|Title of Invention||
DEVICE FOR CULTIVATING PLANT OR ANIMAL TISSUE CULTURES
|Abstract||The present invention relates to a device for cultivating plant or animal tissue. The inventive device comprises a fermenter container (1)/ a supply device for liquid nutrients and gases(S), devices for discharging used nutritive liquids and gases(Sa/7) and solid carrier plates (6) for the plant tissues which are permeable to said liquid nutrients and gases and are arranged within the fermenter container in a stationary manner. According to the invention, the fermenter container consists of a dimensionally stable lid (3) component that carries the various supply and discharge lines as well as the carrier for the cells and consists of a pot- or bag-shaped plastic hose(2). The opening of said hose is sealed against the lid and is fixed.|
Device for the culturing of plant or animal cultures
The subject of the present invention is a new device for the culturing of plant or animal, tissue or organism cultures (in the following called tissues), as well as culturing processes with the use of such devices.
The culturing of cells for the production of cell material and especial of metabolic products of these cells is of continuous importance since the chemical synthesis of such products is frequently difficult or impossible or proves to be uneconomical in comparison with the biochemical production. Besides the already long known culturing to a wide extent of yeasts, moulds and bacteria, the culturing of plant and animal cells is also to an increasing extent of importance for the production of certain products. The investigation of suitable culturing conditions and possibly the genetic change and culturing of plant and animal cells occuriring in nature for the achievement of high yields are thereby urgent objectives of research.
One differentiates plant or animal cell and tissue cultures (preferably cell, suspensions, callus cultures) from plant organ cultures (transformed root hair (hairy root);,-, root or shoot cultures (shooty teratomas)), for the fermentation, hitherto there were used, above all, ceil suspension cultures. Which consist an- undifferentiated individual cells or as cell aggregates. Since the 1980’s, intensive efforts have been underway to culture these cell suspension cultures, in large scale fermenters and to use them for
the commercial production of tissue component materials.
However, apart from a. few exceptions,, it has been shown that cell suspension cultures are genetically frequently unstable (somaclonel variation). This has a serious influence on the active material production since even promising high capacity cell lines can,,
after a few cycles be- subject to great variations or the production of the active materials is completely suppressed. One has attributed this instability to the influence of growth regulators, as well as to the undifferentiated state of the cells which are frequently revealed as not stable without sufficient differentiation of cell organelles or without corresponding cell-cell contact of individual biosynthesis routes,Thus, epi-genetic factors play a quite important influence for the biosynthesis.
Plant organ cultures and here, above all, the commercially interesting transformed root hair cultures form differentiated tissues which also in long-term cultures prove to be genetically clearly more stable than cell suspension cultures. Thanks to the rapid growth of transformed root hair cultures, which frequently display comparable growth rates to cell suspension cultures, these organ cultures are suitable for the fermentative production of commercially interesting. component materials. Above all, transformed root hair cultures - other than cell suspension cultures - can be cultured without growth regulatoxs. By way of example, reference is made to the synthesis of taxoids, podophyllotoxins or rosmaric acidswhich are produced by such processes. Individual growth regulators can inhibit the biosynthesis of secondary metabolites.
However, plant and animal organ cultures .require a completely new fermenter design since a scale-up of the laboratory installations makes difficult the gas and nutrient solution provision due to the inhomogeneous tissues which cannot be mixed up..
For the culturing of tissues,, it is necessary regularly to supply to the cells mineral materials,, growth regulators, carbon sources, normally saccharose,, fructose or glucose, as well as possibly gases, such as oxygen or carbon dioxide, necessary for the nutrition of the cells.
The simplest and most economic form of the culturing of cells is the suspension culture, whereby isolated cells are suspended in a nutrient liquid, consumed nutrient components are regularly supplied to the nutrient solution and possibly a gassing in carried out for the maintenance of the suspension and nutrition of the cells. By means of appropriate growth regulators, a growing together of the cells to give comparatively large aggregates is prevented. It thereby proves to be disadvantageous that many plant or animal cells are not viable for a long time in this form and the formation of metabolites which are difficult to eliminate from the culture liquid requires a frequent transfer of the cells into fresh nutrient solution,
For smaller cultures, it is possible to inoculate the cells on to the surface of nutrient-containing gels, for example on to Petri dishes containing agar, whereby the cells take up the necessary nutrient components from the gel and the surrounding atmosphere. In the case of this method, too,., it proves to be disadvantageous that under these conditions, many cells grow poorly and only slowly and metabolites separate out which, in part, enrich in the surrounding medium and again slow down the growth of the cells and the cell division or even kill, off the cells, By means of the fixing on the gel surfaces, the cells grow to comparatively large heaps which, in turn, has the disadvantage that only the surface is in contact with the gas and the lower side with the nutrient liquid and thus slows down the nutrient supply in the case of growth. Here, too,, in order to maintain a sufficient growth, a frequent inoculating over of the cultures to new nutrient bases is necessary.
