WO2001035737A1

WO2001035737A1 - Device and method for preserving a cell preparation

Info

Publication number: WO2001035737A1
Application number: PCT/EP2000/011108
Authority: WO
Inventors: Horst-Dieter Lemke; Tarja Jonuleit; Margot Hefner; Helmut Völker; Christoph Meyer
Original assignee: Acordis Industrial Fibers Gmbh
Priority date: 1999-11-16
Filing date: 2000-11-10
Publication date: 2001-05-25

Abstract

The invention relates to a device for preserving a cell preparation. The inventive device comprises at least one first membrane module (1) containing at least one first semi-permeable membrane, whereby the membrane module has a first opening (2) and a second opening (3) for directing the cell preparation through the membrane module on one side of the membrane, and has an admission opening (4) and a discharge opening (5) for directing a liquid nutrient medium through the membrane module on the other side of the membrane. The inventive device also comprises a first container (6), which has at least one first container opening (7) and which is provided for containing the cell preparation, comprises a second container (8), which has at least one first container opening (9) and which is also provided for containing the cell preparation, and comprises means for drawing, in an oscillatory manner, the cell preparation out of the first container (6) provided for the cell preparation via the first membrane module (1) and delivering it to the second container (8) for the cell preparation, and vice versa. The first membrane module (1), with its first opening (2) for directing the cell preparation, is fluidically connected via a line to the first container opening (7) of the first container (6) and, with its second opening (3) for directing the cell preparation, is fluidically connected via a line to the first container opening (9) of the second container (8).

Description

Device and method for preserving a cell preparation

Description:

The present invention relates to an apparatus and a method for preserving a cell preparation and the use of the preserved cell preparation.

In the context of the present invention, a cell preparation is to be understood as an aqueous suspension of cells, such as e.g. Blood.

An important prerequisite for the preservation of blood and also for the production of blood preparations is the prevention of blood coagulation through anticoagulation. The two main anticoagulants are citrate and heparin. While the effect of citrate is based on the chelation of Ca ²⁺ ions, heparin intervenes as a cofactor in the inactivation of thrombin by antithrombin III. A disadvantage of citrate is that it is toxic when infused quickly into a patient and at high concentrations. In addition, the pH of blood drops from pH = 7.4 to pH = 7.0 to 7.1 when low citrate concentrations are added. Heparin does not show this disadvantage, so it allows the maintenance of a physiological pH. However, heparin has a limited inhibition of coagulation and leads to the formation of cell aggregates in the blood bank, so that the storage of blood anticoagulated with heparin is only possible for about 24 hours. This makes heparin an anticoagulant for blood with a longer storage period. To obtain erythrocyte and platelet concentrates, erythrocytes (red blood cells) and platelets (platelets) are separated from the other blood components. Erythrocyte concentrates are stored at 5 ± 3 ° C for a maximum permissible storage time of 21 days. Platelet concentrates are stored at room temperature for a maximum storage period of 5 days. After these times, the use of the preparations for the patient can no longer be guaranteed. In addition, during the storage times mentioned, the pH value drops continuously to a pH value of about 6.6 due to the metabolism of glucose to lactate that takes place in the cell preparation, citrate in combination with citric acid and dextran as the anticoagulant ( ACD) is used. This acidification combined with damage to erythrocytes has led to the search for modified preservation solutions for blood. With citrate in combination with phosphate and dextran (CPD blood), for example, the pH value drops to just 6.85. However, a physiological pH value in the range from 7.2 to 7.4 cannot be achieved with this either.

Granulocytes cannot be stored at all and lose their function within a few hours.

Another cell preparation are hematopoietic, ie blood-forming stem cells. These cells are characterized by the fact that they renew themselves, but can also differentiate into other blood cells. Hematopoietic stem cells are used today to accompany or support chemotherapy. However, the hematopoietic stem cells obtained using the haemapheresis procedure should either be transfused within 72 hours or processed and cryopreserved. Temporary storage for a maximum of 72 h should take place at 2 to 6 ° C with temperature control. The cryopreservation of hematopoietic stem cells at - 80 ° C requires the addition of 5 or 10% dimethyl sulfoxide (DMSO) as a cryopreservative (S. Rowley, Journal of Hematotherapy (1992), Volume 1, pages 233-250), whereby DMSO is by no means toxicologically is harmless (RÖMPP CHEMIE LEXIKON, 9th edition (1990), Volume 2, page 987). Haemapheresis is a procedure that places great demands on the qualification and number of medical personnel required and also on the size and equipment of the required rooms, which were laid down in the recommendations of the German Society for Transfusion Medicine and Immunohematology (Infusionther Transfusionsmed ( 1998), volume 25, pages 325-335). Therefore, clinical studies have been conducted using unprocessed CPD blood as a source of hematopoietic stem cells. However, it also emerged from these examinations that the storage time of the blood - as was already the case with the interim storage mentioned at the beginning - was only allowed to be a maximum of 72 h at 4 ° C in order to realize an advantage for the patient (GJ Ossenkoppele et al ., Bone Marrow Transplantation (1994), Volume 13, pages 37-41, GJ Ossenkoppele et al., Bone Marrow Transplantation (1996), Volume 18, pages 427-431).

So far, one has been forced to use an anticoagulant for an anticoagulation of blood in the form of citrate that goes beyond 24 hours and is not able to maintain a physiological pH. Furthermore, blood preparations have hitherto either not been preserved at all (granulocyte concentrates) or at room temperature only for a few days (thrombocyte concentrates) or with cooling for a maximum of 21 days (erythrocyte concentrates). Until now, hematopoietic stem cells have either had to be cryopreserved using the toxicologically harmless DMSO or transfused within a maximum of 72 hours during which the cells have to be kept cool in a controlled manner.

Since, for logistical reasons, a cell preparation, such as blood, has to be stored for a long time after it has been isolated before it can be transfused or otherwise used, there is a need to preserve the cell preparation without the disadvantages described above occurring. The present invention therefore has the task of preserving a cell preparation, the disadvantages described above being overcome or at least substantially reduced.

This object is achieved on the one hand by a device for preserving a cell preparation, comprising at least a first membrane module which contains at least a first semipermeable membrane, the membrane module having a first opening and a second opening for the passage of the cell preparation through the membrane module on one side of the first membrane and has an inflow opening and an outflow opening for the passage of a liquid nutrient medium through the membrane module on the other side of the first membrane, further comprising a first container for the cell preparation with at least one first container opening, a second container for the cell preparation with at least one first container opening and means for oscillating conveyance of the cell preparation from the first container for the cell preparation via the first membrane module into the second container for the cell preparation and vice versa, the first membrane module with its first opening for the passage of the cell preparation is fluidly connected to the first container opening of the first container via a line and its second opening for the passage of the cell preparation is fluidly connected to the first container opening of the second container via a line.

