CA2264206A1

CA2264206A1 - Device with a reaction bag for multi-step culturing/separation operations and/or reactions in several steps

Info

Publication number: CA2264206A1
Application number: CA002264206A
Authority: CA
Inventors: Manfred Kubbies; Stefan Koch; Bernhard Goller
Original assignee: Individual
Current assignee: Roche Diagnostics GmbH
Priority date: 1996-08-16
Filing date: 1997-08-13
Publication date: 1998-02-26
Also published as: JP2000503546A; AU4118497A; JP2000504942A; WO1998007828A1; WO1998007829A2; DE19707497A1; EP0918846A1; CA2264205A1; EP0941307A1; AU4299297A; WO1998007829A8

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

ï»¿CA 02264206 l999-02- 10Reaction bag device for carrying out multistepculture/separation processes and/or reactionsDescriptionThe present invention concerns a reaction bag device forcarrying out multistep culture/separation processesand/or reactions, for example in order to culture anti-tumour cells or cells which stimulate anti-tumour cells.A procedure for treating tumours is to culture anti-tumour cells or cells which stimulate anti-tumour cellsex vivo i.e. outside the human body. After the cultureis completed i.e. after an appropriate intenseproliferation, the anti-tumour cells are then injectedagain into the human body.In order to culture such cells outside the human body ablood sample is taken from a patient in which also asmall number of cells are present which specificallyrespond to a tumour to be treated, the soâcalled anti-tumour cells.These anti-tumour cells that are present in the bloodsample can be stimulated to multiply in a nutrientsolution in a culture vessel by contacting them withwhole tumour cells or soâcalled vesicles of tumourcells.Tumour cells can for example be obtained from the tumourtissue present in a patient. The hereditary informationis removed by lysing the tumour cells and subsequentfiltration so that only the cell membrane or fragmentsï»¿CA 02264206 l999-02- 10thereof remain which form the aforementioned vesicles.These vesicles have the characteristic surface featuresof tumour cells so that they are recognized by anti-tumour cells as tumour cells and lead to a correspondingstimulation of the multiplication of the anti-tumourcells. Since the vesicles only have the surface featuresof the tumour cells and no longer have the hereditaryinformation they cannot influence the proliferatingantiâtumour cells by for example releasing inhibitors.During the stimulation of the growth or multiplicationof the anti-tumour cells by tumour cells or vesicles,the nutrient solution is replaced and the cells aredivided into several culture vessels.If the multiplication has been carried out to anadequate extent, the anti-tumour cells are separatedfrom an undesired background in a selection step. Forthis purpose the anti-tumour cells are for examplelabelled with the aid of lyophilized retroviruses orretroviruses prepared in other ways. These retroviruseslead to the expression of for example the 1-NGFRreceptor on the surface of the anti-tumour cells which,in a subsequent separation step, is used to enrich theanti-tumour cells. After the separation is completedi.e. after enrichment of anti-tumour cells, it ispossible to either subject them to a further separationin order to increase the enrichment rate or to subjectthem to a further growth step for example with additionof a growth-promoting agent.When carrying out such culture/separation processes aswell as other multistep chemical or biologicalreactions, it is important that the individual reactionï»¿CA 02264206 l999-02- 10steps and the transfer of materials between individualreaction steps can be carried out such that, on the onehand, the introduction of contaminants into individualreaction steps is avoided, but, on the other hand, thatit is possible to avoid contamination of the environmentwith reaction products generated in the individualreaction steps. Furthermore a relatively highflexibility in the selection of the various steps isnecessary in order to adapt such multistep processes todiverse requirements in order, for example, to be ableto add additional reaction steps or to omit reactionsteps. For example it is known that reactions can becarried out in reaction bags which have an inlet openingfor a starting material, an inlet opening for thereagents and an outlet opening from which the productproduced by the reaction of the starting material withthe reagents can be removed from the bag. Hence anindividual bag is provided for each reaction, i.e. foreach step, into which the various materials have to beadded or from which materials have to be removed.Especially when adding or removing the respectivematerials there is a risk that the environment becomescontaminated with these materials or that contaminantsare introduced into the material.It is therefore an object of the present invention toprovide a reaction bag device for carrying out multistepculture/separation processes and/or reactions, forexample to culture anti-tumour cells or cells whichstimulate anti-tumour cells which enables an essentiallycontaminationâfree operation with high flexibility withregard to the steps to be carried out.This object is achieved according to the invention by areaction bag device for carrying out multistep culture/ï»¿CA 02264206 l999-02- 10separation processes and/or reactions, for example forthe culture of anti-tumour cells or cells whichstimulate anti-tumour cells, comprising:- a plurality of reaction bag chambers in a reactionbag wherein a starting material for the respectivestep can be located in each reaction bag chamberand can be contacted with reagents for therespective step to produce a reaction product ofthe respective step,- outlet opening means for discharging a reactionproduct from each reaction bag chamber.Hence in the device according to the invention thereactions of the individual steps are carried out in areaction bag containing several reaction bag chambers inwhich, on the one hand, it is possible to arrange astarting material'for the steps in order to carry outthe reaction of the respective step and, on the otherhand, to contact this starting material with a reagentfor the step. Since the reaction therefore can takeplace in a completely closed environment there is norisk of contaminating the environment or of introducingimpurities into the reaction of the respective steps.The device according to the invention is relativelysimple and cheap to produce and provides a great degreeof flexibility in carrying out reactions due to thepossibility of adding or integrating as desiredadditional reaction bag chambers into a process or toremove a reaction bag chamber from a process.The term "starting material" used in the present text isto be understood to include any type of material i.e.cells and other biological or chemical material which itis intended to process, multiply or change in arespective reaction step to form a reaction product ofï»¿CA 02264206 l999-02- 10this step. correspondingly the term "reagents" used inthis text is understood to include any type of materiali.e. biological, chemical or other materials as well ascombinations of various materials or reagents whichinteract with the starting material of the respectivestep and contribute to a further processing,multiplication etc. of the starting material.At least one of the reaction bag chambers can have aninlet opening to introduce a starting material into thisfrom outside with respect to the reaction bag and thereaction bag chambers can be designed as a sequence totransfer the reaction product of a previous reaction bagchamber in the sequence as a starting material into atleast one succeeding reaction bag chamber in thesequence. Since in this embodiment the reaction productis transferred directly between the reaction bagchambers and thus does not have