WO2010130307A1

WO2010130307A1 - Method for detaching adherent cells

Info

Publication number: WO2010130307A1
Application number: PCT/EP2010/001082
Authority: WO
Inventors: Christian Derichs; Ulrike Koropp; Heike Walles; Michaela Kaufmann; Jan Hansmann
Original assignee: Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
Priority date: 2009-05-15
Filing date: 2010-02-20
Publication date: 2010-11-18
Also published as: DE102009022352A1; EP2430147A1; WO2010130307A8

Abstract

The invention relates to methods for the cultivation of adherent cells, in particular for detaching the cells from a surface, to the use of membranes in these methods and to devices for carrying out the methods. A particular area of application is the use of the methods according to the invention in the GMP-compliant, fully automatic cultivation and propagation of cells.

Description

Method for detaching adherent cells

Description

In the technical field of tissue engineering, particularly in relation to regenerative medicine, there is the need to automate in a GMP-compliant manner biological laboratory processes under clean room conditions. A higher yield, higher process safety and also standardisable process optimisation and process control are to be achieved in this way.

Certain standards in laboratory-scale production have become established over the years. Disposable articles made of injection-moulded polypropylene or polystyrene are thus frequently used, as the very cost-intensive cleaning and disinfection of the sample containers is in this way dispensed with. The cells required for building up the tissue constructs are firstly isolated from a biopsate and then cultivated in different sized cell culture dishes, flasks or multiwell plates over a period of several days. For the purposes of cultivation, the isolated primary cells are firstly resus- pended in specific cell culture media. The cell suspension is subsequently pipetted into disposable culture vessels in which the cells adhere in an undefined manner to the specially pretreated plastic surfaces. After an incubation time of several days in an incubator and regularly exchanging the spent cell culture medium with fresh cell culture medium, the cell culture, which has grown with sufficient density, i.e. which is confluent, is detached from the plastics material surface of the cell culture vessels. The detaching process is carried out either purely enzymatically, for example using a trypsin/EDTA-containing solution, or in conjunction with mechanical excitation. In this case, both the enzyme activity and the mechanical stress have negative effects on the vitality of the cells.

This necessary detaching process must be carried out as rapidly as possible in order to avoid a long action time and thus damage to the cells. The action of the enzyme on the upper side of the cell culture is particularly disadvantageous, leading, as it does, to damage to the cells and to a reduced cell vitality.

The method of cultivating cells on PET membranes is used as standard in the laboratory. For this purpose, use is made of cell culture inserts, such as they are known for example from WO 2004/020571 A2. Nevertheless, these are available only in a limited size. The use of insert membranes has in the manual laboratory process the drawback that the growth of the cell culture during the culturing period cannot be monitored under a microscope.

In all the conventional enzymatic detaching methods, an enzyme solution, often a trypsin/EDTA-containing solution, is applied to the adherent cells of the cell culture so that the enzyme can separate the cells from the sur- face to which they are adhering. After the enzyme has acted for a certain time, the enzyme reaction is stopped. An overview of detaching methods of this type is provided, for example, by Hans Jϋrgen Boxberger: ,,l_eitfa- den fur die ZeII- und Gewebekultur: Einfϋhrung in Grundlagen und Tech- niken" Wiley-VCH, Weinheim (2006), pages 132 to 135.

In the conventional detaching processes, the enzyme solution flushes round the cells from all sides. However, in this procedure, the enzyme acts on the membrane proteins of the entire cell surface and thus easily leads to damage to the cells. The complete wetting with the enzyme solution also damages cell surface receptors at the upper side of the cells, which are not involved in the cell adhesion to the surface of the cell culture vessel.

The invention is based on the technical problem of providing methods and devices allowing adherent cells to be detached in a manner which is im- proved and/or simplified over the prior art.

The invention is also based on the technical problem of providing methods and devices allowing cells to be detached from the cultivation surface in a manner that spares the adherent cells.

The invention is also based on the technical problem of providing methods and devices allowing a cultivation and expansion of cells that spares the cells.

The invention is also based on the technical problem of providing methods and devices allowing an automated cultivation and expansion of cells. Ths invention is also based on the technical problem of providing methods and devices which reduce, in particular avoid, damage to cells as a result of a passaging of the cells.