In the context of the present invention, oscillating conveyance means the continuous conveyance of the substantially complete content of the first container via the at least one first membrane module into the second container and vice versa. Essentially completely means that, with the exception of the small fractions of the cell preparation which adhere to the inner walls of the container, the entire volume of the cell preparation is conveyed out of the respective container.

In the context of the device according to the invention, any means that is able to transfer the cell preparation from the first container via the first membrane module into the device is fundamentally suitable as a means for oscillating conveyance of the cell preparation second container and vice versa, whereby it should be noted that the cell preparation are living cells, for which repeated mechanical deformations, which can occur as a result of the promotion, lead to damage.

In a preferred embodiment of the device according to the invention, the means for the oscillating conveyance of the cell preparation are realized by a pump which can be switched with respect to its conveying direction and which is arranged in one of the lines between the first membrane module and one of the containers for the cell preparation. In addition, in the device according to the invention, the means for oscillating delivery can be realized by two pumps, one pump e.g. is arranged in the line between the first membrane module and the first container for the cell preparation and the other pump in the line between the first membrane module and the second container for the cell preparation.

In a further preferred embodiment of the device according to the invention, the means for the oscillating conveyance of the cell preparation are realized by an alternately adjustable first and second configuration of the container for the cell preparation, the first container being arranged above and the second container below the first membrane module in the first configuration and in the second configuration the second container is arranged above and the first container below the first membrane module. In this embodiment, which promotes the cell preparation solely through the action of a hydrostatic gradient, mechanical deformations of the cells, e.g. are unavoidable when using a pump, significantly reduced, so that a device according to the invention results which enables particularly gentle conveyance of the cell preparation.

In the device according to the invention, the first container for the cell preparation is particularly preferably connected to a first transport device and the second container for the cell preparation is connected to a second transport device, and the first and second transport devices are moved vertically in the opposite direction. loaned, whereby the first and the second configuration are adjustable.

In a further particularly preferred embodiment of the device according to the invention, the first container for the cell preparation is connected to the first transport device via a first side unit and the second container for the cell preparation is connected to the second transport device via a second side unit.

In a further particularly preferred embodiment of the device according to the invention, a load cell is rigidly connected to each side unit, on which the respective container for the cell preparation is suspended. The connection between the load cell and the container can be established by a holder. The load cells serve as an automatic fill level indicator for the containers. They also serve to initiate an automatic change of configuration as soon as one container is empty and the other is full. Finally, by means of the registration of the change in weight of the containers over time, the load cells are used to check whether the flow rate of the cell preparation is constant and / or is in the desired range.

In a further particularly preferred embodiment of the device according to the invention, a mixing device is attached to each side unit, which acts on the container located on the respective side unit, as a result of which undesired sedimentation of the cell preparation in the containers is avoided.

Any device that can move the first and second container vertically up and down in the opposite direction, such as chains, toothed belts or a pneumatic or hydraulic cylinder, is basically suitable as the first and second transport device and in the device according to the invention. However, in the device according to the invention, worm threads which can be driven at a constant rotational speed in both directions of rotation are preferred as first and second transport devices, which can be connected to a motor, for example, via a toothed belt. In a preferred embodiment, the first and second containers have flexible walls. Conventional blood bags represent an advantageous embodiment of such containers.

For the application of the device according to the invention, it is advantageous if a first transfusion filter in the line between the first container opening of the first container and the first opening of the first membrane module for the passage of the cell preparation and in the line between the second opening of the first membrane module for the Passing the cell preparation and the first container opening of the second container, a second transfusion filter is installed.

It is also advantageous if a first device for sampling is in the line between the first container opening of the first container and the first opening of the first membrane module for the passage of the cell preparation, and in the line between the second opening of the first membrane module for the passage of the Cell preparation and the first container opening of the second container, a second device for sampling is installed.

In addition, it is advantageous if in the line between the first container opening of the first container and the first opening of the first membrane module for the passage of the cell preparation, a first device for closing the line and in the line between the second opening of the first membrane module for the Passing the cell preparation and the first container opening of the second container, a second device for closing the line is installed.

In a further preferred embodiment of the device according to the invention, the first container for the cell preparation is fluidly connected via a second opening to a line with a means for removing the cell preparation, for example with at least one needle suitable for taking blood, a third inlet in the line direction for the closure of the line is installed. Furthermore, in this embodiment, the second container for the cell preparation is fluidly connected via a second opening to a line with a needle for retransfusion of the cell preparation, a fourth device for closing the line being installed in the line.

It is also advantageous if a third transfusion filter is installed in the line between the second opening of the first container and the means for removing the cell preparation and a fourth transfusion filter is installed in the line between the second opening of the second container and the needle for retransfusion.

In addition, it is advantageous if a third device for sampling in the line between the second opening of the first container and the means for removing the cell preparation and a fourth device for taking samples in the line between the second opening of the second container and the needle for retransfusion is installed.

In the preferred embodiments of the device according to the invention described above, the transfusion filters serve to retain cell aggregates, the presence of which in the two containers is undesirable, or which could lead to a hindrance in the conveyance of the cell preparation in the membrane module. The devices for taking samples open up the possibility of analyzing the condition of the cell preparation at various points in the device according to the invention. The device according to the invention closes the cell preparation sterile from the outside world. The closure devices allow the removal of individual parts of the device according to the invention without the other parts becoming non-sterile. Together with the means for removing or transfusing the cell preparation, the device according to the invention enables sterile transfer of the cell preparation from a storage container or from a donor into the device for the purpose of preservation and, after preservation has ended, the transfer of the cell preparation into a storage container or into a recipient , The membrane contained in the first membrane module of the device according to the invention can in principle be any membrane which is impermeable to the cells of the cell preparation but is permeable to the liquid nutrient medium, with preferably flat or hollow fiber membranes being installed. The membranes are particularly preferably nano, ultra or microfiltration membranes.

In a further preferred embodiment of the device according to the invention, the first membrane module with its inflow opening for the passage of a liquid nutrient medium is fluidly connected to a nutrient medium container for the liquid nutrient medium via a nutrient medium line, so that the cells of the cell preparation are supplied with nutrient medium through the wall of the membranes contained in the first membrane module is made possible. Furthermore, metabolic products of the cell preparation can migrate out of the cell preparation through the wall of the membranes and flow back together with the nutrient medium flow through the outflow opening of the first membrane module into the nutrient medium container or be discarded.