contact with theenvironment, this mode ensures a particularly safe andcontamination/free procedure for the reactions or theculture/separation processes.In order to provide a controlled transfer of reactionproducts or starting materials between the variousreaction bag chambers it is proposed that fluid flowinterruption means that can be opened are providedbetween at least two consecutive reaction bag chambersin the sequence which are preferably destructible fluidflow interruption means that form an outlet opening forthe preceding reaction bag chamber in the sequence.In order to access the reaction product produced aftercarrying out a reaction, it is proposed that at leastone of the reaction bag chambers, preferably the lastï»¿CA 02264206 l999-02- 10reaction bag chamber in the sequence, has an outletopening to discharge the reaction product from this tothe outside with respect to the reaction bag.Furthermore in the case of two consecutive reaction bagchambers in the sequence, it is possible to provide apreceding reaction bag chamber with an outlet openingand a succeeding reaction bag chamber with an inletopening. This is particularly advantageous when thereaction product obtained in the preceding reaction bagchamber is processed further in an external device andsubsequently has to again be introduced into the processcarried out in the reaction bag device i.e. into thefollowing reaction bag chamber.According to one inventive embodiment of the reactionbag device, this can comprise at least one reaction bagchamber which is a reaction bag chamber for a pluralityof consecutive steps in which in each case the reactionproduct of a preceding step forms the starting materialfor a subsequent step. Hence in such a embodiment thematerial to be processed or multiplied etc. remainscontained in the at least one reaction bag chamber sothat also a contamination-free operation is possible.When a reaction bag device is used which containsseveral reaction bag chambers, the liquid, fluid-like orgelatinous material contained in the reaction bagchambers can for example be transported forwards i.e.transported into a next reaction bag chamber by strokingit with the fingers. In order to ensure a very efficienttransfer process without losing any reaction productcontained in a reaction bag chamber it is proposed thatreaction bag chambers that follow one another in theï»¿CA 02264206 l999-02- 10sequence are separated by dividing walls whichessentially funnel together in the direction of fluidtransfer in a liquid transfer region between theconsecutive reaction bag chambers.Especially when using the reaction bag device accordingto the invention to culture anti-tumour cells it isadvantageous when multiplying anti-tumour cells if thestarting material which contains a relatively smallnumber of anti-tumour cells is collected in a relativelysmall volume range since an increase in theconcentration of anti-tumour cells leads to anaccelerated growth. In order to provide such acollection or concentration in at least one reaction bagchamber of the reaction bag device according to theinvention it is proposed that in at least one reactionbag chamber at least one starting material/reactionproduct collection area is formed by wall sections whichrun towards one another preferably in the shape of a"V". In this connection the at least one startingmaterial/reaction product collection area is preferablywidened in the fluid transfer direction.In order to simplify the production of the reaction bagdevice it is proposed that at least partial areas of thepartitions which funnel towards one another form thewall sections of the starting material/reaction productcollection area.If the growth or the multiplication of anti-tumour cellsis carried out in several sequentially arranged reactionbag chambers, for example each with the addition ofvarious growth stimulants, then care must be taken thatthe multiplying anti-tumour cells do not exceed aï»¿CA 02264206 l999-02- 10certain concentration since this would lead to aninhibition of growth or multiplication i.e. as thegrowth process progresses care must be taken that morevolume is available for the proliferating antiâtumourcells. This can be achieved in a simple manner by theconsecutive reaction bag chambers in the sequence havingan increasing chamber volume.In order to be able to contact the starting materialwith the reagents of the respective step in a reactionbag chamber, it is possible according to a particularlysimple and practical embodiment that at least one of thereaction bag chambers is already pre-loaded with thereagents before the starting material is added. Hence insuch a design only the starting material has to be addedwhen it is subsequently used.Furthermore it is possible that at least one reactionbag chamber is provided with at least one reagentchamber which can be brought into reagent transferconnection with the reaction bag chamber by means offluid flow interruption means that can be opened,preferably destructible fluid flow interruption means,and that the reagents for the respective step arecontained in the reagent chamber. In such a design thereagents provided for the respective step can forexample be contacted with the starting material at anydesired time after introduction of the starting materialfor the step.In this connection it is for example possible that atleast one reaction bag chamber is provided with aplurality of reagent chambers for various steps whichcan be successively brought into reagent transferï»¿CA 02264206 l999-02- 10connection with the reaction bag chamber in order tosequentially carry out several steps. Hence thereactions of several successive steps are carried out ina single reaction bag chamber and in each case only thereagents contained in the various reagent chambers aresequentially transferred into the reaction bag chamberaccording to the sequence of the steps i.e. in thisprocess the reaction product generated in a reactionbetween reagents and starting material forms thestarting material for the reaction of the next stepwhich is then contacted with the reagents for the nextstep.Furthermore a tightly closable reagent inlet opening foradding reagents to the reaction bag chamber from anexternal container can be provided in at least onereaction bag chamber.In order to carry out the previously mentionedseparation in the anti-tumour cell culture it ispossible to additionally provide a separation columndevice between at least two consecutive reaction bagchambers in the sequence for fluid transfer throughthis.In a particularly advantageous embodiment of thereaction bag device according to the invention theseparation column device is incorporated into a dividingwall separating at least two reaction bag chambers i.e.even in order to carry out the separation it is notnecessary to convey a reaction product of a step to theoutside, to pass it through a separation column etc. andto again add the concentrated material obtainedtherefrom into a bag for example for further cellï»¿CA 02264206 l999-02- 10-10-multiplication.According to a further aspect of the present invention areaction bag device is provided for carrying out ifdesired multistep culture/separation processes and/orreactions for example to culture anti-tumour cells orcells stimulating anti-tumour cells comprising areaction bag with a reaction bag chamber in which astarting material for a step can be located and can becontacted with reagents for the step in order to producea reaction product of the step, wherein the reaction baghas an inlet opening to introduce starting material intothis as well as an outlet opening to discharge areaction product produced in the reaction bag andwherein the reaction bag device contains the reagentsand together with these is prepared as a reaction unit.In this design of a reaction bag according to theinvention only the starting material has to beintroduced into the reaction bag chamber and can therebe reacted with the reagents that are already kept readyin each reaction bag i.e. the risk of introducingcontaminants via the reagents is also reduced in such areaction bag device.In order to prepare the reaction unit the reaction bagchamber can for example be pre-loaded with the reagents.Alternatively or additionally it is possible that thereaction bag comprises at least one reagent chamberwhich can be brought into reagent transfer connectionwith the reaction bag chamber by means of fluid flowinterruption means that can be opened, preferably adestructible fluid flow interruption means, and that theï»¿CA 02264206 l999-02- 10-11.