Kreislauf;
• 3 eine schematische Schnittzeichnung des teilweise gefülltem Zellkulturbeutels aus 2 mit Quetschrollen und der Nährmittel-Zufuhr; und
• 4 eine schematische Schnittzeichnung eines Ausschnitts aus 1.

The invention is explained in more detail below with reference to the drawings. In it shows

• 1 a schematic representation of the incubator system according to the invention;
• 2 a schematic representation of the cell culture bag according to the invention, framed in a frame, with a cell counter circuit;
• 3 a schematic sectional drawing of the partially filled cell culture bag 2 with squeeze rollers and the nutrient feeder; and
• 4 a schematic sectional drawing of a section 1 .

Detailed description of the invention

Compared to the prior art described, the invention offers the possibility of cultivating several cell cultures separately in a closed system in each case. Both the batch and the fed-batch method can be used here. The cell cultures are not limited to a specific type of cell, such as CAR-T cells. In order to enable joint cultivation, the respective cell cultures must have identical, ideal environmental conditions (e.g. temperature, pH value, humidity, etc.). The core of the invention is the corresponding equipment of an incubator with corresponding automated hardware components, through which the respective environmental conditions are provided and a fully automatic cell cultivation becomes possible without the bags having to be removed from the incubator again and again during the process and manipulated manually . The present invention enables the in situ integration of measurement methods into the ongoing cell cultivation process without increasing the danger or risk of contamination. The inner volume of the incubator, i.e. its entire interior, is adjusted precisely to the cell culture(s) to be processed with the help of appropriate process technology in terms of temperature, humidity and composition of the gas atmosphere.

An incubator system for the simultaneous, automated cultivation of a number of cell cultures is claimed, with the number of cell cultures each being arranged in a closed system and having identical, controllable environmental conditions. The incubator system comprises an incubator with a fixed internal volume; a plurality of flexible cell culture bags floatingly clamped in a respective frame for each of the plurality of cell cultures, the respective frames for the plurality of cell cultures being insertable into the incubator and each frame being receivable at respective mounting points provided in the incubator; a plurality of process airlocks connected to the respective cell culture bags with manually or automatically operable inner and outer airlock doors for connecting components to or sampling from each of the plurality of flexible cell culture bags; an automatic agitation device connected to each of the flexible cell culture bags whereby each of the plurality of cell culture bags is agitatable either individually or in combination; an inner and an outer incubator axis system for automatically connecting multiple hardware components integrated into the incubator system to the multiple cell cultures; a cell counter mounted outside of the incubator on the outer incubator axis system for measuring the cell culture volume or the cell density with a measuring cuvette connected to the incubator by a telescopic rail system, which can be automatically inserted and removed from the cell counter and is provided separately for each cell culture bag, wherein the cell counter can be positioned in front of the process locks using the external incubator axis system; an automatic weighing system with load cells connected to the inner incubator axis system; and a nutrient reservoir associated with each of the plurality of cell cultures, each cell culture bag being associated with its own nutrient reservoir.

1 shows a schematic representation of the incubator system 2 according to the invention. The system 2 consists of an incubator 4, ie, a temperature control device that can be used in biology to create and maintain controlled external conditions for various development and growth processes. The incubator 4 consists of a material known from the state of the art and has a specific internal volume which depends on the desired capacity, ie how many cultures are to be processed in parallel.

One or more cell cultures 6 are placed in a flexible cell culture bag 8 which is clamped in a cell culture bag frame 10 (cf. 2 and 3 ), placed in the incubator 4. All preparatory measures, such as connecting the still empty cell culture bag to the various hose connections, are carried out by the operator in advance, ie before the cell culture bag 8 is filled with the respective cell culture 6 . Each frame 10 is received in the incubator 4 by corresponding mounting points 12 . The material of the cell culture bag 8 is selected from the group consisting of polyethylene, PE; Polypropylene, PP; and polytetrafluoroethylene, PTFE.

In order to provide a suitable volume-to-surface ratio for fed-batch processes ("suitable" in this context means that when the nutrients are refilled, the bag volume is also readjusted or adjusted in such a way that the new process volume is maintained while maintaining the corresponding volume -Surface ratio is sufficiently dimensioned), 10 squeezing rollers 14 are integrated into the frame, which divide the cell culture bag 8 into an adjustable usable volume and a reserve volume. The starting point for the useful and reserve volume of the cell culture bag 8 results from the starting point of the pre-batch method of topping up nutrients in the course of the cultivation. For this purpose, the usable volume is correspondingly increased at the expense of the reserve volume. This is done with the help of the squeezing rollers 14, which separate the useful and reserve volume of the bag from one another. The selected bag size (volume) depends on the desired cultivation process. The present invention can accommodate a variety of different bag sizes common to the industry. The squeezing rollers 14 are raised (opened) to set a new (enlarged) usable volume and are moved to the new position for separating usable and reserve volume by means of actuators. At the new position, the squeezing rollers 14 are then lowered (locked) again in order to achieve the separation of the useful and reserve volume of the respective cell culture bag 8 . The described locked state of the pinch rollers 14 is obtained in a mechanical, electrical, magnetic or other corresponding manner.

