DE19822050A1 - Chemical or biological reactions on surface of particles, retained in container permeable to reaction fluid, especially for growing cells - Google Patents

Abstract

In a process for performing chemical or biological reactions in a vessel, fitted with a stirrer and filled with liquid medium in contact with solid particles on which the react occurs, the new feature is that the particles are enclosed in a container permeable to reaction medium. An Independent claim is also included for a container for retaining solid particles, consisting of a cylindrical casing, a sieve at the base of the cylinder and auxiliary elements that define a free space between the base of the reaction vessel and the underside of the container.

Description

The invention relates to a method for carrying out chemical or biological Reactions, which is carried out in a chamber, which with a stirring device is equipped and has a liquid medium which contains solid particles. The The invention also relates to a container for carrying out the method.

Process which takes place in a liquid medium with solid particles, which is in a Chamber located with stirring device are done in the art fairly common. Since then, such methods have been preferred which are in a completely homogeneous System can run such. B. in one of the starting products and the end products in liquid containing dissolved form, because in such a homogeneous system the constancy and uniformity of the reaction conditions in the reaction system easiest are to be controlled and controlled. In many reactions, however, products have to are used, the solubility under the conditions of the reaction Pose problem. Many larger molecules such as B. protein molecules or antibodies The examiner often experiences such problems, while reactions that are not in a cell-free but must be carried out in a system containing cells, naturally also there is no homogeneous reaction system.

There are several practical ways available to deal with insoluble reactions Components. So it is possible the insoluble components in the liquid to float with practically a suspension. The need is there plan to make constant movement of the liquid, as an insufficient one Movement can lead to a gradient of any ingredients in the liquid, such as a gradient caused by diffusion processes of soluble components or one gravitational gradient of the insoluble components in the liquid. With completely insufficient stirring conditions, the particles will even settle on the bottom of the Put down the reaction vessel, which leads to a completely inadequate control. This Procedures, in particular the presence of a stirring device, often lead to undesirable side effects.  

There are some reactions which place extremely high demands on the reaction conditions and especially the insoluble components in the system remain relatively undisturbed have to. Even small external forces lead to considerable defects in the Reaction event. It is obvious that the shear forces which arise during a stirring process such external forces can already be expected, let alone the direct contact between the stirrer and the insoluble one Components can occur because this contact can be sensitive, insoluble Components regularly lead to destruction.

Another disadvantage is sometimes the fact that at the end of the reaction a Separation of the components must be made. It is in the nature of things that a separation must be made between the starting products and the end product, because usually a pure end product is required and it is also required in in cases where there is no single product but a mixture of different products arises here, too, to carry out a purification, again a pure result End product is available. In processes where insoluble components play a role, it is necessary to make a separation of dissolved and undissolved products. Although As is known, the filtration technology has always been used technically and the specialist A number of useful techniques are available, the disadvantages of which are Implementation can occur, varied and also known to those skilled in the art. Also the The simplest filtration process represents an extra step in the process, which leads to losses Time and material leads.

A special way of carrying out a reaction with a system which contains particles comprises a liquid medium, relates to the cultivation of human cells. Here the cells are kept in a chamber which contains a growth medium and with is equipped with a stirring device, and are located on a carrier material.

There are many reasons for human cells in research and therapeutic medicine to keep alive or even breed outside of the human body, taking this breeding means an increase in human cells under laboratory conditions.  

There are e.g. B. Specific requirements from human therapy where bone marrow or peripheral bone marrow stem cells derived from blood are required:

• (1) In autologous bone marrow transplantation, a patient receives bone marrow removed and it him as part of a later therapy or z. B. after reimplanted chemotherapy treatment,
• (2) in diseases of blood cells one could heal such cells outside the Body to reimplant the healed cells, one such targeted treatment would come e.g. B. in leukemia,
• (3) Allogeneic bone marrow transplantation becomes the bone marrow of a healthy donor implanted in a leukemia patient's own bone marrow instead.

In the examples listed above, the cause of the suffering is a disorder on the Level of the bone marrow and therefore treatment with bone marrow cells is a causal therapy of the disease.

A common option with regard to the implantation of a bone marrow mixture is the Implantation of so-called stem cells. Such stem cells are the precursors all blood cells and are found in the bone marrow and peripheral blood. The very fact that stem cells are found in the blood makes them one attractive option, since a less stressful extraction is possible and through a targeted extraction of the desired stem cells from the donor, no loss of others Blood cells entering.

Thus, the stem cell is valuable for many applications in human medicine Source material for causal therapy viewed and has accordingly many Efforts have been made to keep stem cells alive as long as possible after removal and to develop a method to proliferate the stem cells in vitro, thus for the Therapy is an unlimited or at least a large source of homogeneous material To have available.