-reagents for the respective step are contained in the atleast one reagent chamber.According to a particularly preferred design thereaction bag chamber can be designed as a reaction bagchamber for a plurality of consecutive steps in which ineach case the reaction product of a preceding step formsthe starting material for a subsequent step i.e. also insuch a design a plurality of reaction steps can becarried out successively in a reaction bag i.e. in asingle reaction bag chamber without in each case thereaction product having to be discharged from the bag.In this connection it is advantageous if the reactionbag comprises a plurality of reagent chambers forvarious steps and the said reagent chambers can bebrought successively into reagent transfer connectionwith the reaction bag chamber for sequentially carryingout the several steps.In addition it is possible that a tightly closablereagent inlet opening to introduce reagents into thereaction bag chamber from an external container isprovided in the reaction bag chamber. This also allowsmultistep reactions to be carried out in a reaction bagif for example a first reaction is carried out byintroducing a starting material into a, for example pre-loaded reaction bag, the reaction product of this stepis left in the reaction bag chamber and subsequently asecond reaction step is carried out by introducingreagents form an external container.In order to introduce the starting material into therespective reaction bag chamber in the simplest possibleï»¿CA 02264206 l999-02- 10-12-manner and/or to remove starting material from areaction bag chamber it is proposed that the inletand/or outlet openings are closed by valves or bydestructible or reclosable closing devices etc.The reaction bag device according to the invention ispreferably manufactured from flexible, preferably gas-permeable but liquid-impermeable plastic. This, on theone hand, facilitates a particularly simple and cost-effective manufacture and, on the other hand, the riskof damaging such reaction bag devices made of flexibleplastic, for example by breaking, is practically ruledout.Furthermore the at least one reagent chamber ispreferably designed as an integral part of therespective reaction bag chamber.In order to always be able to carry out themultiplication in a suitable environment when thereaction bag device according to the invention is usedfor cell multiplication, it is proposed that therespective reaction bag chamber is constructed for theintroduction of fresh nutrient solution or for theremoval of consumed nutrient solution.For example the respective reaction bag chamber can havea membrane area that can be penetrated by a cannula etc.which, after removal of the cannula, again automaticallyforms a fluid-tight seal.The present invention also concerns a modular reactionbag system comprising one or a plurality of reaction bagdevices according to the invention. Depending on theï»¿CA 02264206 l999-02- 10-13-application the modular reaction bag system can then beassembled from suitably prepared reaction bag devices inwhich each of the reaction bag devices is for exampleloaded with reagents for the desired reaction steps orthese reagents are contained in certain reactionchambers. A suitable bag system can then be assembledfor a certain multistep process to be carried out from acertain number of different reaction bag devices whichonly contains those reaction bag chambers which areactually required for the process.If it is intended to use the modular reaction bag systemfor cell multiplication, for example to multiply anti~tumour cells, then it is advantageous if this systemadditionally contains at least one separation columnunit.In order to be able to introduce reagents into thereaction chambers from outside if desired it is proposedthat in addition at least one container for reagents isprovided in order to introduce reagents into at leastone reaction bag chamber from outside with respect to areaction bag.Furthermore the present invention concerns the use of areaction bag device according to the invention and/or amodular reaction bag system according to the inventionfor culturing anti-tumour cells or cells which stimulateanti-tumour cells.The present invention additionally concerns a processfor carrying out culture/separation processes with oneor several steps and/or multistep reactions for examplein order to culture anti-tumour cells or cellsï»¿CA 02264206 l999-02- 10-14..stimulating antiâtumour cells.The invention is described in detail in the following onthe basis of preferred embodiments with reference to theattached figures.Fig. 1 shows a schematic view of a reaction bagaccording to the invention which contains fourreaction bag chambers which can each besequentially contacted with one another;Fig. 2 shows an alternative embodiment of a reactionbag according to the invention with two reactionbag chambers in which each of the reaction bagchambers can be brought into contact with aplurality of reagent chambers;Fig. 3 shows a further alternative embodiment of areaction bag according to the invention in whichthe junction between two reaction bag chambersthat follow in sequence is formed by V-shapedwalls that run towards one another; andFig. 4 shows a schematic view of a reaction bag systemin which separate reaction bags are each providedwith a reaction bag chamber.Fig. 1 shows a first embodiment of a reaction bag system10a according to the invention. The reaction bag system10a comprises a single reaction bag 70a which is dividedinto four reaction bag chambers 18a, 26a, 38a and 52a bywall sections 72a, 74a, 76a and 78a. The reaction bagsystem 10a as shown in Fig. 1 is suitable for theï»¿CA 02264206 l999-02- 10-15..expansion of antiâtumour cells. However, it is obviousthat it can also be designed for various other purposes.The first reaction bag chamber 18a in a reactionsequence has an inlet opening 16a for introduction fromoutside, into which â as indicated by an arrow 12a -cell populations which contain a low number of anti-tumour cells can be introduced. A reagent chamber 80a isformed onto a peripheral section of the first reactionbag chamber 18a. The reagents for the first reactionstep are contained in the reagent chamber 80a. Thereagent chamber 80a is separated from the first reactionbag chamber 18a by destructible fluid flow interruptionmeans such as e.g. a destructible i.e. breakablemembrane 81. Either before or after the startingmaterial, i.e. e.g. cells, has been introduced into thefirst reaction bag chamber 18a, the breakable membraneis broken by the finger of a user so that the reagentscan be pressed by stroking with the fingers into thefirst reaction bag chamber 18a and can there react withthe starting material of the first step. The firstreaction step is in turn a stimulation step in which theantiâtumour cells are stimulated to grow by OKT 3 and/orby vesicles.After the stimulation reaction has proceeded to anadequate extent, a further breakable membrane 82a whichis formed in the wall section 72a and which separates thefirst reaction bag chamber 18a from the second reactionbag