The technical problem underlying the invention is solved by the subject matters of the independent patent claims.

In particular, the technical problem underlying the invention is solved by a method according to claim 1.

In particular, the technical problem underlying the invention is solved by a method for the cultivation of adherent cells, containing the steps a) culti- vating the cells on a first surface of a porous material and b) enzymatically detaching the cells from the first surface of the porous material, wherein the enzymatic detaching is carried out by the contacting of a second surface of the porous material with an enzyme-containing liquid, and wherein during step b) only the contact points of the cells that touch the porous material enter into contact with the enzyme-containing liquid.

According to the invention, preferably only the cell branches of the cells, as contact points, enter into contact with the enzyme-containing liquid.

The invention therefore provides in step b) not to apply the enzyme- containing liquid directly to the cells, but to bring the porous material into contact with the enzyme-containing liquid, so that only the contact points of the cells that touch the porous material enter into contact with the enzyme. Thus, in a preferred embodiment, only small partial regions of the cell surface enter into contact with the enzyme, namely the contact points, in particular the cell branches of the cells, and not, as is conventional in the prior art, the entire cell surface. According to the invention, this is achieved in that the enzyme-containing liquid is not applied to the first sur- face of the porous material that directly contact the cells, i.e. on which the cells have settled; instead, the liquid is passed via a second surface of the porous material, through the porous material, to the region of contact between the porous material and the cells.

According to the invention, the detachment of the cells adhered to a porous material, in particular a membrane, is therefore preferably controlled in step b) via an enzyme reaction, wherein the enzyme can act on the cell surface merely through the porous material at the contact of the cells with the porous material.

In relation to the present invention, the term "adherent cells" refers to cells of the type which can be cultured in a cell culture medium on an inert surface, for example a reaction vessel, or on a membrane, as a monolayer or as a multilayer, but in particular as a monolayer. The adherent cells contact the surface and adhere thereto.

The adherent cells therefore have at their surface points of contact with the inert surface which is formed in the present case by the porous material, preferably a membrane. In this case, the adherent cells do not grow with their entire area on the porous material, but form the bond to the inert surface via what are known as cell branches which form this contact to the porous material.

Adherent cells usually form a continuous cell layer. Adherent cells often display a density-dependent proliferation inhibition, also referred to as contact inhibition, which can occur when the confluence is exceeded. Adherent cells often derive from tissues, such as for example skin, muscles, nerves, the liver, kidneys. Examples of adherent cells are fibroblasts, HeLa cells and many tumour cells. The media which are known to the person skilled in the art and are selected depending on the type of cell to be grown are suitable as the cell culture medium for cultivation of the adherent cells.

According to the invention, the adherent cells are preferably eukaryotic cells, in particular mammalian cells. According to the invention, the cells are preferably animal cells. According to the invention, the cells are preferably mammalian cells, in particular human, bovine or murine cells.

Alternatively, however, it is also possible to cultivate prokaryotic cells, plant cells or fungal cells, provided that they are adherent cells.

The cells on the first surface of the porous material can be cultivated in the conventional manner. The person skilled in the art is familiar with a broad range of cultivation methods for adherent cells, from which he can select suitable methods.

According to the invention, the cells are preferably cultivated on an upwardly directed surface of the porous material. According to the invention, the first surface of the porous material is therefore preferably directed upward.

The cultivating is preferably carried out in a cell culture medium. The duration of the cultivation, cultivation temperature, cultivation pressure, pH and oxygen content of the medium and other parameters can be selected by a person skilled in the art from the ranges with which he is familiar.

According to the invention, the cells are preferably applied to the porous material, left to adhere and then further cultivated.

According to the invention, the cells are preferably applied to the porous surface at a cell concentration during seeding of from approx. 4,000 to approx. 20.000 cells/cm², in particular of from approx. 4,000 to approx. 16,000 cells/cm².

According to the invention, the porous material provided as the cell growth area is in this case preferably not wetted in its entirety with cell liquid dur- ing populating; instead, individual cell clusters, distributed uniformly on the cell growth area, are produced.