What has been described above as an example for the stem cell naturally applies equally for other cell types that can be considered for causal therapy. Here is z. B. to think of myocardial cells, brain cells, eye cells, liver cells,  Kidney cells, etc. The need does not have to be limited to the human area the same requirements can arise in the veterinary field or in As part of a targeted development of plant variety protection in the area of developing new ones Plant breeding.

A major problem in many development work is the fact that Many types of cells have a pronounced sensitivity. This sensitivity already prevents sensible handling under pure housing conditions and also has as a result that necessary investigations to optimize posture and Propagation conditions can not be carried out.

In particular also in the case of hematopoietic cells including stem cells practical problems in the development of new therapeutic approaches significantly. The Lines show a particularly high sensitivity to any deviation from the ideal situation, with even low mechanical loads leading to deterioration cell survival rate. This is certainly partly the case with the cells of this cell type attributable to the fact that they have no cell wall and for that reason withstand mechanical loads less well. Already a relatively small one Shear stress is sufficient to cause high mortality in these cells.

Previous approaches have not sufficiently addressed the high sensitivity problem to be able to solve.

In the application P 195 05 109.2 a method is described, human or animal Mixed cell populations in a fluidized bed reactor on microporous microcarriers to settle. In this prior art method, the microcarriers suspended in a growth medium, the flow rate of the Growth medium is kept low to avoid possible losses.

Despite its advantages, the process has not been able to fully solve the problems. It was with certain cell types, especially those that are extremely valuable for research and therapy Stem cells, not possible to drive under routine laboratory conditions, the Cells survived to the desired extent and proliferation could occur among them Conditions are not found at all.  

Another problem with research efforts of this type is that the amount of available material is low. It is the donors, no matter whether patient or subject expected, often taking large amounts of blood or bone marrow material have to tolerate and consequently in the examinations with a small amount Starting material are worked, which are only partially filled from the same source can. This is particularly troublesome in the phase where the research is currently since the work required to optimize many process parameters is only within the scope of an extensive screening program can be meaningfully brought to a conclusion. The availability of a larger quantity is a prerequisite for such a screening program of a homogeneous starting material.

The invention was based on the object of a method for carrying out reactions which are involved in solid particles in a liquid medium is easy to handle and practically easy to carry out, and in particular such Conditions exist in the system that are relatively complex and lead to losses Filtration process can be carried out in a simpler manner. Through the process should improve the cultivation of human or animal cells possible be, the cultivation is carried out under particularly gentle conditions. It is a particular goal to provide a method, with little Amounts of material can be worked on.

The task is accomplished by a process with the characteristic features of the Main claim solved.

It thus relates to a method for carrying out chemical or biological Reactions which are carried out in a liquid medium containing solid particles. The chemical and biological reactions initially include the usual textbook chemistry where from one or more starting products one or more end products End products are formed. It is possible within the scope of the invention that a Starting product or several starting products are presented as solid particles, it is also conceivable that it is not the starting products but the one required for the reaction Catalyst is present as a solid particle or bound to solid particles. The  Be a catalyst of an inorganic nature or very essential in the context of the invention Be enzyme. Enzymes in particular are particularly suitable as high-molecular products for Implementation of the method according to the invention. The enzymes can be used as a single product are used or are present coupled to any carrier material. especially the latter application where the enzyme by means of a chemical reaction or a other mechanism attached to neutral reaction particles, provides in the context of Invention represents a particularly attractive procedure. This is just as feasible Methods for biological reactions which are carried out with the help of an antibody reaction become. The antibody can z. B. as an aid in purification or for a any other use can be used.

The reaction is carried out in a chamber with a stirring device Is provided. The special shape of the chamber is not important, it is meant just any geometry that is capable of containing a liquid. Which Form the chamber has, of what material it is made of, whether it is lockable or is open whether it is equipped with certain supply or discharge lines, whether it what volume of liquid they absorb depends on depending on the type of chemical or biological reaction targeted. In the context of the method according to the invention, it should in no way be ruled out that that it is carried out as part of an industrial production. Also at The process according to the invention can be used to produce on a larger scale find desirable application, perhaps even specifically in such a larger one Benchmark, since it is downright simplification and especially in an industrial application Savings arrive and a relatively small advantage in the industrial environment have a significant impact.

Nevertheless, the primary focus of the inventors is to provide a method that can be used on a laboratory scale and optimally unfolds its advantages where the Examiner who uses the method according to the invention is also forced to do so extremely small amounts of starting material must work.

This primary focus has prompted the inventors to develop the method specifically for such To provide reaction conditions, the amounts of material are extremely small and the  Investigators are constantly keen on the conditions of reaction to the low Align the amount of material.

The problem of the small amount of starting material is e.g. B. encountered when it is a method of culturing human or animal cells such as B. affects hematopoietic stem cells, as is subsequently preferred Embodiment is shown in more detail.