chamber 26a is broken by the user i.e. after openingit provides a discharge opening for the reaction bagchamber 18a. By stroking with the fingers, the startingmaterial for the second reaction step, i.e. the reactionproduct of the first reaction step, can be transferredinto the second reaction bag chamber 26a. The secondï»¿CA 02264206 l999-02- 10-15-reaction bag chamber 26a in turn has a reagent chamber84a whose construction corresponds to the previouslydescribed reagent chamber 80a. Retroviruses are forexample again contained in the reagent chamber 84a which,after being contacted with the starting material in thereaction bag chamber 26a, i.e. the anti-tumour cellswhich are contained therein, initiate a labelling processi.e. result in the incorporation of a selection marker onthe surface of the antiâtumour cells.After this reaction has been carried out, a furtherbreakable membrane 86a is ruptured which is formed inthe wall 74a which separates the second reaction bagchamber 26a from the third reaction bag chamber 38a. Thethird reaction bag chamber 38a also has a reagentchamber 88a which for example contains biotinylatedantibodies for attachment to the 1-NGFR receptor of theantiâtumour cells. The reagent chamber 88a is alsoseparated from the third reaction bag chamber 38a by abreakable membrane or the like.If the reaction in the third reaction bag chamber 38a hasbeen carried out, the reaction product of the thirdreaction step can be introduced via an outlet opening 90avia a tube 92a into a separation column unit 44a. Theanti-tumour cells which are discharged from theseparation column unit 44a which have been freed of aninterfering background can - as indicated by a dashedarrow 94a - be directly introduced into a human body orthey can be introduced into an inlet opening 98a of afourth reaction bag chamber 52a in order to be subjectedthere to a further expansion. For this purpose thereaction bag chamber 52a in turn has a reagent chamber100a which for example contains lyophilized IL2 or IL2prepared in another manner, which together with the anti-ï»¿CA 02264206 l999-02- 10-17..tumour cells obtained from the separation column unit 44aleads to a further growth of the same. Apart from apositive selection, undesired cells can also be removedfrom the cell population within the framework of anegative selection in the column. After carrying out thisfurther expansion step, the reaction product of thefourth reaction step can be discharged through an outletopening 54a and - as indicated by an arrow 56a - beintroduced into a human body.As indicated for the first reaction bag chamber 18a inFig. 1, it is possible that the various reaction bagchambers have an additional inlet opening 102a throughwhich the reagents can be introduced from an externalcontainer such as a bottle.Furthermore, as shown by the dashed line in Fig. 1, aseparation column unit 44aâ can be directly integratedinto the wall section 76a separating the third reactionbag chamber 38a from the fourth reaction bag chamber52a. In this case it is not necessary to remove thereaction product of the third reaction step from thereaction bag 70a and to subsequently introduce it intothis bag. Hence this enables a further reduction of therisk of contamination.Each of the reaction bag chambers can have a membraneregion which can be penetrated by a cannula in order to.be able to add fresh nutrient solutions to the variousreaction bag chambers or to be able to remove consumednutrient solutions wherein after the cannula has beenpulled out from such a membrane region, the membraneregion again forms a fluid-tight seal.ï»¿CA 02264206 l999-02- 10-13-Although not previously described, it is also possibleto subject the entire reaction bag 70a to acentrifugation in order to carry out at least the secondreaction step i.e. initiation of labelling withretroviruses.The various reaction bag chambers with their boundarywall sections can in turn â as described above - beformed by ultrasonic welding, glueing etc. of twooptionally transparent layers of plastic material. Inthis process it is possible to form the variousdestructible membranes which separate the variouschambers from one another as well as the reagentchambers themselves or to form them integrally with thereaction bag 70a.In the figure the reaction chambers are linked togetherlike a square. However, they can be linked together inâsequence in a linear, circular or other manner.Fig. 2 shows an alternative reaction bag system 10b. Thereaction 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 bagchamber 110b in turn has an inlet opening 16b into which- as indicated by the arrow 12b â cells are introducedas the starting material.A plurality of reaction chambers 80b, 81b, 84b, 88b isprovided on a boundary section of the reaction bagchamber 110b which are each in turn separated from thereaction bag chamber 110b by destructible fluid flowinterruption means, for example by membranes that can bedestroyed by finger pressure. After the cell populationsï»¿CA 02264206 l999-02- 10..19._which contain the anti-tumour cells have been passedinto the reaction bag chamber 110b as the startingmaterial for the first step, the reagents which arecontained in the reagent chamber 80b, for example OKT 3and/or vesicles, are passed into the reaction bagchamber 110b and reacted with the starting material i.e.a first growth stimulation is carried out.After completion of the multiplication of the anti-tumour cells, these remain as a starting material forthe second reaction step in the reaction bag chamber110b. Then the reagents contained in the second reagentchamber 81b, for example IL2 i.e. a further growthstimulation agent, are passed into the reaction bagchamber 110b and a further growth step is carried out.The reaction product of this step in turn remains in thereaction bag chamber 110b and subsequently the reagentsfrom the reagent chamber 84b are passed in. These arefor example again retroviruses which initiate alabelling step. Afterwards the reagents from reagentchamber 88b are passed in i.e. e.g. biotinylatedantibodies which attach to the anti-tumour cells andprovide a further label. In addition cells can also belabelled which are to be selectively removed from thecell population. The reaction product obtained is thenfor example by means of wiping with the fingers of auser, passed through a separation column unit 44bâintegrated into the wall sections 106b, 108b which formsan outlet opening for the reaction bag chamber 110b sothat concentrated anti-tumour cells enter the secondreaction bag chamber 112b. Reagent chambers 100b, lolbare in turn integrated within the second reaction bagchamber 112b which can contain growth promoting agentssuch as IL2, IL10, IFNY etc. in order to carry out oneor several additional growth or expansion steps afterï»¿CA 02264206 l999-02- 10_20 -.the separation. After these steps have been carried out,the reaction product i.e. the strongly proliferatedanti-tumour cells, are in turn removed from the outletopening 54b - as indicated by the arrow 56b - andintroduced into a human body.Thus in the embodiment of Fig. 2 the reaction bagchambers 110b, 112b each form reaction bag chambers forseveral reaction steps to be carried out successively inwhich the reaction product of a preceding step forms thestarting material for the subsequent step. Hence theconstruction of the reaction bag is simplified since itis possible to omit the walls separating the variousreaction bag chambers and also the destructible fluidflow interruption means etc. provided in these walls.The reaction bag chambers 110b, 112b of the embodimentof Fig. 2 are also again able to introduce nutrientsolution into these or remove consumed nutrientsolution. Furthermore - like the embodiment of Fig. 1 âadditional inlet openings can be present, into whichreagents can be passed