According to the invention, the cells suspended in a cell culture medium are at the beginning of step a) preferably pipetted dropwise onto the porous material at roughly constant, in particular at constant spacing, using a pipette.

During this process, the following parameters are variable for a person skilled in the art: drop volume, drop spacing, cell concentration in the drop and/or waiting time until the first exchange of media.

These parameters also allow the cultivation behaviour of the respective cells to be altered.

The cell concentration within a drop leads in this case to maintenance of the cell/cell contacts necessary for cell growth while at the same time increasing the size of the available cell growth area to a multiple of the cell growth area of a standard laboratory culture vessel. In addition, this pre- ferred populating method ensures a uniform distribution of the cells over the porous material as the culturing area, whereas in standard culture vessels the cells are distributed randomly and the distribution is often higher in the edge regions than at the centre. A uniform cell distribution has the advantage that the growth conditions on the populated porous material are unitary. As a result of the now sufficiently large cell growth area, in particular additional passaging steps of the cells, in particular in the automated process, can be avoided, thus eliminating the need for a large number of handling steps which otherwise impede the automated process and damage the cells.

According to the invention, the drop volume is preferably between at least 2.5 μl and at most 30 μl.

According to the invention, preferably between 10 and 500, in particular between 25 and 350 cells are present in a drop during seeding.

A uniform dropwise populating of the cell growth area allows the size of the cell growth area to be increased to a multiple without infringing the conditions for successful cell expansion. The size of cell growth area that is necessary for passaging-free cell expansion is in this way achieved.

The additional damaging of the cells during passaging as a result of the action of enzyme reactions or mechanical loads can also be reduced to a minimum, in particular to a single detachment according to the invention of the cells at the end of the cultivation period.

Step b), at the moment of the cell harvest after cultivation has been concluded, thus preferably remains as the single necessary detaching process of the cells. Furthermore, the harmful effect of the enzyme on the cells can be minimised by the new detaching method.

Alternatively, the porous material can also be populated in a manner other than the pipetting of droplets, for example by spraying with cells suspended in liquid or pouring-over with cells in cell culture medium. In 3 preferred embodiment of the invention, adherent cells are left in step a) to adhere to the membrane and then further cultivated while adding liquid, in particular cell culture medium.

In a preferred embodiment of the invention, the cells are cultivated in step a) until they are almost confluent or until they are confluent.

Provision may also be made to exchange in step a) the cell culture medium as required, i.e. to replace spent cell culture medium with fresh cell culture medium.

Provision may, for example, also be made to cultivate the cells in an incu- bator in step a).

The term "confluence" is used to describe the closest possible arrangement of adherent cells at the surface of a culture vessel, i.e., in the present case, of the porous material. The confluence differs from cell line to cell line.

Shortly before or when confluence is reached as a result of the growing and/or the propagation of the cells, the cells are preferably harvested or passaged.

Provision may be made to metrologically test the state of the cells for a broad range of parameters via a process control of step a), for example via electrodes for measuring the TEER value and/or the automated supply of media. In particular, provision may be made to determine the duration of step a) by measuring the TEER value. In this case, step a) can be ended in a preferred embodiment when the desired cell density is reached, in particular when the cells are almost confluent or are confluent. By measuring the TEER value (TEER = transepithelial electrical resistance), the density of the cell population can be determined. Thus, it is possible to determine the point in time at which the cells on the membrane have reached the desired cell density, in particular the point in time at which the cell layer has become almost confluent or confluent.

Provision may also be made to measure, in particular in step a), the opacity of the cell culture medium. Provision may also be made to measure, in particular in step a), the oxygen content of the cell culture medium. Provision may also be made to measure, in particular in step a), the pH of the cell culture medium. Provision may also be made to measure, in particular in step a), the glucose content of the cell culture medium.

In step b) the cells are enzymatically detached after reaching the desired cell density. This is carried out using an enzyme-containing liquid.

In relation to this invention, the term "an enzyme-containing liquid" refers to a liquid containing an enzyme, namely at a concentration at which the enzyme is active. According to the invention, the enzyme is suitable for detaching the adherent cells from a surface, in particular from the porous material.

