WO2014086412A1

WO2014086412A1 - Device and procedure for the long-term supply of cell cultures with medium

Info

Publication number: WO2014086412A1
Application number: PCT/EP2012/074555
Authority: WO
Inventors: Beat Halter; Michaela Aufderheide
Original assignee: Mohr, Ulrich; Japan Tobacco Inc.
Priority date: 2012-12-05
Filing date: 2012-12-05
Publication date: 2014-06-12
Also published as: EP2929010A1

Abstract

Procedure for the supply of cell cultures in a cell culture vessel at the air-liquid interface (ALI), characterized in that a medium level below the cell culture vessel over a period of several weeks (3 to 6 weeks) lies within a few tenths of a millimetre (0.2 to 0.3 millimetres) to a predefined reference value and the medium can be changed and circulated, i.e. mixed.

Description

Device and Procedure for the Long-Term Supply of Cell Cultures with Medium

Introduction

The invention concerns an in vitro device and procedure for the automatic reproducible long-term supply of cell cultures with medium at the air-liquid interface.

The demands and requirements made on cell systems with regard to their complexity (from monocultures right through to 3-dimensional multilayered cultures of at least two different cell types) and their significance in relation to toxicological issues call for the provision of stable, reproducible cell cultures for use in the investigation of active substances, as for example in the prescreening procedure. This is particularly the case for cell cultures that exhibit a comparable cell differentiation for simulation of the in vivo situation. The dominant influencing variable in the setting up and cultivation of the cultures is their medium supply, not only with nutrients for growth, but also with substances that control the cell differentiation. This is of particular importance for cultures to investigate inhalation toxicology-relevant issues at the air-liquid interface (ALI). Here, the cells are supplied basally with nutrients, whilst the apical fraction is in contact with the surrounding atmosphere. An optimal supply of the total cell population - from the basal contact surface right to the apical cell layers - is indispensable for the generation of stable and reproducible cell cultures.

Status of the technology

The medium exchange for long-term cultures under conventional conditions is carried out manually and is dependent on the cell type, e.g. every 24-48 hours over a period of several weeks. The manual exchange of the medium is subject to variation depending on the personnel (particularly due to inaccuracy when pipetting), which with the normally low volume of medium on the base of the cell culture vessels inevitably influences the medium supply (concentration of nutrients). A further instability factor is the reproducible generation of the cell cultures during the incubation phase in the cell incubator. To maintain the vitality of the cells, a temperature of 37°C (± 0.1 °C) at 95% (± 1 %) humidity is necessary. During the incubation phase, which is only interrupted by the renewal of the medium, evaporation of the medium occurs, thus affecting the available nutrient concentrations and noticeably influencing the morphology and physiology of the cells. Similarly, cell cultures react sensitively to mechanical irritations, which result during the handling of the cultures.

In addition to the technical disadvantages mentioned, the manual supply of the cell cultures requires substantial work input.

Purpose of the Invention

The purpose of the invention is to remedy the disadvantages connected with the manual cell supply, so that comparable cultures can be prepared under defined supply conditions and facilitate reproducible toxicological testing. The main elements of the purpose are the stabilization of the medium level more accurate than 0.2 to 0.3 millimetres using a control device, regular mixing of the medium to guarantee a homogeneous nutrient supply and stimulation of the cell growth as well as the complete or partial exchange of the medium at intervals of 12 to 48 hours over a period of 3 to 6 weeks.

Solution of the Task

The invention concerns a device for the supply of cell cultures at the air-liquid interface (ALI). The uniqueness lies in the fact that the medium level below the cell culture vessel is brought over a period of several weeks to a predefined reference value within a tolerance of a few tenths of a millimetre and the medium can be changed and circulated, i.e. mixed. A few tenths of a millimetre means here 0.2 to 0.3 millimetres. The mentioned period of time is preferable 3 to 6 weeks.

In addition, protection by patent is sought separately for a procedure to supply cell cultures at the air-liquid interface (ALI), distinguished by the fact that the medium level below the cell culture vessel is brought over a period of several weeks to a predefined reference value within a tolerance of a few tenths of a millimetre and the medium can be changed and circulated, i.e. mixed. A few tenths of a millimetre means here 0.2 to 0.3 millimetres. The mentioned period of time is preferable 3 to 6 weeks.