So that the packing density of the cell culture bags 8 accommodated in the frame 10 in the incubator 4 can be increased (in the present invention, the usable space inside the incubator should be able to be optimally used for accommodating bags for cell cultivation), there are no permanently installed and/or or removable level metal sheets or grids are integrated, but mounting or holding points 12 for receiving the cell culture bags 8 are attached to the left and right of the side walls in a corresponding grid.

Each cell culture bag 8 clamped into the incubator 4 has its own nutrient reservoir 36 (cf. 3 ). So that larger cell culture bags 8 (e.g. up to 50 liters) can also be held securely in the frame 10, they are held in place by flexible support straps 16 (cf. 2 ), which are in the horizontal plane below the cell culture bags 8 to support them - especially when more and more nutrients are refilled and the bag is becoming heavier as a result. Various types of design are conceivable for the support straps 16, e.g. two crossed ("X"-shaped) straps, as in 2 shown, bands running parallel to each other, bands crossing at right angles or at any angle (“diamonds”), a net or similar.

In order to be able to process even larger cell culture volumes (> 50 liters), the respective cell culture can be automatically transferred from an adjustable volume (intermediate volume - the setting of this volume (parameterization) is carried out via the system control) into a previously empty, correspondingly larger cell culture bag 8, which is already clamped in the incubator and connected in a sterile manner to the starting cell culture bag, can be pumped around (cascading). The cascading process is carried out automatically by the system control when the set value for the intermediate volume is reached. The first cell culture bag 8 in the context of the cascading, usually the bag clamped in the uppermost position of the incubator 4, is referred to as the starting cell culture bag. The initial bag and the bags following it in the cascading series are already connected to one another at the start of the cultivation process via appropriate hose connections, valves and a pump (cf. 2 ) and are placed together in the frame 10 in the incubator.

As described above, the cascading process itself starts automatically when the set intermediate volume in the initial bag is reached.

When pumping over, a valve between the starting bag and the following one is fully automatic (larger, initially empty) bag is opened and the entire liquid contained in the starting bag, including the cell culture(s) contained therein, is pumped into the following bag. The valve is then closed again and the cultivation process is continued or completed in the following larger bag. This process is generally also possible for several cell cultures in parallel, ie it is possible to accommodate several so-called cascades in one incubator. Each of these cascades represents its own, closed system for cultivating the respective cell culture. The only limitations are the size of the incubator interior and the load-bearing capacity of the inner incubator axis system.

The cell culture bag frame 10 and its inlets and outlets have pinch valves (e.g. the in 3 shown pinch valve 48 between the nutrient reservoir 36 and the respective cell culture bag frame 10), which in the normal state, i.e. at all times except when refilling nutrient without external influence of an external actuation, such as by a magnetic switch, a pneumatic axis, an electric drive or The like., are always closed (so-called “normally closed” (nc) valves). These pinch valves securely close the hoses or hose systems 18 used to convey liquid (cf. 2 , valves not shown here) to prevent unwanted fluid flow. They can be both active (electrical or pneumatic) and passive for activation by an external actuator. In this context, passive means that the mechanism for opening and closing does not have its own (permanent) drive. The mechanism is either open or closed, but cannot change this state independently. To change the respective state, a separate active drive must be coupled from the outside. A position signal for constant valve monitoring can be integrated for both active and passive actuation.

As already mentioned, the cell culture bags 8 each have several inlets and outlets (hose system 18, cf. 2 ) Mistake. In order to prevent contamination of the cell cultures 6, the hoses or the hose system 18, which are or are sterilely connected to the cell culture bags 8, are only released when they are used, i.e. only by opening the corresponding pinch valve (active or passive), the nutrient liquid or also (as in the cascading process) the liquid containing the cell cultures can flow into or through the hose previously separated by the respective pinch valve. This means that the liquid only comes into contact with the cell culture suspension when it is actually needed. After use, the respective pinch valve can securely close the hose system 18 again. Inflows and outflows (hose system 18) follow the single-use idea in connection with the pinch valves mentioned, this designation referring to a cell culture as part of its cultivation process. As part of this process, which lasts several days, it may be necessary to top up the nutrient liquid from the relevant reservoir several times. As long as the respective cultivation process is ongoing, all of the above components remain in use. The single-use components are only disposed of after the cultivation process has been completed and the resulting cell culture transferred to another container.