According to the invention, the enzyme is preferably trypsin. However, use may also be made of other enzymes known to the person skilled in the art, such as for example Accutase or rProtease.

According to the invention, the liquid is preferably a buffer. The person skilled in the art is familiar with suitable buffers which can be used for the enzyme. Cell culture media can also be used as the liquid. The enzymatic detaching in step b) can therefore be carried out using enzyme-containing liquids and solutions such as are also used in the prior art for detaching adherent cells. In particular, the person skilled in the art will use trypsin, in particular in an EDTA solution, as the enzyme. The per- son skilled in the art is in this case familiar with suitable trypsin concentrations.

After the cells have been detached, step b) can be stopped in various ways. In particular, the enzyme-containing liquid can be removed again. Alternatively, the detaching process is stopped by adding serum. The po- rous material can also be removed with the cells which rest thereon, but do not adhere thereto.

According to the invention, provision may be made for, in step b), the top of the enzyme-containing liquid to be positioned precisely at the level of the upper side of the porous material.

According to the invention, provision may be made for, in step b), the enzyme-containing liquid to be added under the porous material.

A person skilled in the art is aware for how long he must allow the enzyme to act, i.e. for how long step b) is to be carried out. In particular, the person skilled in the art is also familiar with methods for determining the de- gree of detachment of the cells.

In an alternative embodiment according to the invention, in step b), the cells are detached enzymatically, the detaching being assisted by physical methods. This allows the detaching process to be accelarated.

According to the invention, in step b), the detaching is preferably assisted by mechanical or physical methods, in particular by ultrasound and/or by rising air bubbles below the porous material. The detaching can also be assisted by knocking and/or shaking.

According to the invention, the porous material is liquid-permeable. According to the invention, the porous material has a surface, in particular an inert surface, to which adherent cells, in particular with cell branches, can adhere.

The porous material serves as the growth area of the cells. The porous material preferably has two surfaces, in particular if it is sheet-shaped or disc-shaped, for example if it is a membrane, namely an upper side and an underside. The porous material can however also have more than two surfaces, for example if it is thicker than a membrane, for example if it is sponge-shaped. According to the invention, preferably only one surface of the porous material serves as the growth area of the cells.

If the porous material is used, in accordance with the invention, preferably horizontally, then according to the invention preferably the upper surface, i.e. the upper side, of the porous material serves as the growth area of the cells.

According to the invention, the porous material is preferably a sponge-like material or a membrane.

According to the invention, the porous material is preferably a membrane.

According to the invention, the membrane is preferably part of a membrane unit. According to the invention, the membrane unit preferably consists of a membrane and a frame. Alternatively, the membrane unit can consist only of a membrane. According to the invention, the membrane unit is preferably liquid- permeable through the membrane. According to the invention, the membrane therefore preferably forms the liquid-permeable part, in particular the sole liquid-permeable part of the membrane unit.

The membrane, which is preferred according to the invention, serves as the growth area of the cells. According to the invention, preferably only one of the two surfaces of the membrane serves as the growth area of the cells.

According to the invention, the membrane is preferably used horizontally during the method according to the invention.

If the membrane is preferably used horizontally in accordance with the invention, then, in accordance with the invention, preferably the upper surface of the membrane serves as the growth area of the cells. As the adherent cells adhere to the membrane, provision may also be made for the lower surface of the membrane to serve as the growth area of the cells. The membrane can also be reversed during step a) and/or at the beginning of step b) so that, for example, the cells initially grow on the upper surface, the membrane is reversed and the cells thus continue to grow on the underside. Then, in step b), the enzyme-containing liquid can for ex- ample be applied to the upper surface of the membrane, or the membrane is reversed again, so that the cells rest in step b) against the upper surface of the membrane.

Alternatively, however, in particular in adherent cells, both surfaces of the membrane can also be used as the growth area of the cells. However, in that case, the method according to the invention is applied only for the adherent cells on one of the two surfaces. The other cells can for example serve as feeder cells. !n an alternative embodiment according to the invention the membrane can also be replaced by another suitable porous material, for example by a sponge-like material.

The person skilled in the art is familiar with suitable liquid-permeable membranes which are suitable for cultivating cells, in particular adherent cells.