In order to avoid the suspension culture of isolated cells, one has, therefore, changed over to culturing differentiated cultures, such as "hairy roots" or plant shoots or leaf tissue. In suspension, such comparatively large aggregates tend to dehomogenisation, especially in comparatively large reactors, whereby a change or adaptation
of the process conditions was necessary by means of which a uniform supplying of the cell aggregates with nutrient solution and the necessary gases is achieved,
A widely used process is based on the fixing of the tissues to solid carriers and allowing a thin layer of nutrient liquid to run over the carrier, so that the tissues are continuously supplied with fresh nutrient solution and, at the same time, possess a sufficient contact to the gas atmosphere in the reactor. As carrier bodies, there are used plates or fabrics, especially mesh grids or rod constructions, as substrate which are arranged at a distance parallel to one another appropriate reactors so that the intermediate spaces make possible an impingement with nutrient liquid and gas and a certain growth of the cell culture. The fixing of the cells on the carrier thereby takes place e.g. by squeezing into the gaps or angles of the carrier surface (cf., EP 25-4- 868 and US 5,5855266). For the fermentation of so-called "hairy root cultures", the use of fermenters which contain a system of taut wire& is especially recommended, whereby the distance of the wires at the crossing points is so small that the plant tissue is firmly held at these points or, by special formation of the wires with spikes, are firmly held in these spike axes (cf. WO 85/1G958).
The fermentation vessels of the prior art usually consist, for reasons of stability, of metal, for example steel or aluminum sheet, ar, because of transparency , of glass or acrylic glass and preponderantly have a cubic shape which makes possible a space-saving arrangement of severalp3rallel carrier plate systems in the interior. A removable lid-to which, possibly besides the inlet pipes, are also fixed the carrier plates, permits an access to the container, especially for harvesting of the cell culture and for cleaning. The production of the fermentation vessels from stablematerials permits such reactors to be arranged fixedly next to one another on the bottom but
has the disadvantage that the production and servicing of such vessels is expensive and
does not permit an adaptation to the size of the culture.
Therefore, the task exists to find simple and economic devices for the culturing of tissues, especially of plant tissues, which are simple and economic to produce, are alterable in size and permit simple handling and servicing.
This task is solved by the device described in the main claim and promoted by the features of the subsiduary claims,
In particular, it is the task of the present invention, with the use of such fermentation devices, to make available a process for the culturing of plant tissue of the "hairy root type or plant shoot type".
Surprisingly, it was possible to replace the rigid walls of the fermentation devices of the prior art by cup- or bag-shaped plastic tubes of thin synthetic resin foil when one fixes these via simple known connection devices, especially by squeeze connections, directly to the lid of a conventional fermentation vessel carrying inlet and outlet pipes for the nutrient agent and gas which carries the carrier plates for the fixing of the cells to be cultured.
Holding devices fixed over the lid enable such devices to be hungwithout problems in groups in appropriate Stands, whereby these hanging stands simultaneously also carry the inlet and outlet pipes for the nutrient solutions and gases which can be regulated in their composition by central supply and control units. By means of a modular system of the inlet and removal pipes within the fermentation device of short pipe pieces with intermediately placed connection pieces and an arrangement of the carrier plates on these pipe sections,, fermenters alterable in the length and thus in the capacity can easily be constructed and provided with tube foils of appropriate length as external wall So that it is possible to change the capacity of such a fermenter in definite steps. Usually, the tube foil is
connected below with an inserted plastic bottom or by squeezing together and welding but it is also possible to insert a bottom piece, corresponding in diameter to the lid, of solid work material with an appropriate squeezing seal in which can-possible be fixed further inlet and removal pipes, Furrhermore, the production of the fermentation container from light-permeable plastic foil permits the reactions taking place in the interior to be observed from the outside, as well as to introduce through the wall light or heat radiation which are needed especially for plant tissue culturing. It represents an especial advantagethat for the removal of the cultured tissue for the production subcultures the working up of possible cell component materials, the outer plastic pipes can be cut up or, after loosening of the squeeze connection of the lid, without of the inlet pipes to the lid with the carrier plates fixed thereon, can be pulled up possibly under a sterile laminar protective gas stream so that the culture carrier plate lies free and is available for a simple working up. The cleaning of the container necessary in the case of conventional containers of solid material does not apply since the plastic pipes are discarded after use and are replaced by new ones of suitable size.