Via two sterile hoses of the hose system 18 ( 2 ) a measuring cuvette 20 is connected to the respective cell culture bag 8 in a closed circuit. The measuring cuvette 20 is a transparent vessel that can hold a defined volume of cell culture liquid. The liquid with the cell cultures 6 can be supplied and removed through the hose system 18 . The measuring cuvette 20 can be connected to a cell counter 22 (for its arrangement in the incubator system 2 cf. below) for monitoring the cell cultures 6, ie for measuring the respective cell culture volume or cell density (cf. above). The measuring cuvette 20 is transparent so that the cell culture liquid contained therein can be evaluated using the cell counter 22 .

In addition to the measuring cuvette 20, a pump 24 (membrane or peristaltic pump) is also integrated in the above-mentioned closed circuit, which circulates the cell culture suspension in the measuring cuvette 20. In order to ensure a stable measurement of the cell culture volume or the cell density and thus a corresponding monitoring of the cells or the cell cultivation process, the cell culture suspension is circulated before the measurement for renewal and the pump 24 is deactivated for the measurement as well as the respective pinch valves in the frame 10 closed.

Surfaces that come into contact with the product or cell culture, i.e. all hoses 18, the measuring cuvette 20, the respective nutrient reservoir 36 (cf. 3 ) and the cell culture bags 8 can be used for one-time cell cultivation, which means that all components that come into contact with the product are sterile and are only used once. After the completion of the cell cultivation process, the components are discarded and replaced with new ones. This single-use approach ensures no cross-contamination nation can take place between different cell cultures. However, the drive component of the pump 24 can be reused, since when using a peristaltic pump the actual delivery mechanism does not come into contact with the pumped media (the tube carrying the medium is “flexed”, so to speak).

Any opening of the incubator 4 that changes the internal environmental conditions or atmosphere can affect the cell cultures. In many processes, however, samples of the cell cultures have to be taken again and again in order to be able to carry out further investigations. In order to be able to remove a cell culture and at the same time keep the internal environmental conditions constant, a process lock 26 is provided in the incubator wall for each cell culture 6 introduced into a corresponding cell culture bag 8 . The process lock 26 has an inner and an outer lock door 28 and 30 (so-called double lock—cf. 4 ) on. It is clear to the person skilled in the art that a single lock with only one lock door can also be used. When the inner sluice door 28 is closed, the process sluice 26 is accessible from the outside via the open outer sluice door 30 in order to be able to connect provided hose connections or the like. The lock thus forms the interface to the otherwise closed cell cultivation system inside the incubator 4. Hose connections, valves and similar components can be connected here without the inside of the incubator 4 being exposed to the ambient conditions during this time, as is the case with conventional systems. As soon as the outer lock door 30 is closed again, the atmosphere of the process lock 26 is automatically adjusted to the incubator atmosphere by bringing the lock chamber into contact with the inside of the incubator 4, so that the temperature/humidity and gas atmosphere are adjusted.

The lock doors 28, 30 can be designed to be operated both manually and automatically. The incubator and the locks are controlled by an external system control. A human-machine interface (HMI) connected to the controller is used to exchange information with the worker (display information, make operating elements available).

In order to agitate the cell cultures 6, i.e. to be able to set the liquid in the cell culture bags 8 in motion, the cell culture bag frames 10 are suspended in a floating manner. This makes it possible to agitate the cell cultures 6 individually or in combination by means of rocking movements via the agitation device (not shown) (e.g. by means of a rotary drive with an eccentric disc) in order to prevent cell agglomeration. The amplitude (size of the tilting), frequency (number of tilting movements per time unit) and pause times (either permanent rocking or interruption of the rocking process after each rocking cycle) of the drive that executes the rocking movement can be parameterized via a higher-level controller.

Individual cell culture bag frames 10 can optionally be excluded from the seesaw movement, for example in order to carry out a weighing process (cf. further below). For this purpose, either each cell culture bag frame 10 has its own rocker drive that can be individually activated or deactivated, or there is only one central rocker drive to which all cell culture bag frames can be individually coupled or uncoupled.