The cell growth membrane can for example be made of PET, i.e. polyethylene terephthalate, or a comparable material. The use of polycarbonate membranes is, for example, also possible. The membrane has by defini- tion a porous structure, so that liquids can be exchanged via the membrane.

According to the invention, the membrane is preferably selected from the group consisting of PET membranes, PC membranes, nylon membranes, amphoteric nylon membranes, positively charged nylon membranes, negatively charged nylon membranes, PTFE membranes, cellulose ester membranes, cellulose acetate membranes, cellulose nitrate membranes, cellulose mixed ester membranes, regenerated cellulose membranes, Ny- tran membranes and Nytran SuPerCharge++ membranes.

In an alternative embodiment according to the invention the membrane is a PET membrane. In an alternative embodiment according to the invention the membrane is a PC membrane. In an alternative embodiment according to the invention the membrane is a nylon membrane. In an alternative embodiment according to the invention the membrane is an amphoteric nylon membrane. In an alternative embodiment according to the in- vention the membrane is a positively charged nylon membrane. In an alternative embodiment according to the invention the membrane is a negatively charged nylon membrane. In an alternative embodiment according to the invention the membrane is a PTFE membrane. In an alternative embodiment according to the invention the membrane is a cellulose ester membrane. In an alternative embodiment according to the invention the membrane is a cellulose acetate membrane. In an alternative embodiment according to the invention the membrane is a cellulose nitrate membrane. In an alternative embodiment according to the invention the membrane is a cellulose mixed ester membrane. In an alternative embodiment according to the invention the membrane is a regenerated cellulose membrane. In an alternative embodiment according to the invention the membrane is a Nytran+ membrane. In an alternative embodiment according to the invention the membrane is a Nytran SuPerCharge++ membrane.

A coating of the cell growth membrane or an application of a surface structure is also selectively possible.

According to the invention, the membrane preferably has a pore size of at least 0.01 μm. According to the invention, the membrane preferably has a pore size of at least 0.1 μm. According to the invention, the membrane preferably has a pore size of at least 0.35 μm.

According to the invention, the membrane preferably has a pore size of at most 20 μm. According to the invention, the membrane preferably has a pore size of at most 10 μm.

According to the invention, the membrane preferably has a pore size of at least 0.01 μm, in particular 0.1 μm and at most 20 μm, in particular at most 10 μm.

According to the invention, the membrane preferably has a pore size of at least 0.35 μm, in particular 0.4 μm and at most 9 μm, in particular at most 8 μm. A pore size of 0.4 μm, 0.45 μm, 1.0 μm, 1.2 μm, 3.0 μm, 5.0 μm or 8.0 μm can for example be provided.

The person skilled in the art is familiar with suitable pore sizes of membranes ensuring both a sufficient passage of liquid, in particular a passage of cell culture medium, and at the same time a good cultivation of the cells.

According to the invention, the membrane of the membrane unit preferably has a pore density of at least 10⁵ pores per cm². According to the invention, the membrane of the membrane unit preferably has a pore den- sity of at most 10⁸ pores per cm².

According to the invention, the membrane of the membrane unit preferably has a pore density of at least 10⁵ pores per cm² and at most 10⁸ pores per cm². A pore density of 0.1 x 10⁶, 0,2 x 10⁶, 0.4 x 10⁶, 2 x 10⁶, 22 x 10⁶ or 100 x 10⁶ can for example be provided.

The person skilled in the art is familiar with suitable pore densities of membranes ensuring both a sufficient passage of liquid, in particular a passage of cell culture medium, and at the same time a good cultivation of the cells.

According to the invention, the porous material is preferably located in a vessel.

According to the invention, provision is therefore preferably made for the porous material to be at least partly surrounded by a vessel, in particular by a culture vessel, so that the vessel can receive a liquid. Preferably, the vessel can receive the liquid, the liquid surrounding the porous material, i.e. being located below and above the porous material. A broad range of vessels can be used. Cell culture vessels, for example cell culture flasks, cell culture dishes or multiwell plates, are particularly suitable.

According to the invention, the porous material is preferably located in a cell culture dish. According to the invention, the porous material is preferably located in a well of a multiwell plate, for example a 6-well plate or a 24-well plate.