In a particularly preferred embodiment of the device according to the invention, a second membrane module is installed in the nutrient medium line, which is suitable for gas transfer and contains at least one gas-permeable membrane, the second membrane module having a first opening and a second opening for the passage of nutrient medium through the membrane module on the has one side of the gas-permeable membrane and at least one inflow opening for introducing gases to the other side of the gas-permeable membrane, the inflow opening being fluidly connected to a gas supply. If it is desired to pass gas through the second membrane module in cross-flow mode, the second membrane module also has an outflow opening for gases.

In a further embodiment of the device according to the invention, there is a second one in the line between the first membrane module and the first container or in the line between the first membrane module and the second container .Membrane module for gas transfer installed, which contains at least one gas-permeable membrane, the second membrane module having a first and a second opening for the passage of the cell preparation on one side of the gas-permeable membrane and at least one inflow opening for introducing gases to the other side of the gas-permeable membrane has, the inflow opening being fluidly connected to a gas supply. If it is desired to pass gas through the second membrane module in cross-flow mode, the second membrane module also has an outflow opening for gases.

In a further preferred embodiment, the device according to the invention comprises a third membrane module for separating liquid from the cell preparation, which is arranged in the line between the first container and the first membrane module or in the line between the second container and the first membrane module, the third Membrane module has openings for the passage of the cell preparation through the membrane module on one side of the membrane and on the other side of the membrane has at least one outlet opening for the discharge of liquid separated from the cell preparation. Liquids that can be separated via the third membrane module are e.g. plasma or nutrient medium contained in the cell preparation. For example, the removal of plasma from the cell preparation via the outlet opening of the third membrane module e.g. to set a plasma concentration in the cell preparation that is independent of the donor's plasma portion and that is required by the recipient. The removal of nutrient medium from the cell preparation via the outlet opening can e.g. serve to remove metabolic products that have accumulated in the nutrient medium during preservation and that are toxic to the recipient.

In a particularly preferred embodiment of the device according to the invention, the outlet opening of the third membrane module is fluidly connected to a pump for withdrawing the separated liquid from the membrane module. In a further preferred embodiment, the device according to the invention contains a feed point in the line path between the first and second containers and particularly preferably in the line between the first membrane module and the third membrane module. This can be fluid-connected to the nutrient medium container via a pump. This serves to compensate for the loss of volume in the cell preparation caused by the escape of plasma or nutrient medium via the third membrane module. The feed point can also be used to feed growth or differentiation factors into the cell preparation. For example, the factors mentioned can be fed in together with the nutrient medium.

The possibility of separating liquid from the cell preparation and replacing it with another liquid gives the device according to the invention a high degree of variability in the culture management of the cell preparation to be preserved.

The object of the present invention is further achieved by a method for preserving a cell preparation comprising at least the steps a) making the cell preparation available in a first container for the cell preparation, b) conveying the cell preparation from the first container through at least one first membrane module second container for the cell preparation and c) then conveying the cell preparation from the second container through the at least one first membrane module into the first container, the first membrane module containing at least one semipermeable membrane, via which one during the passage of steps b) and c) The cell preparation is supplied with a liquid nutrient medium and metabolic products of the cell preparation are removed and steps b) and c) are carried out several times in succession.

In principle, all the procedures by which the cell preparation is sterile are suitable for making the cell preparation available in the first container in step a) and can be removed undamaged from a dispenser or from a storage container and placed in the first container. At least one needle is preferably used to remove the cell preparation from a donor. If the cell preparation contains cell aggregates which are undesirable during preservation, the cell preparation removed is brought into the first container via a transfusion filter.

In a preferred embodiment of the method according to the invention, the cell preparation is whole blood, to which a short-term anticoagulant is added, with short-term anticoagulant being an agent such as e.g. Fragmin, it should be understood that blood coagulation is prevented for the time required in the method according to the invention until the calcium concentration in the blood falls below 0.3 mmol / l. This time is in the range <6 h.

In a particularly preferred embodiment of the method according to the invention, the cell preparation is mobilized whole blood. With approx. 20 to 100 stem cells / μl, this has a higher stem cell concentration than non-mobilized whole blood, which only contains around 1 stem cell / μl.

Elevated stem cell concentrations are also found in umbilical cord blood and in leucopheresis preparations. The latter represent the leukocyte fraction of centrifuged blood. Therefore, in further preferred embodiments of the method according to the invention, the cord preparation is umbilical cord blood, to which an anticoagulant has been added, or a leukocyte preparation.

In one embodiment of the device or method according to the invention, the first membrane module additionally contains at least one second membrane for gas transfer, through the wall of which the O ₂ and CO ₂ are supplied to the cell preparation to the extent that the physiologically required values of pO ₂ are found in the cell preparation and pCO ₂ and that the interaction of CO ₂ with the bicarbonate present in the medium of the cell preparation results in the physical sets the logically required pH value. Such a membrane module is described for example in WO 98/33581.

The chronological sequence of steps b) and c) and steps c) and b) takes place in the method according to the invention in such a way that the cells of the cell preparation are sufficient for preservation both with nutrient medium and with O ₂ and CO ₂ are supplied, which sets the physiologically required values of pH, pO ₂ and pCO ₂ , and also in such a way that the cells of their metabolites are disposed of to a sufficient extent.

For this purpose, in a preferred embodiment of the method according to the invention, steps b) and c) take place in direct succession in time.

In two particularly preferred embodiments of the method according to the invention, step b) and then step c) take place, whereupon step b) takes place in direct succession, or both steps b) and c) and steps c) and b) take place directly in succession , In particular, the last-mentioned embodiment provides the cell preparation with a particularly abundant supply of nutrient medium, disposes of its metabolic products in a particularly extensive manner and keeps it in a state in which the physiologically necessary values of pH, pO ₂ and pCO ₂ prevail with particularly high security.

In a preferred embodiment of the method according to the invention, the physiologically required values of pH, pO ₂ and pCO _{2 are} adjusted via the nutrient medium. The nutrient medium can be provided with O ₂ and CO ₂ in a nutrient medium container to such an extent that the required values of pO ₂ and pCO _{2 are} established and that the physiologically required pH is established via the interaction of CO ₂ with the bicarbonate contained in the nutrient medium Value sets. Before entering the first membrane module, however, the nutrient solution preferably passes through a second membrane module for gas transfer, which contains at least one gas-permeable membrane, on one side of which the nutrient solution is passed and on the other side a gas mixture with the physiologically required values of pO ₂ and pCO ₂ flows. Here, O ₂ and CO ₂ get into the nutrient solution through the wall of the gas-permeable membrane, and the physiologically required pH value is established through the interaction with the bicarbonate contained in the nutrient medium.