It is therefore a preferred embodiment of the method according to the invention that the chamber which is intended to carry out the method has a volume of less than 20 ml. A reaction volume of 0.37 to 16.8 ml is preferred, particularly a volume of about 6 ml is preferred. Such a volume is also practical for Attempt where only the smallest amounts of starting material are available, on the other hand, allows one such volume a routine operation. As will be explained later, it is one of the attractive options in a preferred embodiment of the inventive method the inventive method for screening purposes in Specialized investigations to optimize growth conditions for use human blood cells.

As later on based on the description of concrete test series is shown in more detail, it is another particularly preferred embodiment of the method according to the invention as a chamber for carrying out the reaction or cutout of a tissue culture plate.

Such a designation does not automatically define the geometry of a such deepening, it is by no means necessary to select a specific format, since the implementation of the method according to the invention basically with practically everyone Type of deepening is feasible. On the other hand, it is a preferred embodiment the method of taking tissue culture plates which are commercially available. Very good The so-called "Corning® Multiple well plates" from Corning Costar can be used Corporation (Cambridge, MA 02140): Such plates have been used in the biological Research is concerned about its favorable properties and excellent position also represent a preferred working material in the context of this invention. "Corning® Muitiplewell plates "are plates with a different number of wells (also called  referred to as "wells"). Which embodiment when performing the method is chosen primarily by the amount of material available and the Purpose of the test series determined. In principle, the method according to the invention can also be used any available embodiment of the "Corning® Multiple well plates".

The process according to the invention is carried out in a liquid medium. The medium can have any conceivable composition. Here are the theoretical ones There are no limits to the possibilities. Every conceivable possibility varies from one chemical processes in an organic solvent to a biological technique The use of an aqueous buffer solution can be considered, however It should be noted at all times that the selected liquid medium coincides with the others Components in the process tolerated.

The solid particles are not bound to any particular embodiment. Also in there are any number of possibilities with regard to these solid particles. Only one The condition for carrying out the method according to the invention is that the solid particles are not soluble in the selected liquid medium. Otherwise is with the material of the particles and their exact geometry only on their purpose in Procedure to be followed. In the context of the invention, it will certainly be a preferred variant represent starting from more or less spherical particles. These offer one relatively large surface area, can be stacked and allowed in a package nevertheless an undisturbed flow of liquid medium.

As will be discussed later in the context of the embodiment, it is a preferred one Embodiment of the method according to the invention, it as part of the cultivation of to use human body cells. Certain attempts have been made in such attempts Particles shown to be extremely useful. Porous are particularly useful Vertebral bodies with a diameter of 200-1500 µm and a density of at least 1.1 kg / l, whose Pores are filled with medium.

First, favorable experiences were made with Siran® balls (Schott company, Mainz). Such Siran® spheres are porous glass carriers with about 50% porosity and a grain size from 200-1500 µm, preferably from 400-700 µm. The glass supports can be used  pretreated with a diluted gelatin solution. The Siran® balls have repeatedly tried their way through experiments with human or animal cells excellent properties in terms of adhesion and stability.

Another variant which is also preferred in the context of investigations with Cell material is used as collagen microcarriers. These carriers are also in various sizes and designs available. Collagen microcarriers show similarly good results Properties in the experiment have a special property compared to the glass spheres on; which can give them a decisive advantage depending on their needs. It is coming namely, often when using particles with attached components that it is asked for; recover these components at the end of the reaction; with the aim of renewed use or subsequent processing. It can be about chemical molecules such as B. act enzymes or antibodies; this desire is common also in experiments which are carried out with cells, such as when the Particles have been used to cultivate human or animal cells accomplish. In such cultivations, the grown cells serve the purpose of Attempt and it is therefore inevitable they from the particles such. B. the Siran® Loosen balls or the collagen microcarriers. Then the advantage of last-mentioned particles, because the collagen microcarrier is simply a collagenase containing system, with the result that the carrier dissolve and the Cells which are not attacked by the enzyme are isolated from the solution can.

It is a crucial property of the present invention that the solid particles do not float freely in the liquid medium, as is customary in the prior art, but instead be enclosed by a container which is in the chamber in the liquid medium located. This encompassing is to be understood as a separate room in the chamber is provided and the particles only within this space and not at all be found outside the room.

The container can take any conceivable shape and can be designed differently. Good a container that is completely lockable and either floating in the medium or is conceivable is almost set up. Such a solution is e.g. B. recommended if there are solid particles  relates to which have a very low density or to each other a very low Show adhesion. With such a constellation there would be a risk that the effect the stirring device also provided by the method according to the invention causes such action of the liquid medium; that the solid particles are suspended and get into the chamber outside the container. It is absolutely undesirable, because it is precisely one of the advantages of the method that the the solid particle container as a feature of the method a very simple Handling of this procedure allowed. It can be brought into the chamber at will and removed from it, which allows optimal control of the process. At the end of the method, the container can be removed completely from the chamber, the by the Contained particles immediately available for other use and this eliminates the need for a complicated separation or isolation process.

