WO1998007828A1

WO1998007828A1 - Device with a reaction bag for multi-step culturing/separation operations and/or reactions in several steps

Info

Publication number: WO1998007828A1
Application number: PCT/EP1997/004425
Authority: WO
Inventors: Manfred Kubbies; Stefan Koch; Bernhard Goller
Original assignee: Roche Diagnostics Gmbh
Priority date: 1996-08-16
Filing date: 1997-08-13
Publication date: 1998-02-26
Also published as: AU4118497A; JP2000503546A; WO1998007829A8; DE19707497A1; CA2264205A1; EP0918846A1; JP2000504942A; WO1998007829A2; EP0941307A1; CA2264206A1; AU4299297A

Abstract

The invention relates to a device containing a reaction bag for multi-step culturing/separation operations in several steps, particularly of culturing antitumoral cells or cell activating antitumoral cells. In the inventive device, a reaction bag contains a plurality of sections (18a, 26a, 38a, 52a), in each of which an initiating agent may be placed at the corresponding stage and reagents may be brought into contact during that stage to give the reaction product, as well as openings (82a, 86a, 44a', 54a) to collect the reactive product from the respective section (18a, 26a, 38a, 52a).

Description

Reaction bag device for carrying out multistage cultivation / separation processes and / or reactions

description

The present invention relates to a reaction bag device for carrying out multistage cultivation / separation processes and / or reactions, for example for growing antitumor cells or cells stimulating antitumor cells.

One approach to treating tumors is to cultivate anti-tumor cells or anti-tumor cell stimulating cells ex vivo, i.e. outside the human body. After breeding, i.e. according to intensive multiplication, the antitumor cells are injected back into the human body.

In order to be able to cultivate such cells outside the human body, a blood sample is taken from a patient, in which there are also a small number of cells that specifically respond to a tumor to be treated, the so-called anti-tumor cells.

These antitumor cells contained in the blood sample can be stimulated to multiply in a culture vessel in a nutrient solution by bringing them into contact with whole tumor cells or so-called vesicles of the tumor cells.

Tumor cells can be obtained, for example, from the tumor tissue present in a patient. The genetic information is removed by lysis of the tumor cells and subsequent filtering, so that only the cell membrane or fragments thereof are retained, which form the vesicles mentioned above. These vesicles have the surface features that are characteristic of tumor cells, so that they are recognized by tumor cells as tumor cells and accordingly correspond to a appropriate stimulation of the multiplication of the antitumor cells. Since the vesicles only have the surface characteristics of the tumor cells, but no longer have the genetic information of the tumor cells, they cannot act on the multiplying antitumor cells, for example by releasing inhibitors.

During the stimulation of growth or multiplication of the antitumor cells by tumor cells or vesicles, the nutrient solution is exchanged and the cells are portioned into several growth containers.

If the multiplication has been carried out to a sufficient extent, the anti-tumor cells are separated from an undesired background in a selection step. For this purpose, the anti-tumor cells are labeled, for example, with the aid of lyophilized or otherwise prepared retroviruses. These retroviruses lead to expression on the surface of the anti-tumor cells e.g. of the 1 NGFR receptor, which contributes to the enrichment of the antitumor cells in a subsequent separation step. After separation, i.e. Enrichment of the antitumor cells, it is possible to either subject them again to separation in order to increase the enrichment rate, or to subject them to a further growth step, for example with the addition of a growth-promoting agent.

When carrying out such cultivation / separation processes as well as other multi-stage chemical or biological reactions, it is important that the individual reaction stages and the transfer of materials between individual reaction stages can be carried out in such a way that on the one hand the introduction of contaminations into individual ones Reaction stages is avoided, but on the other hand contamination of the environment with reaction products generated in individual reaction stages can be avoided. In addition, in order to adapt such multi-stage To meet different requirements, a relatively great flexibility in the selection of the different stages is required, for example in order to be able to add additional reaction stages or to be able to omit reaction stages. For example, it is known to carry out reactions in reaction bags which have an inlet opening for a starting material, an inlet opening for the reagents and an outlet opening from which the product produced by the reaction of the starting material with the reagents can be removed from the bag . For each reaction, ie each stage, a single bag is therefore provided, into which different materials have to be entered or from which materials can be found. In particular when entering or removing respective materials, there is a risk that the environment will be contaminated by these materials or that contaminations will be introduced into the material.

It is therefore an object of the present invention to provide a reaction bag device for carrying out multi-stage cultivation / separation processes and / or reactions, for example for the cultivation of anti-tumor cells or anti-tumor cell stimulating cells, with high flexibility with regard to the stages to be carried out essentially contamination-free working is made possible.

According to the invention, this object is achieved by a reaction bag device for carrying out multistage cultivation / separation processes and / or reactions, for example for growing antitumor cells or cells stimulating antitumor cells, comprising: a plurality of reaction bag chambers in one reaction bag, wherein in a starting material for the respective stage can be arranged in each reaction bag chamber and can be brought into contact with reagents for the respective stage for producing a reaction product of the respective stage, Dispensing opening means for dispensing a reaction product from each reaction bag chamber.

In the device according to the invention, the reactions of the individual stages are therefore carried out in a reaction bag with a plurality of reaction bag chambers in which, on the one hand, a starting material for the stages can be arranged to carry out the reaction of the respective stage and, on the other hand, this starting material is brought into contact with a reagent for the stage can be. Since the reaction can therefore take place in a completely closed environment, there is no risk of contamination of the environment or introduction of contaminants into the reaction of the respective stages. The device according to the invention is relatively simple and inexpensive to manufacture and, because of the possibility of adding or integrating additional reaction bag chambers in a process or removing a reaction bag chamber from a process, provides a high degree of flexibility in carrying out reactions.