In a further preferred embodiment of the method according to the invention, the physiologically required values of pH, pO ₂ and pCO _{2 are set} via a second membrane module for gas transfer, which is located in the conveying path of the cell preparation between the first container and the first membrane module or in the conveying path between the second container and the first membrane module is arranged, and which contains at least one gas-permeable membrane, on one side of which the cell preparation is passed, while on the other side a gas mixture with the physiologically required values of O ₂ and CO ₂ flows. So O ₂ and CO _{2 get} through the wall of the gas-permeable membrane into the cell preparation and through the interaction with the bicarbonate contained in the cell preparation, the physiologically required pH value is established. The gas mixture is preferably moistened with water. It particularly preferably has a relative humidity of 100%.

In principle, any procedure is suitable for conveying the cell preparation in steps b) and c) of the method according to the invention, by means of which the cell preparation can be conveyed from the first container via the at least one first membrane module into the second container and vice versa. However, it should be noted that the cell preparation consists of living cells, for which repeated mechanical deformations, which can occur as a result of the promotion, lead to damage. In a preferred embodiment of the method according to the invention, a pump which can be switched with respect to its conveying direction is used in steps b) and c) to convey the cell preparation and is installed in the conveying path between the first membrane module and one of the containers for the cell preparation.

In a further preferred embodiment of the method according to the invention, a first configuration of the container for the cell preparation is set in step b) and a second configuration in step c), the first container above and the second container below the first Membrane module is arranged and in the second configuration the second container is arranged above and the first container below the first membrane module. Since in this embodiment the cell preparation is only promoted by a hydrostatic potential, so that no cell deformations occur, as they e.g. when pumping is unavoidable, this type of pumping is particularly gentle on the cells.

In principle, it is irrelevant which method is used to implement the first and second configuration in terms of process technology. However, the following procedure is preferred because it is easy to implement. Both containers are attached to a transport device, with the help of which the containers are brought into the first configuration. The height difference between the containers specifies a hydrostatic potential which, together with the dynamic pressure in front of the at least one first membrane module, enables the desired flow rate of the cell preparation. Thus, in cases in which the cell preparation is blood, it has proven to be advantageous for a blood volume of approximately 40 to 140 ml and a membrane area of approximately 100 to 300 cm ^{2 to have} a flow rate in the range of approximately 2 to 10 ml / min and with a blood volume of about 0.8 to 1.2 l and a membrane area of about 500 to 1500 cm ^{2, set} a flow rate in the range of about 40 to 80 ml / min. In step b), due to the hydrostatic potential, the cell preparation is conveyed from the first container via the at least one first membrane module into the second container until the first container has run empty. Through the choice of membrane areas, the aqueous suspension of the cells can be supplied with a supply of nutrient, O ₂ and CO ₂ just once through the at least one first membrane module, so that it is not absolutely necessary to immediately after the first container has run dry Using the transport devices to set the second configuration. On the other hand, it goes without saying that after the first container has been emptied, the second configuration can only be waited for as long as all cells have enough nutrient solution, O ₂ and CO ₂ , until they pass through the at least one first membrane module to stay vital.

The second configuration is then set to carry out process step c). Then, due to the hydrostatic potential, the cell preparation is conveyed from the second container via the at least one first membrane module into the first container until the second container has run empty, whereupon the first configuration is set again, etc.

These considerations show that in the context of the method according to the invention, for the best possible success of the preservation, it is particularly preferred to change the respective configuration immediately after one of the containers has run dry, and in this way to keep the cells in the best possible supply state during the preservation.

In a further preferred embodiment of the invention, the cell preparation is mixed during the steps b) and c) in the first and / or second container in order to prevent phase separation, ie sedimentation in the cell preparation. In principle, any mixing device which is able to prevent the undesired sedimentation of cells without mechanically damaging the cells can be used for this purpose. This can be done in a simpler and therefore Realize men of the present invention in a particularly preferred manner by executing the container from a plastic bag, for example from a blood bag, and deforming the bags oscillating from the outside to the extent that no separation occurs in the cell preparation.

In a further preferred embodiment of the method according to the invention, the containers for the cell preparation are each suspended from a load cell. The load cells perform two functions, both of which are important for an automated implementation of the method according to the invention. On the one hand, they initiate the change of configuration as soon as this is desired. On the other hand, the load cells measure the flow rate of the cell preparation via their temporal change in weight and check whether the flow rate is constant and / or is in the desired range.

In a further preferred embodiment of the method according to the invention, an exchange of liquid in the cell preparation is carried out while carrying out steps b) and c).

For this purpose, in a particularly preferred embodiment of the method according to the invention, the cell preparation is passed through a third membrane module which is installed in the line between the first membrane module and one of the containers for the cell preparation and which contains at least one semipermeable membrane. The cell preparation is passed through on one side of the membrane and a liquid is drawn off on the other side which permeates through the membrane from the cell preparation.

In a very particularly preferred embodiment of the method according to the invention, the liquid drawn off is replaced by fresh nutrient medium. This can be the same nutrient medium used for preservation. Alternatively, another nutrient medium can also be used, as a result of which a nutrient medium exchange takes place in the cell preparation. These pre It is particularly advisable if the nutrient medium used for preservation is incompatible with the recipient. The fresh nutrient medium can contain growth or differentiation factors.

In a further very particularly preferred embodiment of the method according to the invention, the cell preparation is blood and the liquid is plasma, which is withdrawn from the blood. This allows a cell preparation to be preserved that has a transplant quality tailored to the recipient that is independent of the fluctuations in the plasma properties in the source of the cell preparation, e.g. Proportion and composition of the plasma.

In a further very particularly preferred embodiment of the method according to the invention, the cell preparation is blood and the liquid nutrient medium which is withdrawn from the blood, the nutrient medium containing metabolic products of the cell preparation which are toxic to a recipient.

The possibility of exchanging liquid gives the method according to the invention a high degree of variability in the culture management of the cell preparation to be preserved.

In a preferred embodiment of the method according to the invention, blood is used as the cell preparation and a modified Leibowitz's L15 medium is used as the nutrient medium.

The cell preparations preserved by the method according to the invention can be used advantageously for retransfusion, both an autologous and an allogeneic retransfusion being possible. In addition, the cell preparations preserved by the method according to the invention can also be used for all other purposes, effects and usefulness of preserved cell preparations known to the person skilled in the art. The following surprising effects are achieved with the device and with the method according to the present invention.

Blood or cell preparations from blood can be anticoagulated for at least 7 days without the need to add an anticoagulant.

The physiological pH value can be maintained in the cell preparation for at least 7 days, so that the acidification of the cell preparation which occurs as a result of the metabolism of the cells and the associated cell-damaging effects are avoided.