• - Antriebssystem und Kupplungen für die Quetschrollen 14 zur Teilung des Zellkulturbeutels 8 in ein Nutz- und Reservevolumen;
• - Wägezellen 34 (vgl. weiter unten), welche den im Rahmen 10 gefassten Zellkulturbeutel 8 wiegen können. Hierdurch kann gewichtsgesteuert auf 1 ml genau bspw. Nährmedium dem Zellkultur-Volumen hinzugefügt, bzw. Zellkultur-Suspension abgepumpt werden. Die Wägezellen 34 enthalten einen (unter Einfluss der zu bestimmenden Masse) verformbaren Federkörper. Die bei Belastung mit dem Gewicht auftretende Verformung wird mit Hilfe elektrischer oder optischer Sensoren sehr präzise gemessen. Über eine entsprechende Kalibrierung wird aus der mechanischen Verformung die Masse bzw. Gewichtskraft des Objektes (hier der Zellkulturbeutel 8) ermittelt.
• - Aktuatoren (nicht gezeigt), um passive Quetschventile, d.h., Quetschventile, die die Schläuche abklemmen, wenn sie geschlossen sind, im Zellkulturbeutel-Rahmen 10 zu betätigen;
• - Komponenten zur maschinenlesbaren Identifikation von Zellkulturbeuteln zur GMP (Good Manufacturing Practice) - gerechten Dokumentation, bspw. im medizinischen Sektor.

The incubator system 2 according to the invention is equipped with an inner 32 and an outer incubator axis system 44, each of which allows hardware components to be used multiple times for different cell cultures 6. The inner incubator axis system 32 is arranged inside and the outer incubator axis system 44 outside of the incubator 4 . The following components are integrated in the inner incubator axis system 32:

• - Drive system and couplings for the squeezing rollers 14 for dividing the cell culture bag 8 into a useful and reserve volume;
• - Weighing cells 34 (cf. below), which can weigh the cell culture bag 8 held in the frame 10 . In this way, nutrient medium can be added to the cell culture volume or the cell culture suspension can be pumped out, for example, weight-controlled to within 1 ml. The load cells 34 contain a spring body that is deformable (under the influence of the mass to be determined). The deformation that occurs when the weight is loaded is measured very precisely using electrical or optical sensors. The mass or weight of the object (here the cell culture bag 8) is determined from the mechanical deformation via a corresponding calibration.
• - actuators (not shown) to operate passive pinch valves, ie pinch valves that pinch off the tubing when closed, in the cell culture bag frame 10;
• - Components for machine-readable identification of cell culture bags for GMP (Good Manufacturing Practice) - compliant documentation, e.g. in the medical sector.

The cell counter 22 (for its function cf. further above) is arranged on the outer incubator axis system 44 and is thus integrated into the incubator system 2 .

There are various cell counting methods, mainly based on optical measuring methods. The present invention is preferably based on a measuring microscope, which takes a high-resolution image of the cell culture sample in the measuring cuvette and then evaluates it (number of cells, cell density in the test volume, cell activity over time (several image recordings in succession)). The time in which the measuring cuvette 20 has to be positioned from the incubator 4 into the cell counter 22 is reduced to the time required for image recording. The measuring cuvette 20 can then be transferred back into the incubator 4 immediately afterwards, and the evaluation is carried out asynchronously. The normal position of the measuring cuvette 20, i.e. whenever no cell count is being carried out (basic position), is inside the incubator behind the inner lock door 28. If a measurement process has to be carried out, the inner lock door 28 is opened, the measuring cuvette 20 in moves the interior of the lock and then the inner lock door 28 is closed. Only then does the outer lock door 30 open and the measuring cuvette 20 is moved outwards to the cell counter 22 .

The cell counter 22 is preferably based on a light microscope (there is also the possibility of using methods based on laser technology) and can be used for a number of cell cultures 6 . The arrangement on the axis system 44 mounted outside of the incubator 4 allows the cell counter 22 to be automatically positioned in front of the respective process locks 26 in the incubator wall.

Within the process lock 26, the measuring cuvette 20 is guided on a telescopic rail system (not shown), which is active (with its own permanently installed drive for moving the measuring cuvette 20 in and out on the telescopic rail) or passively (the measuring cuvette 20 sits on the movable telescopic rail - if the cuvette is to be moved into the lock area, a drive must first be coupled to the telescopic rail) can be extended in order to introduce the measurement cuvette 20 into the cell counter 22. After the measurement, the measuring cuvette 20 is moved back out of the cell counter 22 via the telescopic rail system and the outer lock door 30 is closed.

