DE102013113578B4 - Process for the controlled supply of one or more cell cultures in cell culture containers with a medium - Google Patents

Abstract

Method for the controlled supply of one or more cell cultures in cell culture containers (3) with a medium (17), e.g. of the respiratory tract when cultured at an air-liquid interface, characterized in that in particular for improving cell growth and differentiation of the cells in long-term cultures a medium circulation takes place within the cell culture container (3).

Description

introduction

The invention relates to a method according to the preamble of claim 1 and a device therefor.

In particular, it relates to an in vitro device and a method for the automated, reproducible long-term supply of cell cultures on microporous membranes at the air-liquid interface with medium.

State of the art

The demands and requirements on cell systems in terms of their complexity (monocultures up to 3-dimensional multilayer cultures, from at least 2 different cell types) and meaningfulness in relation to toxicological issues require the provision of stable, reproducible cell cultures for drug testing, such as in prescreening procedures. This applies in particular to cell cultures which have a comparable cell differentiation to simulate the in vivo situation. The dominant influencing variable when setting up and growing the cultures is their medium supply via the microporous membrane, not only with nutrients for growth, but also with substances that control cell differentiation. This is of particular importance for cultures used to investigate inhalation-toxicologically relevant issues at the air-liquid interface (ALI). Here the cells are basally supplied with nutrients, while the apical part is in contact with the surrounding atmosphere. An optimal supply of the entire cell population - via the basal contact surface (membrane) to the apical cell layers - is the indispensable prerequisite for the generation of stable and reproducible cell cultures.

The medium exchange for long-term cultures is carried out manually under conventional conditions and, depending 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 person-dependent fluctuations (especially inaccuracies when pipetting), which inevitably influence the medium supply (concentration of nutrients) given the normally small medium volume below the cell culture vessels. Another uncertainty factor is the reproducible generation of cell cultures during the growth phase (incubation) in the cell incubator. A temperature of 37°C (± 0.1°C) with an air humidity of 95% (± 1%) is required to maintain the vitality of the cells. If the medium is changed manually outside of the cell incubator, the temperature and humidity will temporarily drop. During the incubation phase, medium evaporates, further affecting the available nutrient concentrations, which noticeably affects the morphology and physiology of the cells. Likewise, cell cultures react sensitively to mechanical irritations that arise when handling the cultures.

In addition to the technical disadvantages mentioned, the manual care of the cell cultures involves considerable personnel effort.

In the DE19801763 describes a culture device which automatically corrects level deviations by pumping in or pumping out medium or also allows different medium levels under program control. In addition, however, a regular or continuous exchange of medium is necessary, it being important that the entire cell culture is supplied with fresh nutrients evenly and reproducibly. Devices are known, for example from CH 631207, which ensure an even distribution of culture medium flowing through in the case of submerged cultivation. When cultured at the air-liquid interface (ALI), the uniform and reproducible supply of nutrients to the cells is more challenging than with submerged culture, since the culture is naturally located at the interface of the fluid and often only a part of the medium, e.g Third, to be exchanged. A further complication is that the exchanged medium quantities are in many cases only in the range of a few milliliters, so that flow-mechanical effects when the fresh medium is supplied can hardly be used to mix the medium.

object of the invention

The object of the invention is to eliminate the disadvantages associated with the manual cell supply by automated supply of air-liquid cultures of epithelial cells in particular z. B. the respiratory tract, so that comparable cultures can be provided under defined supply conditions and reproducible toxicological studies are possible. In addition to keeping the medium level constant with a control device with an accuracy of 2 to 3 tenths of a millimetre, the central components of the task are the regular, reproducible mixing of the medium to ensure a homogeneous supply of nutrients, the regulation of cell proliferation and cell differentiation by adjusting the duration, frequency and amplitude of biomechanical stress as well as the full constant or partial exchange of the medium at intervals of, for example, 12 to 48 hours over a period of up to 6 weeks without the medium temperature or humidity changing during the medium exchange.

solution of the task

The characterizing part of claim 1 leads to the solution of the problem.

The device should thus be able to circulate the medium for the purpose of homogenization or for stimulating the cells by mechanical stimuli. The circulation takes place by currents being generated in the medium, which can be done by a pump and one or more nozzles or by other conceivable devices. This happens completely independently of the actual supply and removal of the medium.

In a preferred embodiment, the medium is introduced through a perforated plate into a closed chamber that is open to the cell culture. The circulation is generated by forcing a volume change in the closed chamber and the resulting free jet at each hole (synthetic jet). The change in volume is forced by a diaphragm or a piston. The energy required to force a volume change is introduced into the system by an electromagnet or an electric motor. Circulation is achieved by the change in volume or by a moving part in the medium, for example a propeller or a magnetic stirring bar. The circulation process is program-controlled and the parameters of the circulation process are adjustable. Within the scope of the invention, several cell culture containers can also be supplied jointly by one medium. The medium is circulated by a driven disc-shaped body. The medium is pumped around to mix it or pumped out into another container and pumped back in for the purpose of mixing.