The cell preparations are preserved at room temperature or above room temperature, e.g. at 37 ° C, so that cooling and cryopreservation, which is expensive in terms of equipment and energy, is unnecessary. The addition of a cryopreservative such as DMSO is therefore also unnecessary.

During the preservation, the cells are not only supplied with the liquid and gaseous substances necessary for their survival, but also their metabolic products are disposed of without the need to manipulate or disrupt the cell preparation.

The vitality of hematopoietic stem cells can be maintained for at least 7 days.

In addition, tailor-made preparations can be made on the cell preparation during preservation, e.g. the setting of a certain plasma content or the replacement or exchange of the nutrient medium.

The present invention is illustrated by the drawings and examples. In a simplified schematic representation: Figure 1: Side view of an inventive device for preserving a cell preparation in the first configuration;

Figure 2: Side view of a device according to the invention for preserving a cell preparation in the first configuration realized by transport devices;

Figure 3a: Side view of a device according to the invention for preserving a cell preparation in the first (solid lines) and in the second configuration (dashed lines) with nutrient medium and gas supply;

FIG. 3b: side view of a device according to the invention for preserving a cell preparation in the first configuration with nutrient medium and gas supply and with liquid exchange in the cell preparation;

Figure 1 shows a schematic representation of a side view of an inventive device for preserving a cell preparation in the first configuration. The device contains a first membrane module 1 with first semipermeable membranes, the membrane module having a first opening 2 and a second opening 3 for the passage of the cell preparation through the membrane module on one side of the membrane, and an inflow opening 4 and an outflow opening 5 for the passage of a liquid culture medium through the membrane module on the other side of the membrane. Furthermore, the device according to the invention contains a first container 6 for the cell preparation with at least a first container opening 7 and a second container 8 for the cell preparation with at least a first container opening 9, the containers being designed as blood bags. The first membrane module 1 is fluidly connected with its first opening 2 for the passage of the cell preparation to the first container opening 7 of the first container 6 via a line and with its second opening 3 for the passage of the cell Preparation fluidly connected to the first container opening 9 of the second container 8 via a line.

The means for the oscillatory conveyance of the cell preparation from the first container 6 for the cell preparation via the first membrane module 1 into the second container 8 for the cell preparation is the first configuration already mentioned. The device according to the invention also contains in the line between the first container opening 7 and the first opening 2 of the first membrane module 1 for the passage of the cell preparation a first transfusion filter 20, a first device 21 for sampling, a first device 22 for closing the line and in the line between the second opening 3 of the first membrane module 1 for the passage of the cell preparation and the first container opening 9, a second transfusion filter 23, a second device 24 for sampling and a second device 25a for closing the line. Furthermore, in the device according to the invention, the first container 6 for the cell preparation is fluid-connected via a second opening 25, a third closure device 26, a third transfusion filter 27 and a third device 28 for taking samples with a needle 29 for taking the cell preparation via a line, and the second container 8 for the cell preparation is fluidly connected via a line via a second opening 30, a fourth closure device 31, a fourth transfusion filter 32 and a fourth device 33 for taking samples with a needle 34 for retransfusion of the cell preparation. The transfusion filters 20, 23, 27, 32 serve to hold back cell aggregates, the presence of which in the two containers 6 and 8 is undesirable, or which could lead to a hindrance in the promotion of the cell preparation in the first membrane module 1. The devices for sampling 21, 24, 28, 33 open up the possibility of analyzing the condition of the cell preparation at various points in the device according to the invention. The device according to the invention closes the cell preparation sterile from the outside world. The closure devices 22, 25a, 26, 31 enable the device according to the invention to be filled in a controlled manner. Together with the means for removing or transfusing the cell preparation, the device according to the invention enables a sterile transfer of the cell preparation from a storage container or from a dispenser into the device for the purpose of preservation. vation and after the preservation has ended, the transfer of the cell preparation into a storage container or into a recipient.

FIG. 2 shows a schematic side view of a device according to the invention for preserving a cell preparation in the first configuration, which is implemented by a first and a second transport device 42 and 43. The first container 6 for the cell preparation is connected to the first transport device 42 via a first side unit 50 and the second container 8 for the cell preparation is connected to the second transport device 43 via a second side unit 51, and the first and second transport devices 42 and 43 are vertical in opposite direction movable, whereby the first and the second configuration are adjustable. A load cell 52, 53 is rigidly connected to each side unit 50, 51, on which the respective container 6, 8 for the cell preparation is suspended, the connection between the load cell 52, 53 and container 6, 8 being established by a holder 54, 55 , The load cells serve as an automatic level indicator for containers 6 and 8. They also serve to initiate an automatic change of configuration as soon as one container is empty and the other is full. Finally, by means of the registration of the temporal change in weight of the containers 6 and 8, the load cells are used to check whether the flow rate of the cell preparation is constant and / or is in the desired range. A mixing device 56, 57 is attached to each side unit 50, 51 and acts on the container 6, 8, which is in the form of a blood bag and is located on the respective side unit, as a result of which an undesired phase separation, i.e. sedimentation in the cell preparation in the containers is avoided. The first and second transport devices 42 and 43 are designed as a worm thread that can be driven with a constant rotational speed in both directions of rotation. The worm thread is connected to a motor via a toothed belt (not shown in FIG. 2).

FIG. 3a shows a schematic side view of a device according to the invention for preserving a cell preparation in the first (solid lines) and in the second configuration (dashed lines). In the nutrient A second membrane module 13, which is suitable for gas transfer and contains gas-permeable membranes, is installed between the inflow opening 4 for passing the nutrient medium through the first membrane module and the nutrient medium container 11, the second membrane module 13 having a first opening 13a and a second opening 13b for the Passage of nutrient medium through the membrane module 13 on one side of the gas-permeable membrane and an inflow opening 13c and an outflow opening 13d for the passage of gases on the other side of the gas-permeable membrane, the inflow opening 13c being fluidly connected to a gas supply 14, which acts as a gas mixer for the gases N _2> O ₂ and CO _{2 is} carried out. In this device, nutrient medium from the nutrient medium container 11 is passed with the pump 12 through the second membrane module 13 on one side of the gas-permeable membranes and is thereby provided with a gas mixture which contains O ₂ and CO ₂ in a mixing ratio through the wall of the gas-permeable membranes, which corresponds to the physiological values of pO ₂ and PCO ₂ and adjusts the physiological pH value via the interaction of CO ₂ with the bicarbonate contained in the nutrient medium. N ₂ serves as the carrier gas. The solution thus adjusted to physiological values of pH, pθ2 and pCO ₂ leaves the second membrane module through the opening 13b, passes through the opening 4 into the first membrane module, leaves it - depleted in nutrient solution and in the gases added in the second membrane module and enriched with Metabolic products from the cell preparation - via the opening 5 and is returned to the nutrient medium container 11.