The term "starting material" used in the present text is to be understood to mean any kind of material, i.e. Cells and other biological or chemical material, which is to be further processed, multiplied or modified in a respective reaction step to form a reaction product of this step. Similarly, the term "reagents" used in this text should be understood to mean any type of material, i.e. biological, chemical or other materials, as well as combinations of different materials or reagents, which in cooperation with the starting material of the respective stage for further processing, processing, multiplication or the like. of the source material.

At least one of the reaction bag chambers can have an input opening for inputting a starting material into it from the outside with respect to the reaction bag, and the reaction tion bag chambers can be formed in sequence for transferring the reaction product of a reaction bag chamber preceding the sequence as a starting material into at least one reaction bag chamber following the sequence. In this embodiment, since the reaction product is transferred directly between the reaction bag chambers and thus does not come into contact with the surroundings, a particularly safe and contamination-free implementation of the reactions or the cultivation / separation processes is ensured.

For a controlled transfer of reaction products. or to be able to provide starting materials between the different reaction bag chambers, it is proposed that between at least two successive reaction bag chambers in sequence sequential fluid flow interrupting means are provided, preferably destructible fluid flow interrupting means, which form an outlet opening of the reaction bag chamber preceding in the sequence.

In order to gain access to the reaction product generated after a reaction has been carried out, it is proposed that at least one of the reaction bag chambers, preferably the last reaction bag chamber in the sequence, have an outlet opening for discharging the reaction product therefrom with respect to the reaction bag.

It can further be provided that in the case of two reaction bag chambers which follow one another in sequence, a preceding reaction bag chamber has an outlet opening and a subsequent reaction bag chamber has an inlet opening. This is particularly advantageous if the reaction product obtained in the preceding reaction bag chamber is further processed in an external device and subsequently has to be reintroduced into the process to be carried out in the reaction bag device, namely into the subsequent reaction bag chamber. According to an embodiment of the reaction bag device according to the invention, it can comprise at least one reaction bag chamber, which is a reaction bag chamber for a plurality of successive stages, the reaction product of a preceding stage in each case forming the starting material for a subsequent stage. With such a configuration, the processed, increased or the like remains. Contain material in the at least one reaction bag chamber, so that work can be carried out without contamination.

When using a reaction bag device which contains several reaction bag chambers, the liquid, fluid-like or gelatinous material contained in the reaction bag chambers can be transported forward, e.g. by wiping with the fingers. be transported into a next reaction bag chamber. In order to ensure a very efficient transfer process without losing any reaction product contained in a reaction bag chamber, it is proposed that successive reaction bag chambers are separated from one another in the sequence by partition walls which point to a fluid transfer area between the successive reaction bag chambers in a fluid transfer direction in the essentially funnel-shaped s.

In particular when using the reaction bag device according to the invention for the cultivation of antitumor cells, it is advantageous in the multiplication of antitumor cells if the starting material, which contains a relatively small number of antitumor cells, is accumulated in a relatively small volume range, since a Increasing the concentration of antitumor cells leads to accelerated growth. In order to be able to provide such a collection or concentration in at least 5 reaction bag chambers in the reaction bag device according to the invention, it is proposed that at least one through in at least one reaction bag chamber preferably V-shaped wall sections formed starting material / reaction product accumulation area is formed. In this case, the at least one starting material / reaction product collection area is advantageously designed to widen in the fluid transfer direction.

To simplify the manufacture of the reaction bag device, it is proposed that the partition walls tapering in a funnel shape form the wall sections of the starting material / reaction product accumulation area at least in regions.

If the growth or the multiplication of antitumor cells is carried out in a plurality of reaction bag chambers arranged in sequence, for example in each case with the addition of different growth stimulants, care must be taken that the multiplying antitumor cells do not exceed a certain concentration, since this increases inhibits growth or reproduction; i.e. as the growth process progresses, care must be taken to ensure that more volume is available for the multiplying antitumor cells. This can be achieved in a simple manner in that successive reaction bag chambers have an increasing chamber volume in the sequence.

In order to be able to bring the starting material into contact with the reagents of the respective stage in a reaction bag chamber, it is possible according to a particularly simple and practical embodiment that at least one of the reaction bag chambers is already pre-loaded with the reagents before the starting material is entered. With such a configuration, only the starting material has to be entered in the subsequent use.

Furthermore, it is possible for at least one reaction bag chamber to be provided with at least one reagent chamber, preferably by means of apparent fluid flow interruption means destructible fluid flow interrupter, can be brought into reagent transfer connection with the reaction bag chamber, and that the reagents for the respective stage are contained in the reagent chamber. With such a configuration s, the reagents provided for a particular stage can be brought into contact with the starting material at any time, for example after the starting material for the stage has been introduced.

It is possible, for example, that at least one reaction bag chamber is provided with a plurality of reagent chambers for different stages, which can be brought into reagent transfer connection with the reaction bag chamber one after the other in order to carry out the several stages sequentially. The reactions of several successive stages are thus carried out in a single reaction bag chamber and only the reagents contained in the different reagent chambers are sequentially transferred to the reaction bag chamber in accordance with the sequence of the stages, i.e. the reaction product generated in a reaction between reagents and starting material forms the starting material for the reaction of the next step, which is then to be brought into contact with the reagents for the next step.

Furthermore, in at least one reaction bag chamber, a tightly closable reagent input opening can be provided for the input of reagents into the reaction bag chamber from an external container.

To carry out the above-mentioned separation in antitumor cell cultivation, a separation column device for fluid transfer therethrough can also be provided between at least two reaction bag chambers which follow one another in sequence. In a particularly advantageous embodiment of the reaction bag device according to the invention, the separation column device is incorporated into a wall separating the at least two reaction bag chambers, that is to say, even for carrying out the separation, it is not necessary to carry the reaction product out one step, through a separation column or the like. to pass through and to put the resulting concentrated material back into a bag, for example for further cell proliferation.