As a rule, the evaluation of the measurement requires a multiple of the time that the actual measurement requires. The system according to the invention makes it possible to minimize the time in which the measuring cuvette 20 with the cell culture suspension it contains is in a non-optimal environment in the area of the lock 26 (compared to the environment inside the incubator). The evaluation can be carried out when the measuring cuvette 20 has already been separated from the cell counter 22 and is inside the incubator 4 . The measuring cuvette is brought from the inside of the incubator into the lock area—and further to the cell counter 22—only for image recording in order to carry out the cell count. The necessary measurement is then carried out here with the aid of the cell counter 22, after which the measurement cuvette 20 is returned to the interior of the incubator (behind the inner lock door 28). There are no components in the sluice itself - it is only used for the passage of the telescopic rail and the hoses (hose system 18) leading to the measuring cuvette 20 during the counting process.

This arrangement, in which the cell counter 22 is located outside the incubator 4, allows operation under optimal laboratory conditions without the cell culture 6 or the isolator atmosphere, i.e. the gas atmosphere inside the incubator 4, being contaminated.

In its interior, the incubator 4 has an inner incubator axis system 32 for the automatic positioning of a plurality of end effectors 46 in three axes (hereinafter referred to as the x, y and z axis, cf. 1 ). Four end effectors 46 are preferably used (in FIG 1 only two of them are shown), which are advantageously located at the lower corners of the inner incubator axis system 32 and attach to the four corners of the cell culture bag frame 10 and in this way the respective cell culture bag frame 10 to be weighed is safely raised on both sides serve. Load cells 34 are placed on the end effectors 46 and are used to determine the mass of the respective cell culture bag 8 including the frame 10 in which the cell culture bag 8 is inserted. In this case, each end effector 46 is assigned a load cell 34 so that the number of end effectors 46 preferably corresponds to the number of load cells 34 . To weigh a cell culture bag 8 (with frame 10), the end effectors 46 are delivered to the respective cell culture bag frame 10 with the aid of the inner incubator axis system 32, the frame 10 is raised, the weighing is carried out and then the frame 10 is lowered again. Before the cell culture bag 8 is filled for the first time, ie before the start of the first pre-batch process, the net weight (empty cell culture bag 8 including the frame 10) is determined and stored in the system controller. When a certain amount of nutrient solution from the Reservoir 36 has to be pumped into the corresponding cell culture bag 8, the end effectors 46, as described above, are positioned below the corresponding cell culture bag frame 10 with the aid of the inner incubator axis system 32. The units consisting of the respective cell culture bags 8 and associated frames 10 are then lifted out of the holder on both sides by a further movement of the inner incubator axis system 32 in the z-direction. The load cells 34 are then read out by the system controller and the mass of the unit consisting of the cell culture bag 8 and the frame 10 is thus determined. As described above, the pinch valve 48 for the cell culture bag 8 is now opened and nutrients are pumped from the connected reservoir 36 into the respective cell culture bag 8 until the calculated target weight is reached (cf. 3 and below). During the entire supply of nutrient medium from the reservoir 36, the respective cell culture bag is thus lifted in its frame 10 by the inner incubator axis system 32 and the mass of the cell culture bag 8 in its frame 10 is continuously determined or calculated by the system controller by reading out the load cells 34 .controlled.

As already mentioned above, it is necessary from time to time to supply a nutrient medium to the cell cultures 6 in order to carry out the cultivation in a fed-batch process. In order to achieve a longer shelf life, culture media must be kept cool for the most part. In order to be able to regularly supply the cell cultures 6 with nutrient medium, a cooled nutrient medium reservoir 36 of sufficient volume (cf. 3 ) in a direct 1:1 connection sterile connected to the respective cell culture 6. Pinch valves 48 prevent an uncontrolled inflow; a peristaltic pump 38 can be used to add nutrient medium to the cell culture in a weight-controlled manner. The 1:1 connection prevents cross-contamination between different cell cultures 6.

In order to prevent a thermal shock of the cell cultures 6, the invention provides for the nutrient medium to be heated to the incubator temperature in a heating section 40 (cf. 3 ).