In return for the variant of the container just described, this being complete lockable, there is the other variant whereby this complete locking is not he follows. In many reactions, the solid particles will have such a density or have such a connection with one another; that the closing can be omitted because it practically no unwanted suspension of the particles can occur. In In such cases, the variant of the non-lockable container is of course to be preferred, because it will generally be a simpler and less expensive design, and probably also requires fewer actions by the user, because of course that Locking and opening again is not necessary. There are those that cannot be locked Containers still the possibility; that they are a compared to the dimension of the chamber have such construction; that its opening protrudes from the chamber or itself located at least above the surface of the liquid medium as well as the alternative Possibility that the container as a whole is arranged in the liquid medium and the Accordingly, the container opens below the surface of the liquid Medium is located.

Crucial for the functioning of the container in the sense of the invention is the fact that it is permeable to the liquid medium. This means that the container is one Construction; that a free and undisturbed exchange of fluid within and outside the container is possible. The use of a semipermeable is conceivable  Membrane as a complete or partial shell of the container, whereby a free Exchange of molecules up to a certain size can be done. Such one Semipermeable membrane can be beneficial if it is very small and difficult to handle solid particles. Usually, however, such an embodiment is not used, but one where the container has openings; with such a size; that she are not passable for the solid particles. In theory, the container can be a single one Have opening; Usually, however, there will be more because for most chemical and biological reactions is required that the individual components of the Reaction can come into intensive contact with each other, and this intense Contact with only one opening is insufficient. It represents a preferred one Embodiment of the method according to the invention, a plurality of individual Provide openings in the container. This can be done e.g. B. realize very well that one installs a sieve in the container or the container has such a construction that it is wholly or partly constructed from a sieve material. There will be another one below Aspect of the invention will be described; being a specific container available is provided, which due to its simple and practical construction, is ideal for Use in the inventive method is suitable.

It was mentioned in the introductory remarks how harmful in many systems can be a working stirrer. It is obvious that it is one of the Greatest advantages of the method according to the invention is that one very easily spatial separation of the moving parts of the stirring device and the solid particles can reach. The stirring device, which can have any conceivable design, at some point in the chamber sets the liquid medium in motion. Also in the Chamber contains the container that contains the solid particles. It is completely clear that the stirring device is arranged such that its moving parts only with can come into contact with the liquid medium outside the container. This Alignment thus completely prevents the disadvantages described above that the Stirrer leads to significant system malfunctions and either damage or even destruction of the solid particles themselves or impairment of liability of molecules or cells on the solid particles. Every stirring method leads to Movement and can theoretically lead to a disturbance in the system, but it is one of the  great advantages of the method according to the invention, that with the simplest stirring devices a very gentle implementation of a reaction is possible without special effort.

The stirring device according to the inventive method can be as above executed have any shape. However, one is particularly preferred Stirring device with a magnetic stirrer, especially if according to a particular preferred embodiment is operated with a tissue culture plate and more preferred if one in several wells of the tissue culture plate at the same time Reaction takes place, which is one of the attractive possibilities of the invention Represents procedure. Use of a magnetic stirrer with appropriate use of a Multiple full magnetic stirrers in each well where a reaction is taking place certainly the simplest and least complex way of doing the method according to the invention according to the particularly preferred embodiment with a tissue culture plate perform. The tissue culture plate is placed on a multi-position magnetic stirrer positioned, whereby all magnetic stir bars at the same time with a magnetic stirrer can be controlled.

It represents a further variant of the method according to the invention, a temperature control to be provided if it is in the process at a specific temperature that is not compatible with the Ambient temperature or matches the temperature in the incubator. It has also sometimes been shown if according to that described in the previous paragraph Embodiment is worked with a magnetic stirrer that carrying out the reaction leads to undesirable local heating with such a magnetic stirrer. This Of course, heating is most noticeable when the reaction is prolonged Time is running out. Longer response times are e.g. B. in reactions with human or animal cells; like cultivation attempts with such cells, not uncommon. A Response times of more than 24 hours are completely normal. Because the heating too Temperatures in the liquid medium of over 50 ° C, it was inevitable Provision should be made for cooling, especially for the implementation of biological Reactions a deviation from the optimum temperature very quickly to one Deterioration of the reaction processes and when carrying out reactions with human or animal cells can even endanger the survival of this cell material. It is therefore a particularly preferred embodiment when performing the  Process using a magnetic stirrer cooling in the process to provide, this cooling is practically the easiest if between Magnetic stirrer and tissue culture plate, a cooling device is arranged. This Cooling device can be designed as a fairly flat reservoir, which is permanently with Water flows through at 36 ° C. Such a simple form is sufficient, to prevent heating of the reaction mixture in the chamber.