The invention primarily also relates to a device for supplying cell cultures when cultures are carried out at an air-liquid interface (ALI). The medium level under the cell culture container, as known from the prior art, is adjusted to a specified target value within a few tenths of a millimeter over a period of several weeks and the medium is replaced and circulated, i.e. mixed. A few tenths of a millimeter preferably mean 2 to 3 tenths of a millimeter. The period of several weeks mentioned preferably means 3 to 6 weeks.

In this case, the medium level is to be tracked by a control device, as is also the case according to the prior art, to a predetermined desired value. Furthermore, the control device should be an electronic control device. The height of the medium level should preferably be detected by an ultrasonic sensor next to the culture carrier. Next to the culture carrier means that the ultrasonic sensor is not aimed directly at the culture carrier. Next to it means that the ultrasonic sensor is placed in such a way that the medium level can be detected directly next to the circumference of the culture carrier.

The problems associated with the manual supply of an air-liquid culture are solved with a device that records the level of the medium with a distance-measuring ultrasonic sensor and regulates it to a specified target value by pumping in and pumping out medium using two peristaltic pumps. The pumps are also used to pump out the medium or parts of it at predetermined time intervals and to replace it by pumping in fresh medium. A mechanism for generating defined flows in the medium was integrated into the device, since it has surprisingly been shown that this not only achieves a homogeneous distribution of the medium components, but also regulates cell proliferation and cell differentiation. It has proven to be advantageous to arrange the ultrasonic sensor used for level measurement directly next to the cell culture vessels. As a result, greater process reliability is achieved than, for example, with indirect measurement in a communicating pipe or vessel ( DE 198 01 763 C2 ), where foam or air bubbles can easily give false readings. Target values and time intervals are specified and the sensor signals are processed via a programmable logic controller (PLC).

A further development of the devices described consists in preheating fresh medium to the temperature at which the cells are cultivated, usually 37° C., immediately before it is fed in with the aid of a heat exchanger. This allows the medium supply to be provided in a cooled condition, which reduces the risk of an unwanted change in medium components. By preheating the medium, a constant temperature of the cell culture can be guaranteed when changing the medium. A preferred form of heat exchanger consists of a container filled with water in which the water temperature is regulated to a desired value by a heating element. The medium is guided through the water tank in a spiral tube, whereby the medium heats up as it passes through. A particularly good heat transfer is achieved when the water used for temperature control is circulated in the container, for example by means of a magnetic stirring bar.

All the exemplary embodiments have in common that the frequency and intensity of medium changing and mixing processes with the external control (not shown) varies within wide ranges and can be adapted to the requirements of the existing cell culture.

Examples of devices for the automated long-term supply of cell cultures at the air-liquid interface are explained with reference to the drawings.

• 1: Eine Gesamtansicht einer Langzeitversorgungsvorrichtung mit 3 Probenaufnahmemodulen
• 2: Einen Vertikalschnitt durch eine Langzeitversorgungsvorrichtung
• 3: Einen Vertikalschnitt durch ein Probenaufnahmemodul
• 4: Ein Probenaufnahmemodul mit Mischpropeller
• 5: Eine Langzeitversorgungsvorrichtung mit gemeinsamer Mediumversorgung mehrerer Zellkulturbehälter

It shows:

• 1 : An overall view of a long-term supply device with 3 sample receiving modules
• 2 : A vertical section through a long-term care device
• 3 : A vertical section through a sample receiving module
• 4 : A sample intake module with mixing propeller
• 5 : A long-term supply device with common medium supply of several cell culture vessels

1 shows an overall view of an exemplary embodiment of the device according to the invention. The device consists of a supply part 1 and three removable, autoclavable sample receiving modules 2. Each sample receiving module offers space for a commercially available cell culture container 3, which can be protected from foreign bodies and drafts with a removable cover 4. In this version, the three cell cultures can be managed independently of one another.

The supply part 1 has a coupling 5 to which an external reservoir (not shown) with fresh medium is connected. Used medium is pumped via the coupling 6 into an external disposal tank or drain (not shown). The medium is supplied from the supply part 1 to the sample intake modules 2 via the hoses 7, and the medium is removed via the hoses 8. Connectors 9 are used to connect the electrical components to an external control unit (not shown), for example a programmable logic controller (PLC).

2 shows a section through the device according to FIG 1 . A feed pump 10 and a discharge pump 11 are installed in the supply part 1 for the management of each sample receiving module 2 . For reasons of hygiene, peristaltic pumps have proven advantageous. The medium circulation as well as the induction of biomechanical stress through targeted flows in the sample receiving module takes place in the illustrated embodiment by lifting movements, which are generated by the electromagnet 12 installed in the supply part 1 and are introduced into the sample receiving module 2 via the primary plunger 13.