According to a further aspect of the present invention, there is provided a reaction bag device for carrying out desired multi-stage cultivation / separation processes and / or reactions, for example for growing antitumor cells or cells stimulating antitumor cells, comprising a reaction bag with a Reaction bag chamber in which a starting material for a stage can be arranged and brought into contact with reagents for the step for producing a reaction product of the stage, the reaction bag having an input opening for inputting starting material therein and an output opening for outputting one in the Reaction bag produced reaction product and wherein the reaction bag device contains the reagents and is prepared with these to form a reaction unit.

In this embodiment of a reaction bag according to the invention, therefore, only the starting material has to be introduced into the reaction bag chamber and can be brought there to react with the reagents already provided in each reaction bag, i.e. Even with such a reaction bag device, the risk of introducing contamination via the reagents is reduced.

To prepare the reaction unit, it can be provided, for example, that the reaction bag chamber is preloaded with the reagents. Alternatively or additionally, it is possible that the reaction bag comprises at least one reagent chamber which can be brought into reagent transfer connection with the reaction bag chamber by means of apparent fluid flow interruption means, preferably destructible fluid flow interruption means, and that the reagents for the respective stage are contained in the at least one reagent chamber.

According to a particularly advantageous embodiment, it can be provided that the reaction bag chamber is a reaction bag chamber for a plurality of successive stages, the reaction product of a preceding stage in each case forming the starting material for a subsequent stage, i.e. Even with such a design, a reaction bag, i.e. in a single reaction bag chamber, a plurality of reaction stages can be carried out in succession without the reaction product in each case having to be dispensed from the bag.

It is advantageous if the reaction bag comprises a plurality of reagent chambers for different stages, which reagent chambers can be brought into reagent transfer connection with the reaction bag chamber for sequential execution of the plurality of stages.

In addition, it is possible for the reaction bag chamber to also be provided with a sealable reagent entry opening for entering reagents into the reaction bag chamber from an external container. Even in this way, multi-stage reactions can be carried out in a reaction bag if, for example, a first reaction is carried out by introducing a starting material into a - for example pre-loaded - reaction bag, the reaction product of this stage is left in the reaction bag chamber and subsequently by entering reagents from an external one Container a second reaction stage is carried out. In order to be able to introduce the starting material into the respective reaction bag chamber in the simplest possible manner and / or to be able to remove starting material from a reaction bag chamber, it is proposed that the inlet and / or outlet openings be provided by valves, destructible or reclosable closure means o . Like. are closed.

The reaction bag device according to the invention is preferably made of flexible, preferably gas-permeable, but also liquid-impermeable, plastic. On the one hand, this contributes to a particularly simple and inexpensive producibility, and on the other hand the risk of damage to such reaction bag devices made of flexible plastic, for example due to breakage, is practically eliminated.

Furthermore, the respective reaction bag chamber is preferably formed integrally with the at least one reagent chamber. 0

In order to always be able to carry out the multiplication in a suitable environment when using the reaction bag device for cell multiplication according to the invention, it is proposed that the respective reaction bag chamber be designed to enter fresh nutrient solution or to remove used nutrient solution.

For example, the respective reaction bag chamber can be replaced by a. ^{"have a} penetrable membrane area which automatically restores a fluid-tight seal after removal of the cannula.

The present invention further relates to a modular reaction bag system comprising one or a plurality of reaction bag devices according to the invention. Depending on the intended use, the modular reaction bag system can then be composed of suitably prepared reaction bag devices. are provided, the reaction bag device in each case, for example, being loaded with the reagents for the desired reaction stages or containing them in certain reaction chambers. From a certain number of different reaction bag devices, a suitable bag system can then be put together for a specific multistage process to be carried out, in which only those reaction bag chambers are contained which are actually required for the process.

If the modular reaction bag system is to be used for cell multiplication, for example for the multiplication of anti-tumor cells, it is advantageous if this system further comprises at least one separation column unit.

In order to be able to enter reagents from the outside into the reaction chambers, if desired, it is proposed that at least one container for reagents is further provided for entering reagents from outside into at least one reaction bag chamber with respect to a reaction bag.

The present invention further relates to the use of a reaction bag device according to the invention and / or a modular reaction bag system according to the invention for the cultivation of anti-tumor cells or anti-tumor cell-stimulating cells.

The present invention further relates to a method for carrying out cultivation / separation processes with one or more stages and / or multi-stage reactions, for example for the cultivation of anti-tumor cells or anti-tumor cell stimulating cells.

The invention is described in detail below with reference to the accompanying drawings on the basis of preferred embodiments. It shows - '

1 is a schematic view of a reaction bag according to the invention, which contains four reaction bag chambers, each of which can be connected to one another in sequence;

2 shows an alternative embodiment of a reaction bag according to the invention with two reaction bag chambers, each of the reaction bag chambers being connectable to a plurality of reagent chambers;

3 shows a further alternative embodiment of a reaction bag according to the invention, the transition between reaction bag chambers which follow one another in the sequence being formed by walls which converge in a V-shape; and

Fig. 4 is a schematic view of a reaction bag system, in which separate reaction bags are provided, each with a reaction bag chamber.

1 shows a first embodiment of a reaction bag system 10a according to the invention. The reaction bag system 10a comprises a single reaction bag 70a, which is divided by wall sections 72a, 74a, 76a and 78a into four reaction bag chambers 1_, a, 26a, 38a and 52a. The reaction bag system 10a, as shown in FIG. 1, is suitable for the expansion of anti-tumor cells. However, it goes without saying that it can be designed for a wide variety of other purposes.

The first reaction bag chamber 18a in a reaction sequence has an input opening 16a for external input, into which - as indicated by an arrow 12a - cell populations containing a small number of antitumor cells are inserted. will give. A reagent chamber 80a is formed on an edge section of the first reaction bag chamber 18a. The reagents for the first reaction stage are contained in the reagent chamber 80a. The reagent chamber 80a is separated from the first reaction bag chamber 18a by destructible fluid flow interruption means, such as, for example, a destructible, ie breakable, membrane 81. Either before or after the starting material, for example cells, has been introduced into the first reaction bag chamber 18a, the frangible membrane is broken by the fingers of a user, so that the reagents are pressed into the first reaction bag chamber 18a by wiping them with their fingers and there with the Starting material of the first stage can react. The first reaction stage is again a stimulation stage in which the anti-tumor cells are stimulated to grow by OKT 3 and / or by vesicles.