• Zur Vorbereitung des Prozesses wird zunächst das Nährmediums-Reservoir 36 in einen Kälteschrank eingebracht und eine entsprechende Schlauchverbindung nach außen geführt. Nun wird die Schlauchverbindung des Nährmediums-Reservoirs 36 in ein Quetschventil 48, die peristaltische Pumpe 38 und die Wärmestrecke 40 eingelegt bzw. eingeführt (vgl. 3). Der expandierbare Zellkulturbeutel 8 wird anschließend auf einem Tischarbeitsplatz in den Zellkulturbeutel-Rahmen 10 eingespannt, wobei dieser auf einer Wägevorrichtung (bspw. Wägezellen) ruht. Der geschlossene Kreislauf der Messküvette 20 (Küvette incl. Zuführ- und Ablaufschlauch, vgl. weiter oben) für den Zellzähler 22 ist dabei bereits steril mit dem Zellkulturbeutel 8 verbunden. Gewichtsgesteuert (vgl. die Beschreibung des Wägesystems weiter oben) wird anschließend der Zellkulturbeutel 8 mit der initialen Zellkultur 6 über eine sterile Verbindung befüllt. Die beiden Quetschrollen 14, die das Gesamtvolumen des Zellkulturbeutels 8 in ein Nutz- und ein Reserve-Volumen teilen, werden dabei über eine externe übergeordnete Steuerung (das hier beschriebene Gesamtsystem verfügt über einen externen Schaltschrank, in dem alle elektrotechnischen Komponenten untergebracht sind. Hierzu zählt auch die Systemsteuerung bspw. eine speicherprogrammierbare Steuerung) entsprechend des Gewichtssignals aus der Wägevorrichtung (entspricht dem Zellkultur-Suspensions-Volumen) angesteuert, um das gewünschte Nutzvolumen bereitzustellen. Anschließend wird das initiale Zellkultur-Volumen im Zellkulturbeutel 8 über das Schließen des Quetschventils 48 steril vom Nährmediums-Reservoir 36 abgetrennt.

The process of cell cultivation is as follows:

• To prepare for the process, the nutrient medium reservoir 36 is first placed in a cold cabinet and a corresponding hose connection is routed to the outside. The hose connection of the nutrient medium reservoir 36 is now inserted or introduced into a pinch valve 48, the peristaltic pump 38 and the heating section 40 (cf. 3 ). The expandable cell culture bag 8 is then clamped into the cell culture bag frame 10 on a table workstation, with the frame resting on a weighing device (for example load cells). The closed circuit of the measuring cuvette 20 (cuvette including the feed and drain hose, see above) for the cell counter 22 is already sterilely connected to the cell culture bag 8 . The cell culture bag 8 is then filled with the initial cell culture 6 via a sterile connection under weight control (cf. the description of the weighing system further above). The two squeezing rollers 14, which divide the total volume of the cell culture bag 8 into a useful volume and a reserve volume, are controlled via an external higher-level control (the overall system described here has an external control cabinet in which all electrical components are housed. This includes the system controller (e.g. a programmable logic controller) is also controlled according to the weight signal from the weighing device (corresponds to the cell culture suspension volume) in order to provide the desired usable volume. The initial cell culture volume in the cell culture bag 8 is then separated in a sterile manner from the nutrient medium reservoir 36 by closing the pinch valve 48 .

The inner incubator axis system 32 also has an integrated path measuring system (not shown). As a result, the incubator axis system can be positioned with a very high level of accuracy, for example in order to set the new usable volume by moving the squeezing rollers 14 .

The pre-filled cell culture bag 8 is now introduced into the incubator 4, the measuring cuvette 20 is mounted on the telescopic rail of the associated process lock 26 and the sterile connection to the line of the nutrient medium reservoir 36 is established.

In the subsequent actual cultivation process, the cell cultures 6 located in the incubator 4 are first agitated continuously via the agitation device (example by means of a seesaw movement), if provided for in terms of process technology. This function can be freely parameterized or switched off for special cell cultures.

The condition of the respective cell culture 6 is determined via the cell counter 22 . For this purpose, the external cell counter 22 on the outer incubator axis system 44 is delivered to the respective process lock 26, which is opened and the measuring cuvette 20 automatically introduced into the cell counter 22. After the cell count or the image recording (see above), the measuring cuvette 20 is returned to the interior of the incubator and both lock doors 28, 30 are closed. Based on the cell culture monitoring, the next rate of nutrient medium supply is then determined. This is then fed weight-controlled as follows.

First, the inner incubator axis system 32 is delivered to the respective cell culture 6 and the cell culture bag frame 10 is raised by means of the end effectors 46 for weighing by the load cells 34 mounted on the end effectors 46 . The nutrient medium is then pumped into the cell culture bag 8 by the peristaltic pump 38 until the target amount is reached. As already indicated, the nutrient medium is heated by a heat exchanger (heating section 40 in 3 ) brought to incubator temperature. The inner incubator axis system 32 then sets the cell culture bag frame 10 down again. The next cell culture 6 can now be processed in the incubator 4 .

This cultivation process is repeated using the fed-batch method until the desired cell culture volume and/or cell density has been reached.

The automation according to the invention allows seamless data monitoring and logging off in order to generate GMP-compliant reports for the cell cultivation process, with "data logging off" meaning that the current data from the system control is stored in a connected database or in a higher-level software system (control station, MES or ERP) and are therefore available for later evaluations and reports.