The vessel can also be a bioreactor.

According to the invention, the porous material preferably does not cover the bottom of the vessel. According to the invention, the porous material is preferably located in the interior of the vessel, so that there is a free space below and above the porous material.

In an alternative embodiment according to the invention provision may be made for the porous material to be part of a cell culture insert. The cell culture insert can be a conventional cell culture insert from the prior art, such as is offered by various companies.

According to the invention, provision is preferably made for, in step a), the porous material and the adherent cells to be submersed in a cell culture medium and, in step b), the cell culture medium to be removed and the enzyme-containing liquid to be added.

In an preferred embodiment according to the invention the porous material is the bottom of a cell culture insert which is inserted in a cell culture vessel, there being a spacing between the bottom of the cell culture vessel and the porous material. In step a) of the method according to the pre- ferred embodiment the cells are cultivated in a cell culture medium, the vessel being filled with so much cell culture medium that the cell culture medium covers the cells. In step b) the cell culture medium is then removed and the enzyme-containing liquid is introduced into the vessel, only so much enzyme-containing liquid being added that the top of the en- zyme-containing liquid reaches only the points of contact of the cells to the porous material. Depending on the selection of the porous material, it may be sufficient for the enzyme-containing liquid to reach only up to the underside of the porous material, as the material brings the liquid by capillary forces also up to the upper side of the material. However, it is also possi- ble for so much enzyme-containing liquid to be added that the liquid reaches just up to the upper side of the porous material.

The cell culture medium can be supplied in step a) in a broad range of ways.

The enzyme-containing liquid can also be supplied in step b) in a broad range of ways.

For example, the cell culture vessel can have an, in particular closable, opening below the porous material, through which the enzyme-containing liquid can be poured in and drained out. The method according to the invention could therefore use a vessel having an inlet or an outlet below the porous material.

Of course, for removing the cell culture medium and for adding the enzyme-containing liquid, the porous material, for example with the cell culture insert, can also be removed from the vessel, so that the medium and/or the enzyme-containing liquid can be added by pipette, removed by pipette, poured in or poured out. Use may also be made of a specific cell culture insert which comprises the porous material and has a recess through which the bottom of the vessel is accessible, so that the medium can be drawn off using a pipette and the enzyme-containing liquid can be added, alongside the porous material, using a pipette. In this case, the enzyme-containing liquid is therefore pipetted from above, alongside the porous material, under the porous material.

In an automated method the precise amount of cell culture medium and in particular of enzyme-containing liquid can be supplied in a computer- controlled manner in the precisely required amount. In this case, either the amount can be calculated or determined in advance, based on the dimensions of the cell culture vessel and the cell culture insert, or measuring means are provided for measuring the filling level.

In an alternative embodiment according to the invention the method is car- ried out in a bioreactor.

According to the invention, a bioreactor for the cultivation of cells is preferred, the reactor space of the bioreactor being divided by a membrane unit which is liquid-permeable, at least in partial regions, into two reactor regions and each of these two reactor regions having an opening suitable for letting in and/or letting out a liquid.

The method according to the invention can therefore be carried out in a bioreactor with an integrated cell growth membrane. The bioreactor surrounds, as the housing, a reactor space, also referred to as the reactor chamber. This reactor space is split up by the membrane unit, which is liquid-permeable in partial regions, into two reactor regions. The bioreactor therefore preferably has two reactor regions or reactor space regions. According to the invention, the bioreactor preferably contains a reactor lower part, a reactor cover and a membrane unit. According to the invention, the bioreactor preferably consists of a reactor lower part, a reactor cover and a membrane unit. According to the invention, the reactor lower part is preferably upwardly opened. The membrane unit then covers the opening in the reactor lower part in that the membrane of the membrane unit is positioned horizontally on the opening. The reactor cover then has a downwardly directed opening. The reactor cover is then placed onto the reactor lower part, so that the opening in the reactor cover is also covered by the horizontal membrane. The membrane of the membrane unit thus demarcates the reactor region or reactor space formed by the reactor lower part from the reactor region or reactor space formed by the reactor cover.