The medium level is thus fed by a control device to a predefined reference value. The control device should be an electronic control device. The height of the fluid level should preferable be recorded by an ultrasonic sensor next to the cell culture vessel. Next to the cell culture vessel means that the ultrasonic sensor is not directed exactly to the cell culture vessel, but is positioned so that the medium level can be recorded immediately next to the cell culture vessel.

The control device further includes an electronic data processing unit.

The medium level is brought to a predefined reference value by the addition and removal of medium. This in turn means that the predefined reference value is kept constant and an optimal supply to the cells is thus achieved.

The inventive device is so designed that the medium can be exchanged by the addition and removal of medium. This medium exchange can thus be program- controlled.

The addition or removal of medium is effected by a controlled peristaltic pump.

The device should further circulate the medium for the purpose of homogenization or to stimulate the cells by mechanical stimuli. The circulation is effected by a pump and one or more jets producing currents in the medium.

The medium is divided by a perforated plate into a closed chamber and a chamber open to the cell culture and the circulation is effected by forcing a change in volume in the closed chamber, thus producing a synthetic jet from each hole of the perforated plate. The volume change is forced by a membrane or a piston. The energy required to force a volume change is fed into the system by an electromagnet or an electric motor. The circulation is effected by the volume change or a movable part in the medium, for example by a propeller or a magnetic stirrer. The circulatory procedure is program-controlled and the parameters of the circulatory procedure are variable. Within the scope of the device, several cell culture vessels can be supplied with medium from a common pool. The medium is circulated by a moving disc-shaped body. The medium is recirculated by a pump for mixing or pumped into another tank for the purpose of mixing and then pumped back.

With the aid of the figures, examples of devices for the automatic long-term supply of cell cultures are illustrated.

Fig. 1 : General view of a long-term supply device with 3 sample uptake modules Fig. 2: Vertical cross-section of a long-term supply device

Fig. 3: Vertical cross-section of a sample uptake module

Fig. 4: A sample uptake module with a mixing propeller

Fig. 5: A long-term supply device with a common medium supply to several cell culture vessels

Fig. 6: An alternative form of construction of the long-term supply device shown in

Fig. 5

Figure 1 gives a general view of an example of the inventive device. The device is made up of a supply unit (1 ) and three removable autoclavable sample uptake modules (2). Each sample uptake module has space for a commercially available cell culture vessel (3), which can be protected from contaminants and draughts by a removable lid (4). The three cell cultures in this model can all be operated independently.

The supply unit (1 ) has a coupling (5), to which an external storage container (not illustrated) with fresh medium is connected. Used medium is pumped via the coupling

(6) into an external disposal container or outlet (not illustrated). The medium feed from the supply unit (1 ) to the sample uptake modules (2) takes place via the tubes

(7) and the medium removal via the tubes (8). Connectors (9) link the electrical components with an external control unit (not illustrated), for example a programmable logic controller (PLC).

Figure 2 shows a section through the device according to Figure 1 . One supply pump (10) and one removal pump (1 1 ) each is incorporated in supply unit (1 ) for the operation of each sample intake module (2). For hygienic reasons, peristaltic pumps have proved to be advantageous. The medium in the sample uptake module is circulated in the depicted design by strokes, carried out by the electromagnet (12) incorporated in the supply unit (1 ) and directed into the sample uptake module (2) via the primary plunger (13).

Figure 3 shows the sample uptake module (2), whose basic structure is composed of a main body (14), an uptake ring (15) and a glass tube (16) clamped between these two parts. A cell culture vessel (3) is suspended in the uptake ring (15) that is protected by the already mentioned removable lid (4).

The cell culture is situated on a microporous membrane (3a), which is a part of the cell culture vessel (3). The sample uptake module is filled up to the level of the membrane (3a) with medium (17). Depending on the culture, a fluid level several tenths of a millimetre above or below the membrane level can be necessary, whereby a fluid level below the membrane level is docked onto the membranes by prior short- term overfilling. In this case, the medium level thus lies only on the free surface under the membrane level. The surface tension of the medium allows such medium levels in different heights to be set within certain limits.