• - Automatisierte Zellkultivierung
  • ◯ Kontrollieren und Regeln der Umgebungsbedingungen Temperatur (über Heizung) und CO₂-Konzentration (Bestimmung des CO₂-Gehaltes in der Inkubatoratmosphäre mittels Sensor, bei Bedarf Erhöhung des CO₂-Gehaltes durch Öffnen eines Ventils und Zuführung von CO₂ aus einem angeschlossenen Reservoir, bspw. einer Gasflasche);
  • ◯ Automatisierte Zellmessung und Auswertung im Zellzähler 22 nach rezeptierten Einstellungen, global für alle Zellkulturen 6 oder individuell (Intervall der Zeitmessung, Messprotokoll, Auswertung der Ergebnisse zum Ableiten weiterer Maßnahmen, bspw. Zuführung von Nährmedium);
  • ◯ Automatisiertes Zuführen von Nährmedium, gewichtskontrolliert, entsprechend rezeptiver Einstellungen (bspw. basierend auf der Zellmessung oder entsprechend eines Intervalls) bei gleichzeitiger Einstellung des Oberflächen-Volumen-Verhältnisses;
  • ◯ Automatisierte Zell-Agitation entsprechend rezeptiver Einstellungen (Intervall, Frequenz, Amplitude);
  • ◯ Meldung von Abweichungen und Störungen an eine Kundenschnittstelle zu höheren Überwachungssytemen, soweit vorhanden
• - Datenerhebung und Reporting entsprechend GMP-Normen
• - Sichere Speicherung von Los- und Prozessdaten, Rezeptwerten und -änderungen, bspw. Umgebungsbedingungen, Ergebnisse der Zellmessungen, Zuführung von Nährmedium
• - Protokollierung von Abweichungen und Compliance-Problemen, sofern von der Anlage detektierbar
• - ggf. Report-Generierung in Werker-lesbarer Form, d.h., menschenlesbarer Text

The automation of the incubator system according to the invention includes the following areas:

• - Automated cell cultivation
  • ◯ Controlling and regulating the ambient conditions, temperature (via heating) and CO ₂ concentration (determining the CO ₂ content in the incubator atmosphere using a sensor, if necessary increasing the CO ₂ content by opening a valve and supplying CO ₂ from a connected reservoir e.g. a gas bottle);
  • ◯ Automated cell measurement and evaluation in the cell counter 22 according to prescribed settings, globally for all cell cultures 6 or individually (interval of time measurement, measurement protocol, evaluation of the results to derive further measures, e.g. supply of culture medium);
  • ◯ Automated supply of nutrient medium, weight-controlled, according to receptive settings (e.g. based on cell measurement or according to an interval) with simultaneous setting of the surface-to-volume ratio;
  • ◯ Automated cell agitation according to receptive settings (interval, frequency, amplitude);
  • ◯ Notification of deviations and faults to a customer interface to higher monitoring systems, if available
• - Data collection and reporting according to GMP standards
• - Secure storage of batch and process data, recipe values and changes, e.g. environmental conditions, results of cell measurements, supply of culture medium
• - Logging of deviations and compliance problems, if detectable by the plant
• - If necessary, report generation in worker-readable form, ie human-readable text

Reference List

2

incubator system

4

incubator

6

cell cultures

8th

flexible cell culture bags

10

Cell Culture Bag Frame

12

Holding or mounting points

14

pinch rollers

16

flexible support bands

18

hose system

20

measuring cuvette

22

cell counter

24

pump

26

process locks

28

inner lock door

30

outer lock door

32

internal incubator axis system

34

load cells

36

culture medium reservoir

38

pump

40

thermal section

44

external incubator axis system

46

end effector

48

pinch valve

Claims (23)