It has already been stated several times in the description of the method according to the invention, that it is easily possible according to preferred embodiments, various To run reaction approaches in parallel. This possibility is very concrete if Tissue culture plates can be used as it is beneficial in this application To allow reaction to take place in a plurality of individual wells, with it It is of course fundamentally irrelevant whether there are several identical approaches in parallel run like B. in multiple experiments or if by variations in the Reaction parameters in each well a very specific reaction takes place. It is however, one of the crucial advantages of the method according to the invention is that the method can be carried out in a small volume. This is always very large Advantage if the material quantities were not sufficient to run larger batches. Even with small amounts of starting material, e.g. B. if the starting material from a The method according to the invention offers the possibility of procuring humans or an animal Possibility, especially if the preferred variant with the tissue culture plate Small volume wells are used to handle a larger number of individual reactions to run in parallel. This also represents one of the very attractive aspects of the method according to the invention, since this parallel implementation of a larger Trial series allowed a screening program on a larger scale. Such Screening programs are used in studies related to human or animal Cell material is often required, roughly in connection with the question of how certain Cell types can be cultivated. Such investigations where a large number of individuals Parameters can only be varied through extensive screening programs in Be tackled. The method according to the invention is almost predestined for to be used in such screening programs. It is therefore another aspect of the Invention the method according to the invention in the context of the implementation of Use screeeing programs.  

The use of the method according to the invention in the course of the investigations with human or animal cells is not limited to any cell type. Basically all cell types can be used. It is with the invention includes that the cells themselves are solid particles in the sense of the main claim and thus there is a cell culture inside the container. However, such an application comes for many Cell types are out of the question because they cannot survive without a carrier. For such cells are preferred methods which settle the cells under investigation provide for any supports, such as B. the previously discussed Siran® Particles or on collagen microcarriers. It is with such carriers and with other types of Carriers quite possible to put the examined cells on the carriers themselves, because Cells have their own adhesive ability. It is just as possible for liability Tools such as B. Adhesion molecules used.

It is another often-practiced way to use cells in a cell, not alone Bring coculture with another cell type or several other cell types on carrier. Such a combination of different cell types is called a coculture. A Co-culture can have many advantages. First, it is possible that a simultaneous or previously there was adhesion with certain cells, the cell type actually examined one provides a better basis for liability, with the result that the liability of the investigated Cells in a coculture work cheaper because there is simply a better basis. A Another aspect can be the fact that the other cell type has its own influence on the Exerts reaction conditions, such an influence being very specific as production and excretion in the liquid medium of substances involved in metabolism or growth of the cells examined can play a role.

The possibility of using a coculture of different cell types will be examined within the framework of the Invention z. B. used when there are studies related to human plasma cells concerns. Earlier studies have shown that coculture with Stromal cells or fibroblasts is of great advantage, with the importance of the stromal cells for the culture of the stem cells is that the colonized with the stromal cells Particles give the plasma cells improved mechanical stability and the Stromal cells are also believed to improve cytokine production  Functionality of stem cells contribute. It must also be considered in principle that even in the natural situation in the body the cells are not completely isolated and independent of other cells, but always in constant contact and exchange with any other cells. Co-cultivation with other cells is therefore one better approximation of the natural position and the resulting structure is the Bone marrow situation better mimicized. It is therefore expressly an aspect of the present invention, the cells examined together with other cells on the Immobilize carrier material, especially human stem cells together with Fix stromal cells to the solid support.

It is possible to bring the particles into the container untreated and the Make manipulations completely in the container. It is on the other side also possible to treat the particles beforehand and to use them as bring treated particles into the container. The last course of action sometimes Have advantages, but it always has the disadvantage; that it involves more work.

It is yet another aspect of the present invention to have a container available provide that is particularly suitable for performing the method according to the invention. According to the inventive method, it is a prerequisite for using the Container in the process according to the invention which is permeable to a liquid medium, but has such a configuration that the solid particles in the container permanently from be detected, d. H. it must be excluded that the solid particles from the Be washed out of the interior of the container.