3b shows a schematic side view of a device according to the invention for preserving a cell preparation in the first configuration, with the exception of the second configuration, all the device features from FIG. 3a and, in addition, a third in the line between the first membrane module 1 and the first container 6 for the cell preparation Module 14 with semipermeable membranes for fluid exchange. The third membrane module 14 has openings 15, 16 for the passage of the cell preparation on one side of the semi-permeable membranes and on the other side of the semi-permeable membranes Outlet opening 17, through which a liquid, for example plasma or nutrient solution, which was enriched with metabolic products toxic to the recipient during the preservation, can be withdrawn from the cell preparation with a pump 18. The loss of volume of the cell preparation resulting from the removal of the liquid is compensated for by nutrient medium which is introduced from the nutrient medium container 11 with a pump 19 into an opening in the line between the third membrane module 14 and the first membrane module 1.

The following examples show how the device and the method according to the invention can be used for the preservation of cell preparations.

Example 1 :

500 ml of donor blood, which contains 5 units of the short-term anticoagulant Fragmin per ml, are centrifuged in 14 ml tubes with an acceleration of 600 »g for 20 minutes. The plasma fraction is discarded. The ring of thrombocytes and leukocytes (buffy coat) lying on the erythrocytes is added to 40 ml of autologous blood, a leucocyte concentration of 18,500 / μl suspension being set via the mixture of buffy coat and autologous blood.

The resulting 120 ml suspension is brought into the first blood bag 6 of the device according to the invention, which is in the first configuration, via a sterile tube system.

The first blood bag 6 is fastened to the first load cell 52 via the holder 54. The second blood bag 8 is fastened to the second load cell 53 via the holder 55. Due to the hydrostatic potential, the suspension runs at a flow rate of 2 to 10 ml / min through the transfusion filter 20, the first membrane module 1, which contains microfiltration membranes made of polyethersulfone with a membrane area of 200 cm ² , and through the transfusion filter 23 into the second blood bag 8 Here is the first membrane module with its outlet opening 4 connected via a line to the nutrient medium container 11, in which Leibowitz's L15 medium ({without phenol red and calcium chloride, with 320.6 mg / l potassium chloride, 203.3 mg / l magnesium chloride »6H ₂ O, 6311, 52 mg / l Sodium chloride, 2184.26 mg / l bicarbonate}, additives: 0.3 mg / ml glutamine, 10 g / l human serum albumin, 11 μg / ml folic acid, 42 μg / ml i-inositol, 30 μg / ml hoio-transferin , 30 μg / ml L-α-phosphatidylcholine, 7.5 μg / ml cholesterol, 10 μg / ml velosuiin, 1, 6 * 1 O ^ mol monothiolglycerol) and submitted with a pump 12 through the second membrane module 13, which contains gas-permeable membranes made of polypropylene with a membrane area of 45 cm ² , the nutrient solution entering the second membrane module 13 through the inlet opening 13a and flowing on one side of the gas-permeable membrane, while on the other side of the gas-permeable membrane one with water to 100% Relative humidified gas mixture of N ₂ , O ₂ and CO ₂ fl ies that enters the second membrane module through the inflow opening 13c and leaves it through the outflow opening 13d. The gas mixture contains O ₂ and CO ₂ in a ratio that corresponds to the physiological values of pO ₂ and pCO ₂ . O ₂ and CO ₂ permeate through the wall of the gas-permeable membranes and the CO ₂ , in interaction with the sodium hydrogen carbonate present in the nutrient solution, sets a physiological pH. As a result, when the nutrient solution emerges from the second membrane module 13 through the opening 13b, it has the physiological values of pH, pO ₂ and pCO ₂ and transfers these values in the first membrane module 1 through the wall of the semipermeable membranes contained therein to the cell preparation. In the other direction, metabolic products migrate from the cell preparation into the nutrient medium stream, which is passed through the outflow opening 5 of the first membrane module into the nutrient medium container 11.

As soon as the first blood bag 6 has run empty, the second configuration is set by means of the transport devices 42 and 43. As soon as the second blood bag is empty, the transport devices 42 and 43 set the first configuration. This change in configuration is repeated for 168 hours, ie for 7 days, the device according to the invention being at room temperature. FIG. 4 shows the albumin concentration in g / l in the cell preparation over the test period of 168 hours. It can be seen that the albumin concentration drops during the experiment to the value of the albumin concentration in the nutrient medium, which has an albumin concentration of 9.58 g / l. It is thus possible to set not only the third but also the second membrane module, as described at the beginning, during the preservation to a certain content of plasma protein.

FIG. 5 shows the Ca ²⁺ concentration of the cell preparation over the test period of 168 hours. Because the nutrient medium does not contain Ca ²⁺ , the Ca ²⁺ concentration of the cell preparation drops to 0.3 mmol / l within a few hours. This low concentration, at which coagulation is no longer possible, can be maintained over the entire remaining test period.

FIG. 6 shows the pH of the cell preparation over the test period of 168 hours. The gassing of the nutrient medium described above with a gas mixture of 5.0% CO ₂ , 1, 2% O ₂ and 93.8% N ₂ sets a pH in the physiological range and maintains it over the entire duration of the experiment.

FIG. 7 shows the relative proportion of CD34 ⁺ hematopoietic stem cells in the cell preparation compared to the initial value of the cell preparation over the test period of 168 hours. CD34 ⁺ denotes the "Cluster of Designation with the number 34", which is one of the parameters for the detection and quantification of stem cells. The assay from Becton-Dickinson is used. FIG. 7 shows that the proportion of CD34 ⁺ cells remains constant over the entire test period of 168.

FIG. 8 shows the relative proportion of “Burst Forming Units-Erythroid” (BFU-E) in the cell preparation compared to the initial value of the cell preparation over the test period of 168 hours. BFU-E denotes a functional, methyl-based cellulose-based stem cell assay, which measures the ability of the stem cells to differentiate into erythrocytes. Figure 8 shows that after 168 hours 62% of the BFU-E are still present.

FIG. 9 shows the relative proportion of “colony forming units granulocyte / macrophage” (CFU-GM) in the cell preparation compared to the initial value of the cell preparation over the test duration of 168 hours. CFU-GM denotes a functional stem cell assay based on methyl cellulose , which measures the ability of the stem cells to differentiate into granulocytes and macrophages .. Figure 8 shows that after 168 hours 100% of the CFU-GM is still present.