After the stimulation reaction has been carried out to a sufficient extent, the user breaks a further breakable membrane 82a which is formed in the wall section 72a which separates the first reaction bag chamber 18a from the second reaction bag chamber 26a, ie after opening a dispensing opening Reaction bag chamber 18a provides. The starting material for the second reaction stage, ie the reaction product of the first reaction stage, can be transferred into the second reaction bag chamber 26a by wiping with the fingers. The second reaction bag chamber 26a in turn has a reagent chamber 84a, the structure of which corresponds to the previously described reagent chamber 80a. The reagent chamber 84a again contains, for example, retroviruses which, after being brought into contact with the starting material in the reaction bag chamber 26a, ie the antitumor cells contained therein, initiate a marking process, ie lead to the antitumor cells on the surface Cells a selection marker is stored. After this reaction has been carried out, a further breakable membrane 86a is formed, which is formed in the wall 74a, which separates the second reaction bag chamber 26a from the third reaction bag chamber 38a. The third reaction bag chamber 38a in turn also has a reagent chamber 88a, which contains, for example, biotinylated antibodies for attachment to the 1-NGFR receptor of the antitumor cells. The reagent chamber 88a is also separated from the third reaction bag chamber 38a by a frangible membrane or the like.

If the reaction is carried out in the third reaction bag chamber 38a, the reaction product of the third reaction stage can be fed into a separation column unit 44a via a line 92a via an outlet opening 90a. The anti-tumor cells which have been released from the separation column unit 44a and which are freed from a disturbing background can, as indicated by a dashed arrow 94a, be input directly into a human body, or they can be input into an input opening 98a of a fourth reaction bag chamber 52a, to undergo further expansion there. For this purpose, the reaction bag chamber 52a in turn has a reagent chamber 100a, which contains, for example, lyophilized or otherwise prepared IL 2, which in connection with the antitumor cells obtained from the separation column unit 44a leads to a further growth thereof. Apart from a positive selection, unwanted cells can also be removed from the cell population as part of a negative selection in the column. After this further expansion step has been carried out, the reaction product of the fourth reaction stage can be discharged through a discharge opening 54a and - as indicated by an arrow 56a - can be fed into a human body.

As indicated for the first reaction bag chamber 18a in FIG. 1, it is possible that the different reaction bag chambers have a further input opening 102a through which reagents from an external container, for example a bottle or the like, can be input.

5 Furthermore, as shown in broken lines in FIG. 1, a separation column unit 44a ′ can be integrated directly into the wall section 76a separating the third reaction bag chamber 38a from the fourth reaction bag chamber 52a. In this case, there is no need to remove the reaction product of the third reaction stage from the reaction bag 70a and subsequently to enter it. The risk of contamination can thus be further reduced.

Each of the reaction bag chambers can have a membrane area ιε which can be penetrated through a cannula in order to be able to enter fresh nutrient solutions into the various reaction bag chambers or to be able to withdraw used nutrient solutions, the membrane area again having a fluid after pulling out a cannula from such a membrane area creates a tight seal.

Although not described above, it is also possible to use the entire reaction bag 70a to carry out at least the second reaction stage, i.e. introducing the marker 25 with retroviruses to undergo centrifugation.

The various reaction bag chambers with their wall sections delimiting them can in turn - as described above - by ultrasonic welding, gluing or the like.

30 like. Two - possibly transparent - layers of plastic material are formed. In this process, the various destructible membranes that separate the various chambers from one another and the reagent chambers themselves can be molded on or with the reaction bag 70a

35 integrally formed. In the illustration, the reaction chambers are connected to one another in the manner of a square. However, they can also be connected to one another in a linear, circular or other manner in sequence.

An alternative reaction bag system 10b is shown in FIG. The reaction bag system 10b comprises a reaction bag 70b which is divided into two reaction bag chambers 110b, 112b by wall sections 106b, 108b. The first reaction bag chamber 110b in turn has an input opening 16b, into which - as indicated by the arrow 12b - cells are input as the starting material.

A plurality of reagent chambers 80b, 81b, 84b, 88b are provided on an edge section of the reaction bag chamber 110b, each of which is separated from the reaction bag chamber 110b again by destructible fluid flow interruption means, for example membranes that can be destroyed by finger pressure. After introducing the cell populations containing the antitumor cells into the reaction bag chamber 110b as a starting material for the first stage, the reagents contained in the reagent chamber 80b, for example OKT 3 and / or vesicles, are introduced into the reaction bag chamber 110b and for the reaction brought with the starting material, ie a first growth stimulation is carried out.

After the antitumor cells have multiplied, they remain in the reaction bag chamber 110b as the starting material for the second reaction stage. Then the reagents contained in the second reagent chamber 81b, for example IL 2, that is to say a further growth stimulating agent, are introduced into the reaction bag chamber 110b, and a further growth step is carried out. The reaction product of this stage again remains in the reaction bag chamber 110b, and the reagents are subsequently introduced from the reagent chamber 84b. These are, for example, retroviruses that initiate a labeling step. The reagents of the reagent chamber 88b are subsequently introduced, ie, for example, biotinylated antibodies which attach to the antitumor cells and provide a further label. Cells can also be marked which are to be selectively removed from the cell population. This reaction product obtained is then passed, for example, again by wiping with a user's fingers, through a separation column unit 44b 'integrated in the wall sections 106b, 108b, which forms an outlet opening for the reaction bag chamber 110b, so that anti-tumor cells enriched in the second reaction bag chamber 112b enter. Reagent chambers 100b, 101b are in turn assigned to second reaction bag chamber 112b, which can contain growth-promoting agents, for example IL 2, IL 10, IFNγ or the like, in order to carry out one or more further growth or expansion stages after the separation. After these steps have been carried out, the reaction product, ie the greatly increased antitumor cells, is withdrawn again from the discharge opening 54b and, as indicated by the arrow 56b, introduced into a human body.