Such a preferred container consists of a cylindrical base body, which the preferably consists of stainless steel. This cylindrical jacket has two openings, one on the lower side and one on the opposite upper side, the Labels "lower" and "upper" side are used because the container is standing is used d. H. the central axis of symmetry of the cylinder is more or less perpendicular to the surface of the liquid medium in the chamber. The bottom side the container is closed with a sieve. The size of the pores in the sieve will be Chosen as needed, it is based on the size of the solid particles from the container includes are determined. The sieve can be made from any material because  the jacket is preferably made of stainless steel, also a preferred variant provides that the sieve is also made of stainless steel. Aids are ordered this ensures that a stirring process undisturbed underneath the container can take place. Such tools can e.g. B. be small hooks on the outside the cylinder jacket are attached and rest on the outer edge of the chamber. Prefers will be a version with legs which also on the outer edge of the Are attached to the container and what the container as a whole on the bottom of the chamber is positioned. This embodiment is preferred in all cases where the chamber unites has a flat bottom. However, if the floor is not flat, but e.g. B. concave a design of the container with legs leads to a certain instability and is to prefer the execution of the container with hooks. The number of hooks as well as the Legs can be chosen freely. Not only the stability but also the price is an aspect of this consideration, it is considered the optimum of both Considered considerations to provide the container with three hooks or legs.

The dimension of the container relative to the dimension of the chamber is not important exactly on. Even with a very narrow gap between the container and the chamber wall, this will be Procedures work just like a container that is relative to the chamber is small. However, a range is preferred which corresponds to that in P 195 05 109.2 corresponds, namely a ratio of bulk volume of fixed bed body: total volume liquid medium from 0.1-0.6.

Description of the invention using exemplary embodiments.

Fig. 1 shows a container for performing the method in top view (empty).

Fig. 2 shows a container for performing the method in top view (filled).

Fig. 3 illustrates in cross section a schematic representation constitutes a container for performing the method.

Fig. 4 illustrates in cross section a schematic representation constitutes a container for performing the method.

Fig. 5 illustrates the result of a series of experiments represent a stromal cell line.

In Fig. 1, a container is shown in a perspective top view. The container has a cylindrical jacket 1 , which is formed from stainless steel. At the lower end of the cylindrical shell, three legs 2 are attached. A sieve 3 is visible in the container and rests on projections 4 which are arranged on the inside of the cylindrical shell.

In FIG. 2 shows the same container as shown in FIG. 1. Here, however, the container is no longer in an empty state, but rather is filled with spherical particles 5 .

In Fig. 3, the recess 6 of a tissue culture plate is shown in the transverse average.

This depression has a flat bottom 7 . The chamber has a container 8 and a magnetic stir bar 9 .

The container 8 is also shown here in a cross average. The cylindrical jacket 1 and two pillars 2 are visible. The spherical particles 5 are visible in the container.

While Fig. 3 shows the rest position in the present process, in contrast, is shown in FIG. 4, which flow movement starts when the liquid medium (not separately shown in the figure) is agitated by the magnetic stirring bar. 9 As shown in FIG. 4, the magnetic stir bar 9 is located in the middle under the container. The liquid medium is attracted from above by the rotating movement of the magnetic stirring rod 9 . In the present position in the chamber, this means that the liquid medium is conveyed downward from the fixed bed 10 filled with solid particles 5 , is conveyed radially by the movement of the magnetic stirring rod, and is moved upwards along the wall in the direction of arrows 11 and 12 and is guided through the fixed bed 10 through the open entrance of the container.

The flow dynamics shown in FIG. 4 show a number of advantages. First, it makes it clear why the container shown in FIG. 1 does not need a barrier on the upper side. Such a barrier would of course have been easily possible, e.g. B. as a hinged sieve. However, this would have led to more effort in the production of the containers and thereby to a higher cost price, and would also have made working with the containers difficult by some actions. In the method according to the invention, such a shut-off is unnecessary because the flow in the chamber presses the fixed bed into the container and there will be practically no escape of individual solid particles 5 from the container. Even a relatively slight downward movement of the liquid medium will be sufficient to keep the fixed bed intact.

The flow shown in FIG. 4 clearly shows that the solid particles, for example with certain cells, can be loaded simply by adding these cells at an arbitrary point in the chamber. The flow of the liquid medium then leads immediately to the fact that the added cells are guided from above through the fixed bed 10 , the uniformity of the liquid flow through the fixed bed guaranteeing a homogeneous distribution. Of course, the same applies analogously to other components of the reaction which are added to the chamber. The flow automatically leads to such components being fed to the fixed bed quickly and evenly. However, it should be noted that the particles are preferably loaded by direct application to the bed.

FIG. 4 also shows how little the flow conditions in the chamber have on the solid particles. Not only is there no direct contact with the magnetic stir bar, the liquid medium is evenly fed into the fixed bed from above and pulled through the fixed bed with regular measures.

Finally, the use of the container according to the invention is therefore also of This is a great advantage because the fixed bed flows through and creates a three-dimensional structure with a large surface area, which shows a tissue-like cell density.

Description of the invention using exemplary embodiments.

example 1

For the investigation with human hematopoietic cells it was planned to use one To carry out co-cultivation with stromal cells because of such co-cultivation Settlement runs better and the survival rate of the hematopoietic cells is cheaper.  