Incubator system (2) for the simultaneous, automated cultivation of a plurality of cell cultures (6), the plurality of cell cultures (6) each being arranged in a closed system and having identical, controllable environmental conditions, comprising - an incubator (4) with a fixed internal volume; - several flexible cell culture bags (8) clamped floating in a corresponding frame (10) for each of the several cell cultures (6), wherein the respective frames (10) for the several cell cultures (6) can be introduced into the incubator (4), and each frame (10) being receivable at respective mounting points (12) provided in the incubator (4); - Several process locks (26) connected to the respective cell culture bags (8) with manually or automatically operable inner (28) and outer (30) lock doors for connecting components to or for sampling from each of the several flexible cell culture bags (8) ; - an automatic agitation device connected to each of the flexible cell culture bags (8), whereby each of the plurality of cell culture bags (8) can be agitated either individually or in combination; - an inner (32) and an outer (44) incubator axis system for the automatic delivery of a plurality of hardware components integrated into the incubator system (2) to the plurality of cell cultures (6); - a cell counter (22) mounted outside the incubator (4) on the outer incubator axis system (44) for measuring the cell culture volume or cell density by means of a cell counter (4) connected to the incubator (4) by a telescopic rail system and automatically integrated into the cell counter ( 22) measuring cuvette (20) that can be inserted and removed separately for each cell culture bag (8), wherein the cell counter (22) can be positioned in front of the automatically operable process locks (26) with the aid of the external incubator axis system (44); - an automatic weighing system with load cells (34) connected to the internal incubator axis system (32); and - A nutrient reservoir (36) connected to each of the plurality of cell cultures (6), each cell culture bag being assigned its own nutrient reservoir (36). incubator system claim 1 , characterized in that the cell culture bags (8) can be connected to other additional devices via hose connections. incubator system claim 2 , characterized in that the additional devices are analysis devices or devices for introducing material. Incubator system according to one of the Claims 1 until 3 , characterized in that it has a higher-level external control system for acquiring and determining data and for controlling various hardware components. Incubator system according to one of the preceding claims, characterized in that the agitation device consists of a seesaw device. incubator system claim 5 , characterized in that each cell culture bag frame individually has its own rocker drive. incubator system claim 5 , characterized in that a central rocking drive is provided for all cell culture bag frames (10). Incubator system according to one of the preceding claims, characterized in that each of the frames (10) into which the flexible cell culture bags (8) are integrated has two squeezing rollers (14) for dividing the cell culture bag (8) into different volumes. Incubator system according to one of the preceding claims, characterized in that each of the frames (10) into which the cell culture bags (8) are integrated has flexible support straps (16) on its underside. Incubator system according to one of the preceding claims, characterized in that the cell counter (22) is based on a light microscope measurement. Incubator system according to one of the preceding claims, characterized in that the inner incubator axis system (32) has end effectors at its bottom ends for raising or lowering the cell culture bag frame (8). incubator system claim 11 , characterized in that weighing cells (34) for weighing the cell culture bags (8) clamped in the corresponding frame (10) are placed on the end effectors (46). Incubator system according to one of the preceding claims, characterized in that the outer incubator axis system (32) with Components for machine-readable identification of the cell culture bag (8) is equipped. Incubator system according to one of the preceding claims, characterized in that the nutrient medium reservoir (36) can be cooled. incubator system Claim 14 , characterized in that the nutrient medium can be heated to the incubator temperature on an inflow or heating section (40) to the various cell cultures (6). Incubator system according to one of the preceding claims, characterized in that the components which have surfaces which come into contact with the product or cell culture are so-called single-use components. Method for the simultaneous automated cultivation of several cell cultures with an incubator system (2) according to one or more of Claims 1 until 16 , characterized by the following steps: a) filling an expandable cell culture bag (8) with an initial cell culture (6) and dividing the total volume of the cell culture bag (8) into a useful volume and a process volume by means of the squeezing rollers (14); b) clamping the flexible cell culture bag (8) in the cell culture bag frame (10); c) introducing the cell culture bag frame (10) into the incubator (4); d) if necessary, repeating steps a) to c) for further cell cultures (6): e) monitoring the introduced cell cultures by inserting the measuring cuvette (20) into the cell counter (22), measuring the cell culture volume or the cell density of the cell cultures (6), and re-ejecting the measurement cuvette (20) from the cell counter (22); f) determining the amount of the next nutrient liquid addition based on the monitoring in point e); g) feeding the amount of nutrient determined in step f) into the respective flexible cell culture bag (8); and h) repeating steps e) and f) until the desired cell culture volume or cell culture density is reached. procedure after Claim 17 , characterized in that the cell cultures (6) located in the incubator (4) are continuously agitated. procedure after Claim 17 or 18 , characterized in that the monitoring in step e) takes place by means of a light microscope or by laser methods. Procedure according to one of claims 17 until 19 , characterized in that the supply of the amount of nutrient liquid in step g) is weight-controlled by weighing the respective cell culture bag frame (10). procedure after claim 20 , characterized in that for weighing the cell culture bag frame (10) this is raised by means of the end effectors (46) and the load cells (34) mounted thereon with the aid of the inner incubator axis system (32) and is lowered again after weighing has taken place. Procedure according to one of claims 17 until 21 , characterized in that the cell culture bags (8) are connected to other additional devices via hose connections. Procedure according to one of claims 17 until 22 , characterized in that the cell culture (6) is pumped (cascading) into an empty, larger cell culture bag (8) already clamped in the incubator (4) when a preset volume is reached.

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