In the embodiment of FIG. 2, the reaction bag chambers 110b, 112b each form reaction bag chambers for a plurality of reaction stages to be carried out in succession, the reaction product of a previous stage forming the starting material for the subsequent stage. The construction of the reaction bag is thus simplified since the walls separating the various reaction bag chambers and also destructible fluid flow interrupters or the like provided in these walls can be dispensed with. The reaction bag chambers 110b, 112b of the embodiment in FIG. 2 again have the possibility of entering nutrient solution into this or withdrawing the used nutrient solution. Furthermore, similar to the embodiment according to FIG. 1, provision can be made for additional or additional openings, into which external containers, such as eg Reagent bottles or the like, reagents can be introduced. 1, instead of integrating the separation column unit 44b 'into the wall sections 106b, 108b, the first reaction bag chamber 110b has an outlet opening from which the reaction product of the last stage carried out in this reaction bag chamber is drawn off, is passed through a separation column unit which is arranged externally, and is subsequently introduced into the second reaction bag chamber 112b through an input opening.

Fig. 3 shows a further alternative embodiment of an inventive reaction bag system. Reaction bag system 11Oe in turn comprises a reaction bag 70c with a plurality of reaction bag chambers 114c, 116c, 118c. The reaction bag chamber 114c has an input opening 16c through which starting material can be input into it. Furthermore, the reaction bag chamber 114c is assigned two reagent chambers 80c, 80c 'which can be broken by means of frangible fluid flow interruption means, e.g. breakable membranes 120c, 120c ', can be brought into connection with the reaction bag chamber 114c. The first reaction bag chamber 114c is separated from the second reaction bag chamber 116c which follows this in the reaction sequence by wall sections 122c, 124c which converge essentially in the shape of a funnel. A separation column unit 44c 'is arranged in a fluid transfer area 126c, through which the reaction product of the first reaction bag chamber 114c can be transferred into the second reaction bag chamber 116c. Also assigned to the second reaction bag chamber 116c are two reagent chambers 84c, 84c 'which are arranged essentially opposite one another and which can be brought into connection with the reaction bag chamber 116c via destructible membranes 130c, 130c'.

As can be seen in FIG. 3, two wall sections 132c extending from the walls 122c, 124c are formed in the second reaction bag chamber 116c. The wall sections 132c form together with the wall sections 122c and 124c in a fluid transfer device R substantially V-shaped expanding areas. In these areas, for example, the antitumor cells, which form a starting material, can accumulate together with nutrient solution by the force of gravity during the multiplication, so that due to the V-shaped formation, an increased concentration of antitumor cells is present in a lower region of the respective accumulation areas , which leads to an increased multiplication. Corresponding accumulation areas are formed between the walls 122c, 124c and the reaction bag chamber 116c, the walls 136c, 138c of the reaction bag 70c which are delimited towards the outside.

The second reaction bag chamber 116c is separated from the third reaction bag chamber 118c by wall sections 140c, 142c which run towards a fluid transfer region 144c in a substantially funnel shape.

A separation column unit 44c 'is arranged in this fluid transfer region. The third reaction bag chamber 118c is assigned reagent chambers 88c, 88c ', which can again be brought into connection with the reaction bag chamber 118c via breakable membranes or the like. 146c, 146c'. Furthermore, in the third reaction bag chamber 118c, a plurality of wall sections 132c are again arranged, which together with the walls 142c, 140c form accumulation regions which expand in the fluid transfer device R and in which the starting material for cell multiplication or the like can be accumulated.

The exit from the third reaction bag chamber 118c again forms a separation column unit 44c '.

In the embodiment of FIG. 3, the various reaction bag chambers are - as can be seen in the figure - through

V-shaped converging walls. On the one hand, this has the advantage that for fluid transfer between the individual reaction bag chambers in the fluid transfer direction R a funnel effect of the walls is generated, so that the reaction product generated in one reaction bag chamber can be transferred into the next reaction bag chamber with almost no loss. Furthermore, these walls simultaneously serve to form accumulation areas in the different reaction bag chambers, in which cells or the like are then accumulated in order to be able to carry out reactions in order to be able to provide an increased cell concentration.

As can be seen in Fig. 3, the volume of successive reaction bag chambers 114c, 116c, 118c increases. This is due to the fact that, for example, a predetermined cell concentration must not be exceeded during cell multiplication, since this would hinder further multiplication, i.e. for the different steps in which cell multiplication is to occur, more volume must accordingly be made available for the multiplying cells.

Each of the reaction bag chambers can in turn have penetrable membrane means for the introduction or removal of nutrient solution. In addition, it is possible that, in addition to the provision of respective reagent chambers for the various reaction bag chambers, there are provided entry openings through which reagents can be entered from external containers.

FIG. 3 shows the reaction bag 70c shown there for use in a number of expansion steps to be carried out in succession. The various reagent chambers can thus contain growth-stimulating or inhibiting reagents such as OKT 3, IL 2, IL 10, IL 12, IFNγ TNFo ", GM-CSF or the like. In principle, it is possible in all embodiments according to the invention that such reagents contain growth inhibitors, that inhibit the growth of certain cell types. FIG. 4 shows a modular reaction bag system of the type that can be used, for example, to obtain cell transplants for immunotherapy or gene therapy. In particular, the structure of the reaction bag system 10 of FIG. 4 is described below with reference to the cultivation of antitumor cells.