According to the invention, the reactor space is therefore preferably split up by the membrane unit, which is liquid-permeable in partial regions, into a lower reactor region and into an upper reactor region.

The openings, which are suitable for letting in and/or letting out a liquid, can be used in particular for letting in and/or letting out the cell culture medium and/or the enzyme-containing liquid.

According to the invention, in step b), the enzyme-containing liquid is preferably introduced through the opening, which is suitable for letting in and/or letting out a liquid, in the reactor lower part.

In an alternative embodiment according to the invention the method according to the invention is carried out in an automated manner, in particu- lar in an automated device for producing tissue from cell cultures and/or using a robot. According to the invention, the bioreactor preferably has at least one ventilation opening.

According to the invention, the at least one ventilation opening is preferably provided with a sterile filter.

According to the invention, the bioreactor preferably has at least one pipetting opening.

According to the invention, the first reactor housing part preferably has at least one pipetting opening. According to the invention, the first reactor housing part preferably has a pipetting opening.

According to the invention, metrological devices, which allow the measurement of data, for example for measuring TEER values, are preferably integrated into the bioreactor.

The bioreactor can selectively be used as a stand-alone module or as a component in an overall system, for example for producing cell tissue from biopsates.

The present invention also relates to the bioreactor described in the present document.

Provision may be made for, after completion of step b), the cells to be removed from the membrane in a step c). For example, the cells can be taken up in cell culture medium, in particular in serum-containing cell culture medium, and then suction-extracted or removed by pipette.

Provision may also be made, after completion of step b), to introduce in step c) more enzyme-containing liquid and to thereby push the cells away from the porous surface. !t is therefore possible to provide a removal of the cells from the membrane as a result of the use of an excess pressure, in particular via a liquid, for example the enzyme-containing liquid.

Provision may also be made for the cells to be singled out in step c), in particular with the aid of the enzyme-containing liquid.

Examples of an area of application of the cell detaching method according to the invention include in particular automated systems or other laboratory processes in which adherent cells have to be detached from their prior surface for further use. Possible fields are for example the auto- mated cell expansion of: - various cell types for producing artificial skin as an in-vitro test system or transplant; - cells for producing artificial cartilage transplants; - generally cells used in the cell culture or for tissue engineering.

According to the invention, the application of the method according to the invention in the GMP-compliant, fully automatic cultivation and propagation of cells is preferred.

The present invention also relates to the use of a porous material, in particular a membrane, in a method according to the invention.

The present invention also relates to the use of a cell culture container, in particular a cell culture dish, a cell culture flask or a multiwell plate, in a method according to the invention.

The present invention also relates to the use of a cell culture insert, in particular a cell culture insert with a bottom which is formed from a porous material, in particular a membrane, in a method according to the invention. The present invention also relates to the use of a bioreactor in a method according to the invention.

Embodiments, which are preferred and alternative in accordance with the invention, of the method according to the invention are also to be under- stood as being embodiments, which are preferred and alternative in accordance with the invention, of the uses according to the invention and as being embodiments, which are preferred and alternative in accordance with the invention, of the devices according to the invention.

Particular embodiments of the invention are disclosed also in the depend- ent claims.

Further advantages of the invention will emerge from the figure described hereinafter.

Figure 1 shows, in an illustration which is not true to scale, an embodiment, which is preferred in accordance with the invention, of a bioreactor (100) for carrying out the method according to the invention. The bioreactor consists of a reactor lower part (20), a reactor upper part (30) and a membrane unit formed from a membrane (10) and a frame (15). The reactor lower part (20) and reactor upper part (30) have support elements (21 , 31) which fix the membrane (10). The reactor lower part (20) and reactor upper part (30) are connected in an air-tight manner, so that only the openings (24, 34), which are in the form of connections, connect the reactor chambers (22, 32) to the environment of the bioreactor (100) via the guidance systems (23, 33). The guidance systems (23, 33) ensure a continuous and uniform supply of the cell growth membrane over the entire area. The reactor lower part (20) and reactor upper part (30) both have electrodes (26, 36) for measuring the TEER value of cultivated cells. The growth process can be monitored and the optimum harvest moment determined via the TEER values which are measured.