in from external containers suchas reagent bottles etc. in addition to or alternative tothe introduction of reagents from the reagent chambers.Furthermore, on the other hand, instead of integratingthe separation column unit 44bâ into the wall sections106b, 108b, it is possible like the embodiment of Fig. 1that the first reaction bag chamber 110b has an outletopening from which the reaction product of the last stepcarried out in this reaction bag chamber is removed,passed through a separation column unit which isarranged externally and subsequently passed into thesecond reaction bag chamber 112b through an inletopening in this.Fig. 3 shows a further alternative embodiment of aï»¿CA 02264206 l999-02- 10-21-.reaction bag system according to the invention. Thereaction bag system lloe again comprises a reaction bag70c with a plurality of reaction bag chambers 114c, 116c,118c. The reaction bag chamber 114c has an inlet opening16c through which the starting material can be introducedinto this. Furthermore two reagent chambers 80c, 80câ areassigned to the reaction bag chamber 114c which can beconnected with the reaction bag chamber 114c by means ofbreakable fluid flow interruption means e.g. breakablemembranes 120c, 120c'. The first reaction bag chamber114c is separated from the subsequent second reaction bagchamber 116c in the sequence by wall sections 122c, 124cwhich run towards one another substantially in the shapeof a funnel. A separation column unit 44câ is arranged ina fluid transfer region 126c through which the reactionproduct of the first reaction bag chamber 114c can betransferred into the second reaction bag chamber 116c.Again two reagent chambers 84c, 84câ which are arrangedessentially opposite to one another are assigned to thesecond reaction bag chamber 116c which can be connectedto the reaction bag chamber 116C by means of destructiblemembranes 130c, 130c'.As shown in Fig. 3, wall sections 132c are formed in thesecond reaction bag chamber 116c which extend away fromthe walls 122c, 124c. The wall sections 132c togetherwith the wall sections 122C and 1240 form essentially V-shaped collection areas expanding in the fluid transferdirection R. During multiplication, the antiâtumourcells which form a starting material can for exampleaccumulate in these areas together with nutrientsolution by the effect of gravity so that due to the V-shaped design an increased antiâtumour cellconcentration is present in a lower region of therespective collection zones which leads to an increasedï»¿CA 02264206 l999-02- 10-22-multiplication. Corresponding collection zones areformed between the walls 122c, 124c and the walls 136c,138c of the reaction bag 70c which form the outerboundary of the reaction bag chamber 116c.The second reaction bag chamber 116c is separated fromthe third reaction bag chamber 118c by wall sections140c, 142c which essentially funnel towards a fluidtransfer region 144c.A separation column unit 44câ is located in this liquidtransfer region. Reagent chambers 88c, 88câ areallocated to the third reaction bag chamber 118c whichagain can be connected with the reaction bag chamber118c by means of breakable membranes or the like 146c,146c'. Furthermore several wall sections 132c arelocated in the third reaction bag chamber 118c whichtogether with the walls 142c, 140c form collection zoneswhich expand in the fluid transfer direction R in whichthe starting material for cell multiplication etc. canbe collected.A separation column unit 440' again forms the exit fromthe third reaction bag chamber 118c.In the embodiment of Fig. 3 â as shown in the figure -the various reaction bag chambers are separated by wallswhich run towards one another in the shape of a "V". Oneadvantage of this is that a funnel effect of the wallsis produced for the liquid transfer between theindividual reaction bag chambers in the direction offluid transfer R so that the reaction product producedin a reaction bag chamber can be transferred almostwithout loss into the next reaction bag chamber.ï»¿CA 02264206 l999-02- 10-23-.Furthermore these walls at the same time are used toform the collection zones in the various reaction bagchambers in which cells etc. for reactions are collectedin order to provide an elevated cell concentration.As can be seen in Fig. 3 the volume of successivereaction bag chambers 114c, 116c, 118c increases. Thisis due to the fact that for example in cellmultiplication a predetermined cell concentration shouldnot be exceeded since this would impede a furthermultiplication i.e. correspondingly more volume must beprovided for the multiplying cells in the various stepsin which cell multiplication is to occur.Each of the reaction bag chambers can again havepenetrable membrane devices for adding or removingnutrient solution. In addition inlet openings can beprovided through which reagents can be added fromexternal containers in addition to the provision of therespective reagent chambers for the different reactionbag chambers.In the diagram of Fig. 3, the reaction bag 70c shownthere can be used in several expansion steps to becarried out sequentially. Hence the various reagentchambers can contain growth-promoting or growth-inhibiting reagents such as OKT3, IL2, IL10, IL12, IFNy,TNFa, GM-CSF etc. In all embodiments of the invention itis fundamentally possible that such reagents containgrowth inhibitors which inhibit the growth of certaincell types.A modular constructed reaction bag system is shown inFig. 4 which can be used for example to obtain cellï»¿CA 02264206 l999-02- 10-24-transplants for immunotherapy and gene therapy. Theconstruction of the reaction bag system 10 of Fig. 4 isdescribed in the following on the basis of a culture ofanti-tumour cells.Firstly for example a cell population e.g. a bloodsample is collected from a human body which contains alow proportion of the antiâtumour cells to be cultured.This sample is placed in a first reaction bag 14 throughan inlet opening 16 as indicated by an arrow 12 in Fig.4. A growth stimulation agent for the antiâtumour cellssuch as lyophilized or otherwise prepared OKT3 and/orthe previously described vesicles is already present inthe first reaction bag 14 which forms a stimulation bagi.e. a bag in which the anti~tumour cells contained inthe blood sample are to be multiplied. The blood sampletogether with the anti-tumour cells that are containedtherein form the starting material for the reaction stepwhich is to be carried out in the reaction bag 14; theOKT3 and/or the vesicles with which the first reactionbag 14 is pre-loaded form the reagents for the reactionin the first step. In addition the first reaction bag 14as well as the subsequently described other reactionbags has a membrane in its wall which can for example bepenetrated by a cannula so that nutrient solution can beadded to or consumed nutrient solution can be removedfrom the reaction bag 14 i.e. a reaction bag chamber 18integrated into this.After the first step has been completed i.e. the anti-tumour cells have adequately multiplied in the firstreaction bag, these are transferred to the outsidethrough an outlet opening 20 for example via a flexibletube 22 and an inlet opening 28 and passed into a secondreaction bag 24. In the second reaction bag theï»¿CA 02264206 l999-02- 10-25-multiplied anti-tumour cells are contacted with aretrovirus as the reagent in this second step in orderin this way to treat the cells by genetic engineeringand, if necessary, to initiate a cell marker expression.In order to achieve an amplified or accelerated reactionof the retroviruses with the antiâtumour cells in thereaction bag chamber 26 of the second reaction bag 24,this bag can be subjected to a centrifugation process ina centrifuge which is not shown in the figures.Subsequently the contents of the second reaction bagchamber 26 are introduced