3 shows the sample receiving module 2, the basic structure of which consists of a base body 14, a receiving ring 15 and a glass tube 16 clamped between these two parts. A cell culture container 3 is suspended in the receiving ring 15 and is protected by the cover 4 that can be lifted off, as already mentioned.

The cell culture is located on a microporous membrane 3a, which is part of the cell culture container 3. The sample receiving module is filled with medium 17 up to the level of the membrane 3a. Depending on the culture, a medium level that is several tenths of a millimeter above the membrane level or below the membrane level can be advantageous, with a medium level below the membrane level being docked to the membrane by a brief overfilling beforehand. In this case, the medium level is only below the membrane level on the free surface. The surface tension of the medium allows the medium level to be set at different heights within certain limits.

It is of great importance that the medium level can be set with an accuracy of 2 to 3 tenths of a millimetre. Deviations, for example due to evaporation, must be recognized and compensated for by readjustment. It has proven particularly useful to record the height of the medium level using a displacement-measuring ultrasonic sensor and to regulate the medium level to the desired value with the aid of the feed or discharge pump. In the embodiment according to 3 an ultrasonic sensor 18 is mounted on a sonic nozzle 19. The sonic nozzle allows the delimitation of the scanned area.

Periodic mixing of the medium is also important, eg at intervals of 60 minutes. Mixing is necessary on the one hand to mix used medium from the medium layer close to the cell with the remaining medium, which is even more nutrient-rich, and to mix the fresh portion with the remaining portion after a partial renewal of medium. On the other hand, it has also surprisingly turned out that on the basis of fluid mechanics Ways the growth and differentiation of the cells can be decisively regulated. In this case, however, it is essential that the cell cultures are flown on in a reproducible manner.

• Das Medium 17 befindet sich in einer oberen Kammer 17a und einer unteren Kammer 17b. Zwischen den beiden Kammern befindet sich ein Lochblech 20, welches beispielsweise 36 Löcher mit Durchmesser 0.8 mm enthält. Im Boden der unteren Kammer 17b befindet sich ein in vertikaler Richtung beweglicher Sekundärstössel 21, welcher mit Hilfe einer Membrane 22 dicht eingebaut ist. Durch Betätigung des Sekundärstössels 21 nach oben wird Medium aus der unteren Kammer 17b verdrängt und strömt durch die Lochplatte 20 nach oben in die Kammer 17a. Die Löcher wirken dabei als Düsen und lassen das verdrängte Medium bis gegen die Mediumoberfläche bzw. Membrane 3a strömen (synthetic jet). Bei Bewegung des Primärstössels nach unten wird Medium von der oberen Kammer 17a in die untere Kammer 17b gesaugt, wobei das angesaugte Medium naturgemäss aus der unmittelbaren Umgebung der Löcher stammt und somit nicht dem vorher von unten nach oben geförderten Medium entspricht. Auf diese Weise entsteht durch wenige Hübe des Sekundärstössels 21 eine gute Durchmischung. Die strömungsmechanischen Effekte können durch Anzahl und Durchmesser der Löcher in der Lochplatte, sowie durch Einstellung von Dauer, Frequenz und Amplitude der Hübe in weiten Bereichen variiert werden, was für die entdeckte Regulierbarkeit der Zellproliferation und Zelldifferenzierung durch biomechanischen Stress von besonderer Bedeutung ist.

In the embodiment according to 3 the generation of defined flows in the medium takes place 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 contains, for example, 36 holes with a diameter of 0.8 mm. In the bottom of the lower chamber 17b there is a secondary ram 21 which can be moved in the vertical direction and which is tightly installed with the aid of a membrane 22. By actuating the secondary plunger 21 upwards, medium is displaced from the lower chamber 17b and flows upwards through the perforated plate 20 into the chamber 17a. The holes act as nozzles and allow the displaced medium to flow against the medium surface or membrane 3a (synthetic jet). When the primary ram moves downwards, medium is sucked from the upper chamber 17a into the lower chamber 17b, the sucked medium naturally originating from the immediate vicinity of the holes and thus not corresponding to the medium previously conveyed from the bottom upwards. In this way, thorough mixing is achieved by a few strokes of the secondary ram 21 . The flow-mechanical effects can be varied over a wide range by the number and diameter of the holes in the perforated plate, as well as by adjusting the duration, frequency and amplitude of the strokes, which is of particular importance for the discovered controllability of cell proliferation and cell differentiation by biomechanical stress.

The secondary plunger 21 is actuated as in the description 2 mentioned by the electromagnet 12 and the primary plunger 13 connected to it. The resetting of the secondary plunger is ensured by springs (not visible in the figures). The lifting movements can also be generated by a connecting rod/crankshaft, for example, instead of by an electromagnet. Medium is supplied and discharged through bores (not visible in the figures) in the base body 14. The bores lead into the lower chamber 17b.