The membrane (10) has an upper side (11) and an underside (12). Preferably, adherent cells are cultivated on the upper side (11) of the mem- brane. In this case, the reactor chambers (22, 32) are flooded with cell culture medium via one of the openings (24, 34). When the TEER value measurement via the electrodes (26, 36) reveals that an optimum cell density has been reached, for example that the cells have grown confluent, the cell culture medium can be drained out via the lower opening (24). A trypsin-containing buffer can then be introduced into the bioreactor (100) through the opening (24). So much of the buffer is introduced that the buffer just obtains contact to the membrane (10). As a result, the cells are detached from the membrane surface (11); however, only the membrane contact points, i.e. the cell branches, of the cells enter into contact with the trypsin, so that the cells are damaged less than during a conventional detaching of the cells. Afterwards, the buffer can be drained out again through the opening (24). The cells can be removed by pipette or be flushed out through the opening (34) with the aid of cell culture medium.

For example, provision may be made to remove the cell culture medium via the guide system and to fill both chambers with PBS/EDTA and to incubate them for 1 to 30 min. This process can be repeated 1 to 2 times, depending on the cell type. Both chambers can then be completely emptied and the lower chamber can subsequently be filled with an enzyme solution. For a defined range of from 0.2 to 10 min and by application of a defined pressure in the range of from 50 to 2,000 Pa, the enzyme solution can be passed through the pores, having a diameter in the range of from 0.1 to 10 μm, of the membrane, even to the points of contact of the cells to the upper side of the membrane. Subsequently, an incubation step hav- ing a duration in the range of from 0.5 to 10 min can be carried out. In order to assist the detaching process, a pressure in the same pressure range can subsequently, i.e. after the detaching, be applied in the lower chamber. Said pressure can additionally be modulated with a frequency of from 0.1 to 200 Hz. In addition, the process can be intensified by way of mechanical excitation of the entire reactor. The pressures which are applied produce volume flows through the membrane in the range of from 0.5 to 20 ml/(min cm²).

Claims

1. Method for the cultivation of adherent cells, comprising the steps

a) cultivating the cells on a first surface of a porous material;

b) enzymatically detaching the cells from the first surface of the porous material,

wherein the enzymatic detaching is carried out by the contacting of a second surface of the porous material with an enzyme-containing liquid, and wherein, in step b), only the contact points of the cells that touch the porous material enter into contact with the enzyme- containing liquid.

2. The method according to one of the preceding claims, wherein the porous material is a membrane.

3. The method according to one of the preceding claims, wherein the cells are cultivated in step a) on the upper side of the porous material.

4. The method according to one of the preceding claims, wherein the top of the enzyme-containing liquid enters in step b) into contact with the porous material.

5. The method according to one of the preceding claims, wherein, in step b), the top of the enzyme-containing liquid is positioned precisely at the level of the upper side of the porous material.

6. The method according to one of the preceding claims, wherein the enzyme-containing liquid is a trypsin/EDTA solution.

7. The method according to one of the preceding claims, wherein the porous material is located in a vessel.

8. The method according to one of the preceding claims, wherein, in step a), the porous material and the adherent cells are submersed in a cell culture medium and wherein, in step b), the cell culture medium is removed and the enzyme-containing liquid is added.

9. The method according to one of the preceding claims, wherein, in step b), the enzyme-containing liquid is added under the porous material.

10. The method according to one of the preceding claims, wherein the method is carried out in a bioreactor.

11. The method according to one of the preceding claims, wherein the adherent cells are eukaryotic cells, in particular mammalian cells.

12. The method according to one of the preceding claims, wherein the method is carried out in an automated manner, in particular in an automated device for producing tissue from cell cultures and/or using a robot.

13. Use of a porous material, in particular a membrane, in a method ac- cording to one of the preceding claims.

14. Use of a cell culture container, in particular a cell culture dish, a cell culture flask or a multiwell plate, in a method according to one of claims 1 to 12.

15. Use of a ecu culture insert, in particular a cell culture insert with a bottom formed from a membrane, in a method according to one of claims 1 to 12.

16. Use of a bioreactor in a method according to one of claims 1 to 12.