For example, a cell population, for example a blood sample, is first taken from a human body and contains a small proportion of the antitumor cells to be grown. As indicated by an arrow 12 in FIG. 4, this sample is introduced into a first reaction bag 14 through an input opening 16. In the first reaction bag 14, which forms a stimulation bag, i.e. A bag in which the antitumor cells contained in the blood sample are to be replicated contains a growth stimulating agent for the antitumor cells, for example lyophilized or otherwise prepared OCT 3 and / or the ones described above, before the blood sample is entered Vesicle. The blood sample with the antitumor cells contained therein form the starting material for the reaction step which is to be carried out in the reaction bag 14; the OCT 3 and / or the vesicles with which the first reaction bag 14 is preloaded form the reagents for the reaction of the first stage. In addition, the first reaction bag 14, like the further reaction bags described below, has a membrane in its wall, which can be penetrated, for example, by a cannula, so that in the reaction bag 14, i. nutrient solution can be entered into a reaction bag chamber 18 formed therein, or used nutrient solution can be removed.

After the first stage has been completed, ie the antitumor cells have increased sufficiently in the first reaction bag, they are passed through an output opening 20, for example via a flexible line 22 and an input opening 28, and into a second reaction bag 24. wear. In the second reaction bag, the multiplied antitumor cells are brought into contact with a retrovirus as the reagent in this second stage, in order to treat cells genetically and, if necessary, to initiate a cell marker expression.

In order to achieve an intensified or accelerated reaction of the retroviruses with the antitumor cells in the reaction bag chamber 26 of the second reaction bag 24, this bag can be subjected to a centrifugation process in a centrifuge (not shown in the figures). The content of the second reaction bag chamber 26 is subsequently examined. an output opening 30 of the second reaction bag 24, for example again by means of a flexible line 32, is entered into a third reaction bag 36 via an input opening 34. Antibodies are contained as reagents in the reaction bag chamber 38 of the third reaction bag 36. Using immuno-adsorptive separation columns based on avidin or sreptavidin, these can be, for example, biotinylated antibodies. The contact of the anti-tumor cells in the reaction bag chamber 26 of the second reaction bag 24 e.g. With retroviruses, a receptor for cell separation is expressed on a cell surface of the antitumor cells. This can e.g. be a low affinity NGF receptor. The biotinylated antibodies attach to this receptor in the reaction bag chamber 38 of the third reaction bag 36 and thus lead to an increased labeling effect of the antitumor cells.

The anti-tumor cells marked in this way are introduced via an outlet 40 of the third reaction bag 36 via a line 42 into a separation column unit 44. The separation process carried out in the separation column unit 44 is designed in such a way that the labeled anti-tumor cells can pass through the separation column unit 44 unhindered, whereas unlabeled cells are retained. At the outlet of the separation column unit 44, a Directional valve, not shown, can be arranged, which leads in a first way via a line 46 to an input opening 48 of a fourth reaction bag 50. A further growth stimulating agent, for example lyophilized or otherwise prepared IL 2, is contained in the reaction bag chamber 52 of the fourth reaction bag 50, which leads to a further cell expansion. After cell expansion has been carried out, the antitumor cells can then be obtained through an outlet opening 54 of the fourth reaction bag 50, as indicated by an arrow 56, and can be injected, for example, into a human body.

A second way of the directional valve, which connects to the separation column unit 44, leads via an input opening 60 into a fifth reaction bag 58. The reaction bag 58 can be designed such that it essentially corresponds to the third reaction bag 36, i.e. e.g. contains biotinylated antibodies in order to mark the anti-tumor cells obtained from the separation column unit again in its reaction bag chamber 62 and then to guide them again via an output opening 64 and a line 66 shown in broken lines to the entrance of the separation column unit 44.

The reaction bag system 10 shown in FIG. 4 comprises a plurality of different reaction bags 14, 24, 36, 50, 58, each of which has a reaction bag chamber 18, 26, 38, 52, 62, which contains the reagents for the different stages of the multi-stage reaction are loaded; i.e. the reagents are introduced into the respective reaction bag chambers of the reaction bags during the manufacture thereof, in order then to prepare them for reaction with the starting material which has been introduced into the respective reaction bags. Of course, the system can also comprise only a single such bag for different reactions.

Each of the reaction bags preferably consists of a flexible plastic material or layers of flexible plastic materials which are connected to one another in an edge region forming the various reaction bag chambers by welding, gluing or the like. To observe the reactions, the material of the bags can be transparent at least in some areas. The reaction bags are generally rectangular, but can take any other configuration.

The input and output openings of the various reaction bags can be such that they are already connected to the respective connection tubes between the individual reaction bags or have means for connection to such tubes. Furthermore, in the area of input or Dispensing openings or the connecting hoses can be provided by means of which the input or output openings can each be closed; These can be valves, clamps or the like, for example. In addition, it is possible to design the input or output openings to be weldable, so that, for example, once an initial material has been introduced into a reaction bag, the associated input opening is welded and the further input or escape of material through this input opening is no longer possible. The same applies to the dispensing openings.

The contacting of the respective starting material with the reagents contained in the bags can be carried out, for example, by swiping the fluid-like or liquid starting material forward through the flexible bags with the fingers of a user. Furthermore, it is possible to shake the bags or to centrifuge them, as described above with reference to the second reaction bag 24.

The reagents in the various reaction bags can be contained there in liquid or gelatinous form. In addition, with different reactions, it is possible arrange the reagents in dried form on the inner surface of the reaction bags, so that they are only dissolved when a liquid starting material is added. In particular when using liquid reagents in the various reaction bags, it is important that both the input and the output openings are normally closed and can only be brought into an open state when they are connected to a line hose, a needle or the like. For example, it is possible for membranes to be provided in the area of the input and output openings, which can be penetrated by needles and which produce a tight seal again after the needles have been removed.

It goes without saying that various design features of the various previously described embodiments may be applied to other embodiments. For example, it is possible to construct the reaction bags of FIG. 4 in such a way that they contain several reaction bag chambers or that they have one or more 20 reagent chambers 17, which contain reagents for subsequent contacting with starting materials and from the reaction chambers through destructible membranes 19 o. the like are separated. Furthermore, additional input openings for the input of reagents from the outside can also be provided there.