via the inlet opening 34 intoa third reaction bag 36 through an outlet opening 30 ofthe second reaction bag 24 for example again by means ofa flexible tube 32. Antibodies are present as reagentsin the reaction bag chamber 38 of the third reaction bag36. When using immunoâadsorptive separation columnsbased on avidin or streptavidin, these can for examplebe biotinylated antibodies- The contact between theanti-tumour cells in the reaction bag chamber 26 of thesecond reaction bag 24 with for example retrovirusesleads to the expression of a receptor for cellseparation on a cell surface of the anti-tumour cells.This can for example be a low affinity NGF receptor. Thebiotinylated antibodies attach to this receptor in thereaction bag chamber 38 of the third reaction bag 36 andhence lead to an increased labelling effect of the anti-tumour cells.The antiâtumour cells labelled in this manner areintroduced via a tube 42 into a separation column unit44 via an outlet opening 40 of the third reaction bag36. The separation process carried out in the separationcolumn unit 44 is designed such that the labelled anti-tumour cells can pass unhindered through the separationï»¿CA 02264206 l999-02- 10-26..column unit 44 whereas non-labelled cells are retained.A directional control valve which is not shown can belocated at the outlet of the separation column unit 44which in a first path leads via a tube 46 to an inletopening 48 of a fourth reaction bag 50. A further growthstimulation agent, for example lyophilised or otherwiseprepared IL2 is contained in the reaction bag chamber 52of the fourth reaction bag 50 as a reagent which leadsto a further cell expansion. After the cell expansionhas been carried out, the anti-tumour cells can then beobtained through an outlet opening 54 of the fourthreaction bag 50 as indicated by an arrow 56 and forexample be injected into a human body.A second path of the directional valve which isconnected to the separation column 44 leads via an inletopening 60 into a fifth reaction bag 58. The reactionbag 58 can be designed such that it essentiallycorresponds to the third reaction bag 36 i.e. it forexample contains biotinylated antibodies in order toagain label the anti-tumour cells obtained from theseparation column unit in its reaction bag chamber 62and these are then again conducted via an outlet opening64 and a tube 66 shown as a dashed line to the inlet ofthe separation column unit 44.The reaction bag system 10 shown in Fig. 4 comprises aplurality of different reaction bags 14, 24, 36, 50, 58which each have a reaction bag chamber 18, 26, 38, 52,62 which are pre-loaded with the reagents for thevarious steps of the multistep reaction i.e. alreadyduring manufacture of the reaction bags, reagents areintroduced into their respective reaction bag chambersin order to prepare them for reaction with the startingmaterial introduced into the respective reaction bag. ofï»¿CA 02264206 l999-02- 10-27-course the system can also only comprise a single suchbag for various reactions.Each of the reaction bags is preferably composed of aflexible plastic material or of layers of flexibleplastic material which are bonded together in a boundaryzone forming the various reaction bag chambers bywelding, glueing etc. In order to observe the reactions,the material of the bags can be transparent at least incertain areas. The reaction bags have an essentiallyrectangular shape but can, however, adopt any otherconfigurations.The inlet and outlet openings of the various reactionbags can be such that they are already connected withthe respective connecting tubes between the individualreaction bags or have devices for connection with suchtubes. Furthermore devices can be provided in the areaof the inlet and outlet openings or of the connectingtubes by means of which the inlet and outlet openingscan be respectively closed; these can for example bevalves, clamps and the like. Furthermore the inlet andoutlet openings can be designed to be weldable so thatfor example after a starting material has once beenintroduced into a reaction bag the respective inletopening is welded and hence it is no longer possible formaterial to be introduced or escape from this inletopening. The same applies to the outlet openings.Contacting the respective starting material with thereagents contained in the bags can for example becarried out by stroking the fluid-like or liquidstarting material forwards through the flexible bag bythe fingers of an operator. In addition it is possibleï»¿CA 02264206 l999-02- 10_28...to shake the bag or subject it to a centrifugation aspreviously described with reference to the secondreaction bag 24.The reagents in the various reaction bags can be presentthere in a liquid or gelatinous form. Moreover forvarious reactions it is possible to arrange the reagentsin a dry form on the inner surface of the reaction bagso that they are not dissolved until a liquid startingmaterial has been introduced. Especially when usingliquid reagents in the various reaction bags it isimportant that the inlet as well as the outlet openingsare normally closed and can only be brought into an openstate when they are connected with a lead tube, a needleor the like. For example it is possible in turn toprovide membranes in the area of the inlet and outletopenings which can be penetrated by needles and againform a tight seal after removing the needles.It is obvious that the various design features of thedifferent previously described embodiments can beapplied to other embodiments. Thus for example it ispossible to construct the reaction bags of Fig. 4 insuch a way that they contain several reaction bagchambers or that they have one or several reagentchambers 17 which contain reagents for the subsequentcontact with starting materials and are separated fromthe reaction chambers by destructible membranes 19 orthe like. Furthermore additional inlet openings forintroducing reagents from outside can also be providedthere.Also in the embodiment of Fig. 1 it is possible totransfer the reaction product of one of the reaction bagï»¿CA 02264206 l999-02- 10-29 ..chambers via an outlet opening to the outside, toseparate it in an externally located separation columnunit and then again to pass it into a reaction bagchamber.In all embodiments according to the invention butespecially in those in which a reaction bag containsseveral reaction bag chambers so that the entirereaction sequence can be practically carried out in onereaction bag, it is possible to operate almostcompletely free of contamination.The provision of various types of reaction bags whichfor example are pre-loaded with various reagents and/orare combined with various reagent chambers, enables, bycombining various pre-loaded reaction bags or usingreaction bags combined with reagent chambers, theprovision of systems for numerous different multistepprocesses or reactions. For example it is also possibleto provide a reaction bag with at least one reactionchamber which has a plurality of various reagentchambers in which case only those reagent chambers haveto be opened for use which are required for a certainreaction type.Furthermore it is also possible that in the embodimentsof Fig. 1 to 3 at least one of the various reaction bagchambers is already pre-loaded with the reagents as isthe case in the embodiment of Fig. 4.In addition to the immuno-adsorptive separation shownfor the embodiments, it is also possible to use animmunomagnetic separation.ï»¿CA 02264206 l999-02- 10-30-In addition to or instead of the previously describedreaction agents it is also possible to use other cell-activating or/and cell growth-stimulating substances.In all previously described embodiments the reagents canalso be introduced through the inlet openings for thestarting material which are then preferably designed tobe resealable.