Example 2:

500 ml of donor blood containing 5 units of the short-term anticoagulant Fragmin per ml are centrifuged in 14 ml tubes at an acceleration of 600 g for 20 minutes. The plasma fraction is discarded. The ring of thrombocytes and leukocytes (buffy coat) lying on the erythrocytes is added to 40 ml of autologous blood, a leucocyte concentration of 23,400 / μl suspension being set via the mixture of buffy coat and autologous blood.

The first blood bag 6 is fastened to the first load cell 52 via the holder 54. The second blood bag 8 is fastened to the second load cell 53 via the holder 55. Due to the hydrostatic potential, the suspension runs at a flow rate of 2 to 10 ml / min through the transfusion filter 20, the third membrane module 14, the microfiltration membranes made of polyethersulfone with a Contains membrane area of 175 cm ² , the first membrane module 1, which contains dialysis membranes made of polyethersulfone with a membrane area of 290 cm ² , and through the transfusion filter 23 into the second blood bag 8. The first membrane module with its outlet opening 4 is connected via a line to the Nutrient media container 11 connected, in which the modified Leibowitz's L15 medium already described in Example 1 is placed and fed with a pump 12 through the second membrane module 13, which contains gas-permeable membranes made of polypropylene with a membrane area of 324 cm ² , the nutrient solution through the Inlet opening 13a enters the second membrane module 13 and flows on one side of the gas-permeable membrane, while on the other side of the gas-permeable membrane a gas mixture of N ₂ , O ₂ and CO ₂ moistened with water at 100% relative humidity flows through the inflow opening 13c enters the second membrane module u nd leaves it through the outflow opening 13d. The gas mixture contains O ₂ and CO ₂ in a ratio that corresponds to the physiological values of pO ₂ and pCO ₂ . O ₂ and CO ₂ permeate through the wall of the gas-permeable membranes and the CO ₂ interacts with the sodium hydrogen carbonate in the nutrient solution to set a physiological pH. As a result, the nutrient solution has the physiological values of pH, PU ₂ and PCO ₂ when it emerges from the second membrane module 13 through the opening 13b and transfers these values in the first membrane module 1 through the wall of the semipermeable membranes contained therein to the cell preparation. In the other direction, metabolic products migrate from the cell preparation into the nutrient solution stream which is passed through the outflow opening 5 of the first membrane module into the nutrient medium container 11.

As already mentioned, the cell preparation flows through the third membrane module 14 along one side of the microfiltration membranes made of polyethersulfone contained therein. On the other side of these membranes, plasma is drawn off via the outlet opening 17, which is fluidly connected to the pump 18 via a line. The resulting loss of volume of the cell preparation is compensated for by Leibowitz 'L15 medium, which flows from the nutrient medium container 11 with the pump 19 is introduced into the line between the first membrane module 1 and the third membrane module 14. The plasma is therefore exchanged for the modified Leibowitz 'L15 medium.

As soon as the first blood bag 6 has run empty, the second configuration is set by means of the transport devices 42 and 43. As soon as the second blood bag is empty, the transport devices 42 and 43 set the first configuration. This change of configuration is repeated for 4 hours, the device according to the invention being at room temperature. The pump 18 is then switched off and the plasmapheresis is thus ended by means of the third module. Immediately afterwards, the preservation of the cell preparation is carried out as in Example 1 for a period of 168 hours at room temperature.

FIG. 10 shows the albumin concentration in g / l in the cell preparation over a 4 h plasmapheresis on the basis of two experiments carried out independently of one another. It can be seen that the albumin concentration after 4 h of plasmapheresis corresponds to the albumin concentration of the nutrient medium. The plasma was thus replaced by the nutrient medium without having to take blood cells from the cell preparation.

FIG. 11 shows the Ca ²⁺ concentration of the cell preparation after plasmapheresis over the test period of 168 h in two experiments carried out independently of one another. The Ca ²⁺ starting value in the cell preparation is represented by a black rectangle. After the 4-hour plasmapheresis, the Ca ²⁺ concentration dropped to 0.3 mmol / l due to the use of a nutrient medium without Ca ²⁺ . This low concentration can be kept largely constant over the entire duration of the experiment and ensures that no coagulation occurs in the cell preparation.

FIG. 12 shows the pH value of the cell preparation after 4 hours of plasmapheresis in two tests carried out independently of one another over a test period represented by 168 hours. By gassing the nutrient medium with a gas mixture as in Example 1, a pH in the physiological range is set and kept constant over the entire remaining test period.

FIG. 13 shows the pθ2 value of the cell preparation after 4 hours of plasmapheresis. It can be seen that with 25 mm Hg at 25 ° C a physiological pθ ₂ value can be set and kept constant within the 168 h test period.

FIG. 14 shows the lactate concentration of the cell preparation after 4 hours of plasmapheresis in two experiments carried out independently of one another over a test period of 168 hours. The metabolic product lactate is disposed of via the membranes of the first membrane module. It can be seen that a lactate concentration of less than 1 mmol / l can be set and kept constant over the test period of 168 hours.

FIG. 15 shows the relative proportion of CD34 ⁺ hematopoietic stem cells in the cell preparation compared to the initial value of the cell preparation after 4 hours of plasmapheresis in two experiments carried out independently of one another over a test period of 168 hours. It can be seen that the proportion of CD34 ⁺ hematopoietic stem cells only decreases by 10% over the entire duration of the experiment. This shows that hematopoietic stem cells can be preserved over longer periods of time using the device and the method according to the present invention.

Claims

claims:

1. Device for preserving a cell preparation comprising at least a first membrane module (1) which contains at least a first semipermeable membrane, the membrane module having a first opening (2) and a second opening (3) for the passage of the cell preparation through the membrane module on the has a side of the first membrane and an inflow opening (4) and an outflow opening (5) for the passage of a liquid nutrient medium through the membrane module on the other side of the first membrane, further comprising a first container (6) for the cell preparation with at least one first Container opening (7), a second container (8) for the cell preparation with at least a first container opening (9) and means for oscillating delivery of the cell preparation from the first container (6) for the cell preparation via the first membrane module (1) into the second container (8) for the cell preparation and vice versa, the first membrane module ( 1) with its first opening (2) for the passage of the cell preparation with the first container opening (7) of the first container (6) via a line and with its second opening (3) for the passage of the cell preparation with the first container opening ( 9) of the second container (8) is fluid-connected via a line.

2. Device according to claim 1, characterized in that the means for oscillating conveying of the cell preparation are realized by a pump which can be switched with respect to their conveying direction and which is located in one of the lines between the first membrane module (1) and one of the containers (6) or (8 ) is arranged for the cell preparation.