2S

1, it is also possible to transfer the reaction product of one of the reaction bag chambers to the outside via an outlet opening, to separate them in an externally arranged separation column unit and then to introduce them back into a reaction bag chamber.

In all of the embodiments according to the invention, but particularly in those in which a reaction bag contains a plurality of reaction bag chambers, so that the entire reaction sequence can be practically carried out in one reaction bag, it is possible to work almost contamination-free. By providing different types of reaction bags, which are, for example, pre-loaded with different reagents and / or combined with different reagent chambers, it is possible to provide systems for a multitude of different multi-stage processes or reactions by combining different pre-loaded s or reaction bags combined with reagent chambers. For example, it is also possible to provide a reaction bag with at least one reaction chamber which has a multiplicity of different reagent chambers, but only those reagent chambers which are required for a specific reaction type are then opened for use.

Furthermore, it is also possible that in the embodiment 5 according to FIGS. 1 to 3 at least one of the various reaction bag chambers is already pre-charged with the reagents, as is the case with the embodiment according to FIG. 4.

In addition to the immunosorbent separation shown in the embodiments, the use of an immunomagnetic separation is also possible.

In addition to or instead of the reaction agents described above, the use of other cell-activating and / or cell growth-stimulating substances is also possible.

In all of the embodiments described above, the reagents can also be input through the input openings for the starting material, which are then preferably designed to be closable again.

Claims

claims

1. A reaction bag device for carrying out multistage cultivation / separation processes and / or reactions, for example for growing antitumor cells or cells stimulating antitumor cells, comprising: a plurality of reaction bag chambers (18a, 26a, o 38a, 52a; 110b, 112b; 114c, 116c, 118c) in a reaction bag, wherein in each reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) a starting material for a respective stage can be arranged and with reagents for the respective study 5 fe can be brought into contact to produce a reaction product of the respective stage,

Dispensing opening means (82a, 86a, 54a; 54b; 44c) for dispensing a reaction product from each reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c).

2. Reaction bag device according to claim 1, characterized in that at least one (18a) of the reaction bag chambers (18a, 26a, 38a, 52a) has an input opening (16a) for entering a starting material into it from the outside with respect to the reaction bag and that the reaction bag chambers ( 18a, 26a, 38a, 52a) in sequence for transferring the reaction product of a reaction bag chamber preceding the sequence as a starting material into at least one reaction bag chamber following the sequence.

3. Reaction bag device according to claim 2, characterized in that between at least two sequential reaction bag chambers (18a, 26a, 38a) fluid flow interruption means (82a, 86a) are provided, preferably destructible fluid flow breaking means which form a discharge opening of the reaction bag chamber preceding the sequence.

4. Reaction bag device according to claim 2 or 3, characterized in that at least one (52a) of the reaction bag chambers (18a, 26a, 38a, 52a), preferably the last reaction bag chamber in the sequence _. (52a), has a discharge opening (54a) for discharging the reaction product therefrom with respect to the reaction bag.

5. Reaction bag device according to claim 4, characterized in that in the case of two reaction bag chambers (38a, 52a) which follow one another in sequence, a preceding reaction bag chamber (38a) has an outlet opening (90a) for dispensing to the outside and a subsequent reaction bag chamber (52a) one Has input opening (98a) for input from the outside.

6. Reaction bag device according to one of the preceding claims, characterized in that at least one reaction bag chamber (110b, 112b) is a reaction bag chamber for a plurality of successive stages, the reaction product of a preceding stage in each case forming the starting material for a subsequent stage.

7. Reaction bag device according to one of claims 2 to 6, characterized in that in the sequence successive reaction bag chambers (110b, 112b; 114c, 116c, 118c) are separated from one another by partition walls (106b, 108b; 122c, 124c, 140c, 142c) which run towards a fluid transfer area (126c, 144c) between the successive reaction bag chambers in a fluid transfer direction (R) in an essentially funnel shape.

8. Reaction bag device according to one of the preceding claims, characterized in that in at least one reaction bag chamber (116c, 118c) at least one starting material / reaction product accumulation area is formed by wall sections (132c) which preferably converge in a V-shape.

9. Reaction bag device according to claim 8, characterized in that the at least one starting material / ιo reaction product accumulation area is designed to expand in the fluid transfer direction (R).

10. Reaction bag device according to claim 7 and one of claims 8 or 9, characterized in that the

15 funnel-shaped dividing walls (122c, 124c, 140c, 142c) form the wall sections of the starting material / reaction product - accumulation area at least in regions.

11. Reaction bag device according to one of claims 2 to 10, characterized in that in the sequence successive reaction bag chambers (114c, 116c, 118c) have an increasing chamber volume.

25 12. Reaction bag device according to one of the preceding claims, characterized in that at least one of the reaction bag chambers (18, 26, 38, 52, 62) is preloaded with the reagents.

30 13. Reaction bag device according to one of the preceding

Claims, characterized in that in at least one reaction bag chamber (18a, 26a, 38a, 52a; 110b,

112b; 114c, 116c, 118c) at least one reagent chamber

(80a, 84a, 88a, 100a; 80b, 81b, 84b, 88b, 100b, 101b;

3 80c, 80c ', 84c, 84c', 88c, 88c ') is provided, which is preferably destroyed by apparent fluid flow interruption means (120c, 120c', 130c, 130c ', 146c, 146c'). care fluid flow can be brought into the reagent transmission connection with the reaction bag chamber (18a, 26a, 38a, 52a, 110b, 112b; 114c, 116c, 118c), and that in the reagent chamber (80a, 84a, 88a, 5 100a; 8C-D, 81b, 84b, 88b, 100b, 101b; 8Cc, 80c ', 84c, 84c', 88c, 88c ') the reagents for the respective step are included.

14 reaction bag device according to claim 6 and 13, characterized in that at least one reaction bag chamber (110b, 112b) is provided with a plurality of reagent chambers (80b, 81b, 84b, 88b, 100b, 101b) for different stages, which are used for sequential Carrying out the several stages in succession in reagent transfer

15 can be brought to the reaction bag chamber (110b, 112b).