Claims

1. Reaction bag device for carrying out multistep culture/separation processes and/or reactions, for example for the culture of anti-tumour cells or cells which stimulate anti-tumour cells, comprising at least one reaction bag, characterized in that the at least one reaction bag has a plurality of reaction bag chambers (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c 118c), the reaction bag chambers (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) are separated by dividing walls (72a, 74a, 76a, 78a; 106b, 108b; 122c, 124c, 140c, 142c) formed in the at least one reaction bag from adjacent reaction bag chambers (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c), fluid flow interruption means (82a, 86a) that can be opened, preferably destructible fluid flow interruption means (82a, 86a) are provided between two consecutive reaction bag chambers (18a, 26a 38a, 52a; 110b, 112b; 114c, 116c, 118c) in a transfer sequence which are integrated into the dividing wall (72a, 74a, 76a, 78a; 106b, 108b; 122c, 124c, 140c, 142c) separating these two reaction bag chambers (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c), at least one reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) has allocated thereto at least one reagent chamber (80a, 84a, 88a, 110a; 80b, 81b, 84b, 88b, 100b, 101b; 80c, 80c', 84c, 84c', 88c, 88c') containing reagents which is separated from the reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b, 114c, 116c, 118c) by a wall, fluid flow interruption means (81a; 120c, 120c', 130c, 130c', 146c, 146c') preferably destructible fluid flow interruption means (81a;
120c, 120c', 130c, 130c', 146c, 146c') are provided integrated into the wall and a starting material for a reaction step that is to be carried out in this reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) can be located in the at least one reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) and contacted with the reagents contained in the at least one reagent chamber (80a, 84a, 88a, 100a; 80b, 81b, 84b, 88b, 100b, 101b; 80c, 80c', 84c, 84c', 88c, 88c') by opening the fluid flow interruption means (81a; 120c, 120c', 130c, 130c', 146c, 146c') provided in the wall in order to produce a reaction product for this reaction step which can be transferred into a next reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) in the transfer sequence or which can be discharged from the reaction bag chamber (18a, 26a, 28a, 52a; 110b, 112b; 114c, 116c, 118c) of this reaction step to the outside of the reaction bag chamber.

2. Reaction bag device as claimed in claim 1, characterized in that at least one (18a) of the reaction bag chambers (18a, 26a, 38a, 52a) has an inlet opening (16a) to introduce a starting material into this from outside with respect to the reaction bag.

3. Reaction bag device as claimed in claim 2, characterized in that tble openable fluid flow interruption means (82a, 86a) provided between at least two of the reaction bag chambers (18a, 26a, 38a) form an outlet opening for the preceding reaction bag chamber in the sequence.

4. Reaction bag device as claimed in 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 (52a) in the sequence, has an outlet opening (54a) to discharge the reaction product from this to the outside with respect to the reaction bag.

5. Reaction bag device as c:laimed in claim 4, characterized in that in two consecutive reaction bag chambers (38a, 52a) in the sequence, a preceding reaction bag chamber (38a) has an outlet opening (90a) for discharge to the outside and a succeeding reaction bag chamber (52a) has an inlet opening (98a) for introcluction from the outside.