For such cocultivation, it was important to understand the immobilization properties of the to characterize individual cell types. This is the only way to be targeted Adjust concentrations of lines on the carrier material.

For the characterization of the immobilization properties of the murine stroma cell line M2-10B4 (Lemoine et al, Expl Hematol, 1988; 16: 718-726) have been different Inoculum densities compared in parallel approaches.

The stainless steel container shown in FIG. 1 was used. The containers were sterilized together with the associated magnetic stirrers and tweezers by autoclaving. A magnetic stir bar and a sterile container were first inserted into each well in a sterile 12-hole tissue culture plate (Corning Costar Corp., Cambridge) and then covered with 3 ml of culture medium. RPMI 1640 with 10% fetal calf serum (each Gibco BRL, Eggenstein) was used as the culture medium. 1 ml of sterile carrier was pipetted into each of the containers and the container was then completely covered with medium. The tissue culture plate prepared in this way was placed in the incubator (36.5 ° C., 5% CO ₂ ) on a 12-position magnetic stirrer (Telesystem HP 12 p; H + P Labortechnik, Oberschlossheim) and put into a sterile test for 25 hours.

The cells were precultivated in tissue culture plates and locked at 80 for growth arrest Gy irradiated. After centrifugation, the desired amount of inoculum was in 100 ul Culture medium was added and carefully layered over the carrier.

For the investigation of the immobilization properties of the stromal cell line, four different inoculum densities (5.10 ⁵ , 1.10 ⁶ , 5.10 ⁶ and 1.10 ⁷ cells / batch) were each investigated in a triple batch. After 24 hours of cultivation, the individual batches were harvested. For this purpose, the containers were removed from the wells using sterile tweezers and each transferred to a 50 ml centrifuge tube (Greiner, Frickenhausen) in which 5 ml of PBS (phosphate buffered saline) were placed. After washing once with PBS, the number of immobilized cells was determined by nuclear staining with crystal violet (Merck, Darmstadt).

The results of the test series are shown in FIG. 5.

The Fig. 5 is a unique linear relationship between the number of inoculated and the number of immobilized cells thereof. It follows that a constant immobilization rate of stromal cells on collagen carriers of about 45% can be assumed in the concentration range examined. The high degree of reproducibility of these results can be seen from the extremely low standard deviations of the triple approaches, which are additionally shown as error bars in FIG. 5.

Example 2

Another attempt was made to investigate the possibility of cocultivation hematopoietic cells. In this experiment, a direct comparison was made Micro carrier made of borosilicate glass (Siran®, Schott Glaswerke, Mainz) and collagen (Cellex Bioscience Inc., USA).

The cultivation system was equipped as described in Example 1. Half of the Containers were filled with borosilicate carriers, the other half with collagen carriers. As Culture medium IMDM was used, the 12.5% horse serum (Boehringer Mannheim GmbH, Mannheim), 10 ng / ml IL-3 (produced by a baculo expression system, Insect cell line Sf 9 and virus AcNPV, with recombinant human IL-3), 10 ng / ml G-CSF (Amgen, Munich) and 50 ng / ml murine SCF (produced by E. coli SG 13009, Expression vector pDS (0), the extracellular part of the murine kit is expressed Ligands) contained.

After a sterility test, the supports were populated with 2.10 ⁶ growth-locked M2-10B4 cells (stromal cell line) in each well. The conditions corresponded to the information given in Example 1.

After an immobilization time of 24 hours, 2.10 ⁵ CD34 + hematopoietic cells from umbilical cord vein blood were inoculated into each well and cultivated in an incubator at 36.5 ° C. and 5% CO ₂ . Two inserts with borosilicate carriers and two with collagen carriers were harvested on days 3, 5 and 7 after the start of coculture according to the method described above. On day 5 an additional half change of media was carried out.

For this purpose, medium was withdrawn from the gap between the container and the wall of the depression (position 13 in FIG. 3) using a syringe with a cannula (Braun, Melsungen) and replaced in the same way by fresh medium.

In the case of the borosilicate carrier, the cells were detached with trypsin for the analysis.

In contrast, the collagen carriers were dissolved enzymatically. This was largely the withdraw all liquid over the carriers and 1 ml collagenase solution (2 mg / ml Collagenase type 1A, C 9891, Sigma, Deisenhofen) added. After an hour at 37 ° C the carriers were lysed and the enzymatic reaction was started by adding 5 ml PBS / FDTA solution (0.5 mM) stopped. The cells thus suspended were centrifuged off and taken up in 1 ml of fresh culture medium.