4 shows an alternative solution to the in 3 shown mixing principle. The medium is mixed and the cells are stimulated by a propeller 23 which is seated on a shaft 24 and is driven by a driver pin 25 . The drive is provided by an electric motor (not shown), which is installed in the supply part 1 instead of the electromagnet 12 .

5 shows a further variant of a device according to the invention. In contrast to the statements according to 1 until 4 Here, a large number of cell culture containers 3 - 24 pieces in the exemplary embodiment - are hung in a receiving plate 26 and supplied by a common medium pond 27 . The mixing of the medium and the stimulation of the cells for cell proliferation and cell differentiation is carried out by a permanently or periodically rotating mixing disk 28. The mixing effect can be intensified by recesses 28a in the mixing disk 28. To ensure that all cell culture containers have the same conditions when the mixing disk is stationary, it is recommended that the mixing disk should have a recess pattern that corresponds to the cell culture container arrangement. However, other disc shapes can also be used, in which case the same conditions can also be created for all cell culture containers by frequently moving the mixing disc briefly or permanently slowly.

In this exemplary embodiment, the mixing disc 28 is driven by an electric motor 29. In addition, the receiving plate 26 can be adjusted in height via columns 30, which, for example, allows the cell culture container 3 to be lowered briefly after the medium has been filled and thus ensures reliable wetting while at the same time using medium sparingly.

Reference List

1

supply part

2

sample acquisition module

3

cell culture container

3a

membrane

4

lid

5

coupling

6

coupling

7

Hose

8th

Hose

9

connector

10

feed pump

11

drain pump

12

electromagnet

13

primary lifter

14

body

15

recording ring

16

glass tube

17

medium

17a

upper chamber

17b

lower chamber

18

ultrasonic sensor

19

sonic nozzle

20

perforated plate

21

secondary tappet

22

membrane

23

propeller

24

Wave

25

driving pin

26

recording plate

27

medium pond

28

mixing disc

28a

recesses

29

electric motor

30

pillar

Claims (20)

Method for the controlled supply of one or more cell cultures in cell culture containers (3) with a medium (17), for example the respiratory tract when cultivating at an air-liquid interface, characterized in that in particular to improve cell growth and differentiation of the cells in long-term cultures medium is circulated within the cell culture container (3). procedure after claim 1 , characterized in that a biomechanical stress is caused in the medium by means of a defined flow in the medium. procedure after claim 1 or 2 , characterized in that the cell culture container (3) is divided into an upper chamber (17 a) with the cell culture and a lower chamber (17 b) with the medium. procedure after claim 3 , characterized in that the lower chamber (17 b) is open to the cell culture through a perforated plate (20) and flows by forcing a change in volume of the lower chamber and the resulting free jet is generated through each hole (synthetic jet). procedure after claim 4 , characterized in that the change in volume is forced by a membrane (22) or a piston (21). procedure after claim 4 or 5 , characterized in that the energy required to force a change in volume is introduced into the system by an electromagnet or an electric motor. procedure after claim 1 or 2 , characterized in that the medium circulations or defined flows are effected by a moving part in the medium. procedure after claim 7 , characterized in that the medium circulation or defined currents are carried out by a part rotating in the medium. procedure after claim 8 , characterized in that the part rotating in the medium is a propeller (23). procedure after claim 7 , characterized in that the body located in the medium is disc-shaped and medium circulations or defined currents are generated by rotation of the disc-shaped body. procedure after claim 10 , characterized in that the generation of medium circulation or defined currents is reinforced by recesses in the disc-shaped body. Method according to one of the preceding claims, characterized in that several cell culture containers (3) are supplied by a common medium pond (27). Method according to at least one of the preceding claims, characterized in that the medium supplied is preheated in a heat exchanger. procedure after Claim 13 , characterized in that the heat exchanger is a spiral tube heat exchanger. procedure after Claim 13 , characterized in that supplied medium is preheated in a continuous process. Device for carrying out the method according to at least one of Claims 1 - 15 , characterized in that the cell culture container (3) in an upper chamber (17 a) in which the cell culture is located, and a lower chamber (17 b), in which the medium is divided. device after Claim 16 , characterized in that a circulation or flow can be generated between the lower chamber (17 b) and the upper chamber (17 a). device after Claim 16 or 17 , characterized in that a membrane or wall permeable to the medium is arranged between the lower chamber (17 b) and the upper chamber (17 a). device after Claim 18 , characterized in that a perforated plate is provided between the lower chamber (17 b) and the upper chamber (17 a). Device according to at least one of Claims 16 - 19 , characterized in that in the lower chamber (17 b) at least one movable body (22, 23, 28) is provided.

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