15 reaction bag device according to one of the preceding claims, characterized in that at least

20 a reaction bag chamber (18a) has a closable reagent delivery opening (102a) for the input of reagents m the reaction bag chamber (18a) is provided from an external container.

25 16 reaction bag device according to one of the preceding claims, characterized in that between at least two reaction bag chambers (38a, 52a; 110b, 112b; 114c, 116c, 118c) which follow one another in sequence, a separation column device (44a, 44a ', 44b';

30 44c ') for fluid transfer therethrough

17. A reaction bag device according to claim 16, characterized in that the separation column device 3s (44a ';44b'; 44c ') m separates the at least two reaction bag chambers (110b; 112b; 114c, 116c, 118c) Wall (76a; 106b, 108b; 122c, 124c, 140c, 142c) is incorporated.

18. Reaction bag device for performing desired multi-stage cultivation / separation processes and / or reactions, for example for growing anti-tumor cells or cells stimulating anti-tumor cells, comprising a reaction bag with a reaction bag chamber (18, 26, 38, 52, 62), in which a starting material for a stage can be arranged and brought into contact with reagents for the stage for producing a reaction product of the stage, the reaction bag (14, 24, 36, 50, 58) having an input opening (16, 28, 34, 48, 60) for feeding raw material into it

15 and a dispensing opening (20, 30, 40, 54, 64) for dispensing a reaction product generated in the reaction bag (14, 24, 36, 50, 58), and wherein the reaction bag device contains the reagents and prepares them with them to form a reaction unit is.

20th

19. Reaction bag device according to claim 18, characterized in that the reaction bag chamber (18, 26, 38, 52, 62) is preloaded with the reagents.

20. Reaction bag device according to claim 18 or 19, characterized in that the reaction bag comprises at least one reagent chamber (17), which by means of apparent fluid flow interrupting means (19), preferably destructible fluid flow interrupting means, in reagents.

30 transfer connection can be brought to the reaction bag chamber, and that the reagents for the respective stage are contained in the at least one reagent chamber.

35 21. Reaction bag device according to one of claims 18 to 20, characterized in that the reaction bag chamber has a reaction bag chamber for a plurality of mutually at subsequent stages, the reaction product of a previous stage forming the starting material for a subsequent stage.

5 22. Reaction bag device according to claim 21, characterized in that the reaction bag comprises a plurality of reagent chambers for different stages, which reagent chambers can be brought into connection with the reaction bag chamber for the sequential implementation of the several stages one after the other in reagent transfer ιo.

23. Reaction bag device according to one of claims 18 to 22, characterized in that in the reaction bag chamber there is further provided a sealable reagent inlet entry for the entry of reagents into the reaction bag chamber from an external container.

24. Reaction bag device according to one of the preceding 20 claims, characterized in that the input and / or the output openings are closed by valves, destructible or reclosable closure means or the like.

25 25. Reaction bag device according to one of the preceding claims, characterized in that it is made of flexible, preferably gas-permeable but liquid-impermeable, plastic.

26. Reaction bag device according to claim 13, 20 or 22 and, if desired, another of the preceding claims, characterized in that the respective reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) came with the at least one reagent - 5 mer (80a, 84a, 88a, 100a; 80b, 81b, 84b, 88b, 100b, 101b; 80c, 80c ', 84c, 84c', 88c, 88c ') is integrally formed.

27. Reaction bag device according to one of the preceding claims, characterized in that the respective reaction bag chamber (18, 26, 38, 52, 62; 18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) for input fresher

5 nutrient solution or is designed to remove used nutrient solution.

28. Reaction bag device according to claim 27, characterized in that the respective reaction bag chamber ιo (18, 26, 38, 52, 62; 18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) one through a cannula or the like has penetrable membrane area, the _. after removal of the cannula automatically restores a fluid-tight seal.

15

29. A modular reaction bag system comprising one or a plurality of reaction bag devices according to any preceding claim.

: o 30. Modular reaction bag system according to claim 29, further comprising at least one separation column unit (44b).

31. Modular reaction bag system according to claim 29 or 30, further comprising at least one container for reagent

25 to input reagents into at least one reaction bag chamber from the outside with respect to a reaction bag.

32. Use of a reaction bag device according to one of claims 1 to 28 and / or a modular reaction bag system according to one of claims 29 to 31 for the cultivation of anti-tumor cells or anti-tumor cell stimulating cells.

3 33. Method for carrying out cultivation / separation processes with one or more stages and / or multi-stage reactions, in particular by means of a reaction 28, and / or a modular reaction bag system according to one of claims 29 to 31, comprising the steps: a) providing at least one reaction bag chamber (18, 26, 38, 52, 62; 18a, 26a, 38a, 52a ; 110b, 112b;

114C, 116c, 118c); b) introducing a starting material into the at least one reaction bag chamber (18, 26, 38, 52, 62; 18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c); c) contacting the starting material in the at least one reaction bag chamber (18, 26, 38, 52, 62; 18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c), ^* with reagents for at least one stage by: - previously loading the at least one reaction bag chamber (18, 26, 38, 52, 62) with the reagents and / or

Bringing into contact one of the at least one reaction bag chamber (18a, 26a, 38a, 52a, 110b, 112b; 114c, 116c, 118c) associated reagent chamber (80a, 84a, 88a, 100a; 80b, 81b, 84b, 88b, 100b, 101b; 80c, 80c ', 84c, 84c', 88c, 88c ') with the reaction bag chamber (18a, 26b, 38a, 52a, 62a; 110b, 112b; 114c, 116c, 118c) and / or - introducing reagents into the at least one reaction bag chamber from an external container through a reagent inlet opening (102a), d) withdrawing the reaction product of one stage from the reaction bag chamber through an outlet opening, if appropriate with previous or subsequent

Perform a separation step.