6. Reaction bag device as claimed in one of the previous claims, characterized in that at least one reaction bag chamber (110b, 112b) is a reaction bag chamber for a plurality of successive steps in which in each case the reaction product of a preceding step forms the starting material for a subsequent step.

7. Reaction bag device as claimed in one of the claims 1 to 6, characterized in that consecutive reaction bag chambers (110b, 112b; 114c, 116c, 118c) in the sequence are separated from one another by dividing walls (106b, 108b; 122c, 124c, 140c, 142c) which essentially funnel together in the direction of fluid transfer (R) in a liquid transfer region (126c, 144c) between the consecutive reaction bag chambers.

8. Reaction bag device as claimed in one of the previous claims, characterized in that in at least one reaction bag chamber (116c, 118c) at least one starting material/reaction product collection area is formed by wall sections (132c) which run towards one another preferably in the shape of a "V".

9. Reaction bag device as claimed in claim 8, characterized in that the at least one starting material/reaction product collection area widens in the fluid transfer direction (R).

10. Reaction bag device as claimed in claim 7 and one of the claims 8 or 9, characterized in that at least partial areas of the dividing walls which funnel towards one another (122c, 124c, 140c, 142c) form the wall sections of the starting material/
reaction product collection area.

11. Reaction bag device as claimed in one of the claims 1 to 10, characterized in that the successive reaction bag chambers in the sequence (114c, 116c, 118c) have an increasing chamber volume.

12. Reaction bag device as claimed in one of the previous claims, characterized in that at least one of the reaction bag chambers (18, 26, 38, 52, 62) is pre-loaded with the reagents.

13. Reaction bag device as claimed in claim 6 or one of the claims 7 - 12 if they refer to claim 6, 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 various steps which can be successively brought into reagent transfer connection with the reaction bag chamber (110b, 112b) in order to sequentially carry out several steps.

14. Reaction bag device as claimed in one of the previous claims, characterized in that a tightly closable reagent inlet opening (102a) for adding reagents to the reaction bag chamber (18a) from an external container is provided in at least one reaction bag chamber (18a).

15. Reaction bag device as claimed in one of the previous claims, characterized in that a separation column device (44a, 44a'; 44b'; 44c') is provided between at least two consecutive reaction bag chambers (38a, 52a; 110b, 112b; 114c, 116c, 118c) in the sequence for fluid transfer through this.

16. Reaction bag device as claimed in claim 15, characterized in that the separation column device (44a'; 44b'; 44c') is incorporated into a dividing wall (76a; 106b, 108b; 122c, 124c, 140c, 142c) separating at least two reaction bag chambers (110b; 112b; 114c, 116c, 118c).

17. Reaction bag device as claimed in one of the previous claims, characterized in that the inlet and/or outlet openings are closed by valves or by destructible or reclosable closing devices or such like.

18. Reaction bag device as claimed in one of the previous claims, characterized in that they are manufactured from flexible, preferably gas-permeable but liquid-impermeable plastic.

19. Reaction bag device as claimed in one of the previous claims, characterized in that the at least one reagent chamber (80a, 84a, 88a, 100a; 80b, 81b, 84b, 88b, 1000b, 101b; 80c, 80c', 84c, 84c', 88c, 88c') is formed as an integral part of the respective reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c).

20. Reaction bag device as claimed in one of the previous claims, characterized in that the respective reaction bag chamber (18, 26, 38, 52, 62; 18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) is designed for the introduction of fresh nutrient solution or for the removal of consumed nutrient solution.

21. Reaction bag device as claimed in claim 20, characterized in that the respective reaction bag chamber (18, 26, 38, 52, 62; 18a, 26a, 38a, 52a;
110b, 112b; 114c, 116c, 118c) has a membrane area that can be penetrated by a cannula or such like which, after removal of the cannula, again automatically forms a fluid-tight seal.

22. Modular reaction bag system comprising one or a plurality of reaction bag devices as claimed in one of the previous claims.

23. Modular reaction bag system as claimed in claim 22, additionally comprising at least one separation column unit (44b).

24. Modular reaction bag system as claimed in claim 22 or 23, additionally comprising at least one container for reagents in order to introduce reagents into at least one reaction bag chamber from outside with respect to a reaction bag.

25. Use of a reaction bag device as claimed in one of the claims 1 to 21 and/or a modular reaction bag system as claimed in one of the claims 22 to 24 for culturing anti-tumour cells or cells which stimulate anti-tumour cells.

26. Process for carrying out culture/separation processes with one or several steps and/or multistep reactions preferably by means of a reaction bag device as claimed in one of the claims 1 to 21 or/and a modular reaction bag system as claimed in one of the claims 22 to 24, comprising the steps a) preparing at least one reaction bag with a plurality of reaction bag chambers (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c);
b) introducing a starting material into one of the reaction bag chambers (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c);
c) contacting the starting material in the one reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) with reagents for at least one step by making a fluid connection between the one reaction bag chamber and at least one reagent chamber (80a, 84a, 88a, 100a; 80b, 81b, 84b, 88b, 100b, 101b; 80c, 80c', 84c, 84c', 88c, 88c') allocated to the one reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b;
114c, 116c, 118c) by opening openable fluid flow interruption means provided in a wall separating the one reaction bag chamber (18a, 26a, 38a, 52a;
110b, 112b, 114c, 116c, 118c) from the at least one reagent chamber (80a, 84a, 88a, 100a; 80b, 81b, 84b, 88b, 100b, 101b; 80c, 80c', 84c, 84c', 88c, 88c') d) after carrying out t:he reaction of the starting material with the reagents, transferring the reaction product obt:ained into a reaction bag chamber (18a, 26a, 38a, 52a; 110b, 112b; 114c, 116c, 118c) which follows directly in the transfer sequence by opening openable fluid flow interruption means provided in a dividing wall (72a, 74a, 76a, 78a; 106b, 108b; 122c, 124c, 140c, 142c) which separates the two reaction bag chambers or discharging the reaction product to the outside of the reaction bag.