A live cell count with trypan blue (Sigma, Deisenhofen) was carried out in a Neubauer counting chamber. For the phenotypic characterization of the cells in the flow cytometer (FACScalibur, Becton Dickinson, Heidelberg), these antibodies were stained with PE-conjugated antibodies against CD34 and FTTC-conjugated antibodies against CD45 (both Dako, Hamburg). The biological activity of the hematopoietic cells was examined using the methyl cellulose assay (MC) for the detection of colony-forming cells (colony-forming cells) and the long-term culture initiating cell assay (LTC-IC) for the analysis of previous progenitor cells. In the MC assay, 500 CD34 + cells were used per plate and MethoCult ™ (Cellsystems, St. Katharinen) with 10 ng / ml IL-3 and 100 ng / ml SCF was used as medium (manufacturer information see above). After two weeks of cultivation, the assay was evaluated. The LTC-IC was carried out in a 96-well tissue culture plate, 7.10 ³ M2-10B4 cells (irradiated with 80 Gy) being seeded as well in each well. In 10-fold replicates, 3, 6, 12, 24, 48 and 96 CD34 + cells per well were inoculated and fed with fresh medium once a week over a period of 6 weeks (IMDM, 12.5% FCS, 12.5% HS , 10 ng / ml IL-3 and 10 ng / ml G-CSF, manufacturer information see above). After the subsequent overlaying of the cells with methyl cellulose medium, the assay could be evaluated after two weeks.

Example 3

The choice of media additives is crucial in cultivation hematopoietic cells.

The influence of individual cytokines on the Growth of CD34 + hematopoietic cells in coculture examined. This was the Container as described in Example 1 equipped with collagen carriers and sterility checked. Already in this phase of the investigation, the various specializations of the Tissue culture plate used different media. Basically, as a medium Uses IMDM, which contains 12.5% fetal calf serum (both Gibco BRL, Eggenstein) and Contained 12.5% horse serum (Boehringer Mannheim GmbH, Mannheim).

The following were used as cytokines:

• - 10 ng / ml IL-3,
• - 100 ng / ml IL-6,
• - 50 ng / ml SCF,
• - 50 ng / ml GM-SCF and
• - 100 ng / ml flt-3

(Supplier of all cytokines: Genzyme, Rüsselsheim).

Four of these cytokines were added to the medium in duplicate. A Double batch contained all five cytokines.

After a sterility test, the supports of each well were populated with 2.10 ⁶ growth-locked M2-10B4 cells (Stromal cell line) (as described in Example 1). After an immobilization time of 24 hours, 2.10 ⁵ CD34 + hematopoietic cells from umbilical cord vein blood were inoculated into each well and cultured in an incubator at 36.5 ° C. and 5% CO ₂ . Half a change of media was carried out after 4 days. After 7 days the cells were harvested. A live cell count with trypan blue, a phenotypic characterization of the cells in the flow cytometer, and MC and LTC-IC assays were carried out (as described in Example 2).

Claims (15)

1. A process for carrying out chemical or biological reactions in a chamber equipped with a stirrer and containing a liquid containing solid particles, the reaction taking place on the solid particles, characterized in that these solid particles are permeable to the liquid medium by one Rehälts be included. 2. The method according to claim 1, characterized in that the chamber has a volume below 20 ml. 3. The method according to claim 1 or 2, characterized in that the chamber Well of a tissue culture plate. 4. The method according to any one of claims 1 to 3, wherein the particles have antibody functions have. 5. The method according to any one of claims 1 to 3, wherein the particles contain enzymes. 6. The method according to any one of claims 1 to 3 for culturing cells human or of animal origin, with the human or animal cells settling the particles are. 7. The method according to claim 6, characterized in that the cells are human Are stem cells. 8. The method according to any one of claims 6 or 7, characterized in that the particles Glass carriers from 400 to 700 µm or collagen micro carriers are. 9. The method according to any one of claims 6 to 8, characterized in that in all Chambers of tissue culture plate, e.g. B. 6 or more, an investigation with the same Cell material expires.   10. The method according to claim 9, characterized in that the stirring device in each single chamber is a magnetic stir bar, which is made by a single magnetic stirrer is driven. 11. The method according to any one of claims 6 to 10, characterized in that the examined cells together with other cells immobilized on the carrier material become. 12. Use of the method according to one of claims 6 to 11 for establishing a Screening method for the investigation of reaction conditions for cultivation human or animal cells, such as human stem cells. 13. Container for holding solid particles for use in a process according to one of claims 1 to 12, wherein the container is a cylindrical shell, a Sieve which completes the lower side of the cylindrical jacket and aids so that a free between the bottom of the chamber and the lower side of the container Space arises, embraces. 14. Container according to claim 13, characterized in that the container and the sieve are made of stainless steel. 15. Container according to one of claims 13 or 14, characterized in that the Aid three or more legs attached to the lower outer edge of the container are.