3. Device according to claim 1, characterized in that the means for the oscillating conveyance of the cell preparation are realized by an alternately adjustable first and second configuration of the container (6, 8) for the cell preparation, in the first configuration the first container (6) Above and the second container (8) are arranged below the first membrane module (1) and in the second configuration the second container (8) above and the first container (6) are arranged below the first membrane module (1).

4. The device according to claim 3, characterized in that the first container (6) for the cell preparation with a first transport device (42) and the second container (8) for the cell preparation with a second transport device (43) is connected and the first and second transport device (42) and (43) are vertically movable in the opposite direction, whereby the first and second configuration are adjustable.

5. The device according to claim 4, characterized in that the first container (6) for the cell preparation on the first transport device (42) via a first side unit (50) and the second container for the cell preparation (8) with the second transport device (43 ) are connected via a second side unit (51).

6. The device according to claim 5, characterized in that with each side unit (50, 51) a load cell (52, 53) is rigidly connected, on which the respective because the container (6, 8) for the cell preparation is suspended.

7. The device according to claim 5 or 6, characterized in that a mixing device (56, 57) is attached to each side unit (50, 51), which acts on the container (6, 8) located on the respective side unit.

8. The device according to one or more of claims 5 to 7, characterized in that the first and second transport devices (42) and (43) are screw threads drivable in both directions of rotation with a constant rotational speed.

9. The device according to one or more of claims 1 to 8, characterized in that the first membrane contained in the first membrane module (1) is a flat or hollow fiber membrane.

10. The device according to claim 9, characterized in that the flat or hollow fiber membrane is a nano, ultra or microfiltration membranes.

11. The device according to one or more of claims 1 to 10, characterized in that the first membrane module (1) with its inflow opening (4) for the passage of a liquid nutrient medium with a nutrient medium container (11) for the liquid nutrient medium is fluid-connected via a nutrient medium line.

12. The device according to claim 11, characterized in that a second membrane module (13) is installed in the nutrient medium line, which is suitable for gas transfer and contains at least one gas-permeable membrane, the second membrane module (13) having a first opening (13a) and one second opening (13b) for the passage of nutrient medium through the membrane module (13) on one side of the gas-permeable membrane and at least one inflow opening (13c) for introducing gases to the other side of the gas-permeable blen membrane, wherein the inflow opening (13c) is fluidly connected to a gas supply, or wherein the second membrane module (13) also has an outflow opening (13d) for gases.

13. The apparatus according to claim 11, characterized in that in the line between the first membrane module (1) and the first container (6) or in the line between the first membrane module (1) and the second container (8), a second membrane module for Gas transfer is installed, which contains at least one gas-permeable membrane, the second membrane module having a first and a second opening for the passage of the cell preparation on one side of the gas-permeable membrane and at least one inflow opening for introducing gases to the other side of the gas-permeable membrane, wherein the inflow opening is fluidly connected to a gas supply, or wherein the second membrane module also has an outflow opening for gases.

14. The device according to one or more of claims 1 to 13, characterized in that it comprises a third membrane module (14) for separating liquid from the cell preparation, which in the line between the first container (6) and the first membrane module (1 ) or in the line between the second container (8) and the first membrane module (1), the third membrane module having openings (15, 16) for the passage of the cell preparation through the membrane module on one side of the membrane and on the on the other side of the membrane has at least one outlet opening (17) for discharging liquid separated from the cell preparation.

15. The apparatus according to claim 14, characterized in that the outlet opening (17) of the third membrane module (14) is fluid-connected to a pump (18) for withdrawing the separated liquid from the membrane module.

16. A method for preserving a cell preparation comprising at least the steps a) making the cell preparation available in a first container for the cell preparation, b) conveying the cell preparation from the first container through at least one first membrane module into a second container for the cell preparation and c) then conveying the cell preparation from the second container through the at least one first membrane module into the first container, the first membrane module containing at least one semipermeable membrane, via which, during the steps b) and c), the cell preparation is supplied with a liquid nutrient medium and metabolic products of the cell preparation are removed and steps b) and c) are repeated several times in succession.

17. The method according to claim 16, characterized in that steps b) and c) are carried out directly in time.

18. The method according to claim 16 or 17, characterized in that steps c) and b) are carried out directly in time.

19. The method according to one or more of claims 16 to 18, characterized in that the nutrient medium is used to adjust the physiologically required values of pH, pO ₂ and pC0 ₂ in the cell preparation.

20. The method according to claim 19, characterized in that the nutrient solution passes through a second membrane module for gas transfer before entering the first membrane module.

21. The method according to one or more of claims 16 to 18, characterized in that the adjustment of the physiologically required values of pH, pO ₂ and pCO ₂ takes place via a second membrane module for gas transfer, which is arranged in the conveying path of the cell preparation between the first container and the first membrane module or in the conveying path between the second container and the first membrane module.

22. The method according to one or more of claims 16 to 21, characterized in that in step b) and c) a pump which can be switched with respect to its conveying direction is used for conveying the cell preparation and which is in the conveying path between the first membrane module and one of the containers for the Cell preparation is installed.

23. The method according to one or more of claims 16 to 21, characterized in that to promote the cell preparation in step b) a first and in step c) a second configuration of the container for the cell preparation is set, the first configuration in the first configuration Container is arranged above and the second container below the first membrane module and in the second configuration the second container is arranged above and the first container below the first membrane module.

24. The method according to one or more of claims 16 to 23, characterized in that the cell preparation is mixed in the first and / or second container during the passage of steps b) and c).

25. The method according to one or more of claims 16 to 24, characterized in that an exchange of liquid in the cell preparation is carried out while carrying out steps b) and c).

26. The method according to claim 25, characterized in that the cell preparation is passed through a third membrane module, which is installed in the line between the first membrane module and one of the containers for the cell preparation and contains at least one semipermeable membrane, on one side of which the cell preparation is passed through and on the other side Liquid is drawn off.

27. The method according to claim 26, characterized in that the withdrawn liquid is replaced by fresh nutrient medium.

28. The method according to claim 26 or 27, characterized in that the cell preparation is blood and the liquid is plasma which is withdrawn from the blood.

29. The method according to claim 26 or 27, characterized in that the cell preparation is blood and the liquid is nutrient medium which is withdrawn from the blood, the nutrient medium containing metabolites of the cell preparation which are toxic to a recipient.

30. The method according to one or more of claims 16 to 29, characterized in that blood is used as the cell preparation and a modified Leibowitz's L15 medium is used as the nutrient medium.

31. Use of the cell preparation preserved according to one or more of claims 16 to 30 for retransfusion.