It is extremely important that the medium level can be set more accurate than 0.2 to 0.3 millimetres. Deviations, for example due to evaporation, must be recognized and balanced out by readjustment. It has been proven to be advisable that the height of the medium level is recorded by an ultrasonic distance measurement and the medium level is adjusted to the reference value with the aid of the supply or removal pump. In the design example in Figure 3, an ultrasonic sensor (18) is fitted on a sonic nozzle (19). The sonic nozzle allows the limitation of the scanned surface.

Also important is a periodic circulation of the medium, e.g. at 60-minute intervals. The circulation is necessary to mix more exhausted medium from the membrane environment with less exhausted medium as well as to mix the fresh portion of medium with the remaining portion after a partial renewal of the medium. A targeted circulation also allows the generation of mechanical stimuli on the cells, which stimulates their growth. In the design example in Figure 3, the medium is circulated as follows:

The medium (17) is located in an upper chamber (17a) and a lower chamber (17b). Between the two chambers there is a perforated plate (20), which has, for example, 36 holes with a diameter of 0.8 mm. In the base of the lower chamber (17b), there is a secondary plunger (21 ), that moves in a vertical direction, and which is sealed with the aid of a membrane (22). By rapid upward activation of the secondary plunger (21 ), medium from the lower chamber (17b) is displaced and streams upwards through the perforated plate (20). The holes act as jets and allow the displaced medium to flow up to the medium surface or membrane (3a). Moving the primary plunger (13) downwards, medium is drawn from the upper chamber (17a) into the lower chamber (17b), whereby the drawn medium comes naturally from the immediate environment of the holes and thus need not to be the same medium previously pushed upwards . In this way, a good mixture is achieved with only a few strokes of the secondary plunger (21 ).

Activation of the secondary plunger (21 ) is achieved, as explained in the description of Figure 2, by the electromagnet (12) and the primary plunger (13) connected to it. Resetting of the secondary plunger is ensured by springs (not visible in the figures). The supply and removal of medium occurs via holes (not visible in the figures) in the main body (14). The holes lead into the lower chamber (17b).

Figure 4 shows an alternative solution to the mixing principle visible in Figure 3. Mixing is done by a propeller (23), which sits on a shaft (24) and is operated by a driving pin (25). The propulsion is effected by an electric motor (not illustrated), which is installed in place of the electromagnet (12) in the supply unit (1 ).

Figure 5 shows a further variant of an inventive device. In contrast to the designs shown in Figures 1 to 4, here a number of cell culture vessels (3) - in the example shown 24 vessels - are suspended in a sample plate (26) and supplied from a common medium pool (27). Mixing of the medium is done by a periodically rotating mixing disc (28). Notches (28a) in the mixing disc (28) lead to a stronger mixing effect. In order to ensure that all cell culture vessels have the same conditions when the mixing disc is resting, it is recommended that the mixing disc has a notch pattern corresponding to the cell culture vessel positioning. However, other forms of disc can be used, whereby the same conditions for all cell culture vessels can also be created by frequent short or permanent slow movement of the mixing disc.

Propulsion of the mixing disc (28) is effected in this design example by an electric motor (29). In addition, the sample plate (26) is vertically adjustable via columns (30), which allows, for example, after filling with medium, a short-term lowering of the cell culture vessels (3) and thus guaranteed wetting with simultaneous economical use of medium.

Figure 6 shows a further variant of the inventive device. Like the example in Figure 5, this device too is suitable for use with a number of cell culture vessels and has a common medium supply. In contrast to Figure 5, in a base plate (31 ) for each cell culture vessel, there is a cavity (31 a) which is filled with medium (not illustrated) via a central medium connection (32) and ducts moulded (31 b) in the base plate (31 ). To mix the medium, in this design example the medium is pumped out temporarily into an external mixing tank (not illustrated) and then pumped back again into the base plate (31 ).

All the design examples have in common that the frequency and intensity of medium change and mixing procedures can be varied using external controls (not illustrated) in wide areas and can be adapted to the requirements of the respective cell culture.

Reference List

1 Supply unit

2 Sample uptake module

3 Cell culture vessel

3a Membranes

4 Lid

5 Coupling Coupling

Tube

Connector

Supply pump Removal pump Electromagnet Primary plunger Main body

Uptake ring

Glass tube

Medium

a Upper chamberb Lower chamber Ultrasonic sensor Sonic nozzle Perforated plate Secondary plunger Membranes Propeller

Shaft

Driving pin

Sample plate Medium pool Mixing disca Notches

Electric motor Column

Base platea Cavity

b Ducts

Medium connection

Claims

Claim 1

Claim 2

Procedure as in Claim 1 , characterized in that the medium level is fed by a control device to a predefined reference value.

Claim 3

Procedure as in Claim 2, characterized in that the control device is an electronic control device.

Claim 4

Procedure as in one of the Claims 1 to 3, characterized in that the height of the medium level is determined by an ultrasonic sensor related to a base and next to the cell culture vessel.

Claim 5

Procedure as in Claim 3 or 4, characterized in that the control device includes an electronic data processing unit.

Claim 6

Procedure as in one of the previous Claims, characterized in that the medium level is brought to a predefined reference value by the addition and removal of medium.

Claim 7

Procedure as in one of the Claims 1 to 6, characterized in that the medium can be changed by addition and removal of fluid. Claim 8

Procedure as in Claim 7, characterized in that the medium exchange is program- controlled.

Claim 9

Procedure as in Claim 6 or 7, characterized in that the addition and removal of medium is effected by a controlled peristaltic pump.

Claim 10

Procedure as in one of the Claims 1 to 9, characterized in that the medium can be circulated for the purpose of homogenization or to stimulate the cells by mechanical stimuli.

Claim 1 1

Procedure as in Claim 10, characterized in that the circulation is effected when a pump and one or more jets produce currents in the medium.

Claim 12

Procedure as in Claim 1 1 , characterized in that the medium is divided by a perforated plate into a closed chamber and a chamber open to the cell culture and the circulation is effected by forcing a change in volume in the closed chamber, thus producing a synthetic jet from each hole of the perforated plate.

Claim 13

Procedure as in Claim 12, characterized in that the volume change is forced by a membrane or a piston.

Claim 14

Procedure as in Claim 12 or 13, characterized in that the energy required to force a volume change is fed into the system by an electromagnet or an electric motor.

Claim 15 Procedure as in one of the Claims 10 to 14, characterized in that the circulation is effected by a movable part in the fluid, for example by a propeller or a magnetic stirrer.

Claim 16

Procedure as in one of the Claims 10 to 15, characterized in that the circulatory procedure is program-controlled and the parameters of the circulatory procedure are variable.

Claim 17

Procedure as in one of the previous Claims, characterized in that several cell culture vessels are supplied with medium from a common pool.

Claim 18

Procedure as in Claim 17, characterized in that the medium is circulated by a rotating disc-shaped body.

Claim 19

Procedure as in Claim 17, characterized in that the medium is recirculated by a pump for mixing or pumped into another tank for the purpose of mixing and then pumped back.

Claim 20

Device for the supply of cell cultures in a cell culture vessel at the air-liquid interface (ALI), characterized in that there is a control device which regulates that the medium level below the cell culture vessel over a period of several weeks is constant, within a few tenths of a millimetre of a predefined reference value and that the medium can be changed and circulated, i.e. mixed.

Claim 21

Device as in Claim 20, characterized in that the control device is an electronic control device. Claim 22

Device as in one of the Claims 20 to 22, characterized in that there is an ultrasonic sensor that determines the medium level related to a base and next to the cell culture vessel.

Claim 23

Device as in Claims 20 to 22, characterized in that the control device further includes an electronic data processing unit.

Claim 24

Device as in one of Claims 20 to 23, characterized in that the addition or removal of medium is effected by a controlled peristaltic pump.

Claim 25

Device as in one of Claims 20 to 24, characterized in that the circulation is effected by a pump and one or more jets.

Claim 26

Device as in one of Claims 20 to 25, characterized in that the medium is divided by a perforated plate into a closed chamber and a chamber open to the cell culture and the circulation is effected by forcing a change in volume in the closed chamber, thus producing a synthetic jet from each hole of the perforated plate.

Claim 27

Device as in Claim 26, characterized in that there is a membrane or a piston in the chambers.

Claim 28

Device as in Claim 26 or 27, characterized in that the energy required to force a volume change is introducible into the system by an electromagnet or an electric motor.

Claim 29 Device as in one of Claims 20 to 28, characterized in that the circulation is effected by a movable part in the medium, for example by a propeller or a magnetic stirrer.

Claim 30

Device as in one of Claims 20 to 29, characterized in that the medium is circulated by moving a disc-shaped body.