The plastic pipes can consist of all commercially usual foils, polyethylene, polypropylene,, polvinyl chloride or poly-ester foils are especially preferred since they are not only especially economic to produce but also correspond in their chemical stability, elasticity, light permeability and strength to all necessary requirements. In the case of the choice of the plastic foil, only care has to be taken that these contain no plasticisers or other adjuvants from the production which can possibly act as poisons for the fermentation when they are dissolved out from the foil by the nutrient solution.
As carriers for the tissue to be cultured, there preferably serve grids or parallel-arranged rods of biologically inert materials, e.g. stainless steel, sythetic resins or a taxtile fabric stretched in a frame which, as radial plates, project from the central supply pipe of the nutrient solution, whereby this supplying preferably consists of several short tube pieces to which are fixed the correspondingly wide plates and the tube pleces are connected by intermediate places which serve as outlet nozzles for the nutrient liquid so that nutrient liquid can be sprayed at- regular intervals into the fermentation container and thus bring about a uniform moistening of the culture surfaces from the middle. Alternatively, however, a dosing in of the nutrient solution can also take place via separate supply pipes in the outer region between the radial plates, whereby the central pipe then only functions as carrier of the plates,,
By fixing of correspondingly dimensioned lattices of steel wire or synthetic materially a firm outer frame;, which preferably consists of stainless steel, the carrier system, on the one hand, can be adapted to the plant tissue to be cultured in the size and formation of the contact places (crossover points), on the other hand, taken apart for the harvesting of the cell tissue and for cleaning.
Alternatively to a radial arrangement, it is possible also to arranged the plates transversely to the direction of the central guide whereby a 0 - 90 angle between plate and guide permits the rate of flow-off of the nutrient liquid over the plate to be influenced which is the higher that steeper the plate stands in the reactor. Preferably, therefore, this angle can be adjusted via appropriate tilting devices, for example hinges. Positioning and cleaning of the device is also simplified by this tilting possibility.
The production of the nutrient liquid in the form of fine mist droplets is per se known (EP 0 234 868 Bl) and has proved to be especially useful for the nutrient supply for the plant cells since,, at the same time, a high gas exchange is brought about. Excess or spent nutrient solution can, on the basis of gravity, drop off from the plates to the bottom of the vessel from where, by an appropriate sucking off device, the liquid can, after regeneration, i.e. the provision of consumed nutrients and the separating off of the metabolites formed, be returned into the fermenter. The sucking off pipes are also provided with appropriate modular units, whereby the intermediate pieces are naturally not formed as nozzles but rather as continuous connections in order, in this respect, to make possible a change of size of the fermenter
The supplying of the nutrient solution usually takes place via 1 or 2 substance nozzles Preferably, however, a liquid stream emerging from a bore.to the nutrient solution inlet pipe is swirled via gas currents which emerge from neighbouring gas pipes and bring about a gas exchange in the system and break it into fine droplets. Bore size, pressure of liquid and gas and the geometry of the streams coming together can control the atomizing in wide limits.
In the simplest case, in the case-of the device according to the invention, all elements, such as carrier plates, nutrient run in and run off dievices and gas pipes are fixed centrally on the lid. In order to simplify the harvesting of the tissue or a culture exchange, it can,, however, be advantageous to separate the lid part into an outer ring which carries the supply pipes and a middle lid part to which the carrier plate system is fixed. Thus, by means of lifting systems, the carrier plate system can be lifted out from the fermenter without loosening the connections of the supply pipes..
Preferably, in the case of such a dividing up, all pipes are attached to one side of the outer ring and
passed on by ring tubes in the circumference of the outer ring. It is thereby made easier to engage from the other sideinto the opened reactor and to prevent a contamination during tha engagement by passing over of a laminar,sterile air current.
The fermenters according to the invention usually have a volume of 10 - 250, preferably 50 - 100 1 and are equipped with four cross-standing carrier plates.
A 100 1 fermenter has, for example, a height of 85 cm in the case of a diameter of 28 cm and contains? two plate crosses arranged over one another with 4-0 cm height and 20 cm breadth.
Insofar as horizontal or sloping plate systems are used, their distance is to be chosen as 5 -- 25 cm, preferably about 10 cm. Thus,, in the case of the present reactor, 4- 10 plates are used.
In addition, this arrangement makes it possible to arrange two different cultures over one another, e.g. a root cell culture in the upper region and a leaf shoot culture in the lower region which cav then possibly be additionally illuminated. Thus, more strongly formed intermediate products in the root cell region can be brought into the nutrient liquid on to the leaf culture where they are further reacted to the desired end products. By means of the vertical mounting of the carriers over one another, the nutrient medium drops off passively and makes unnecessary an additional, transport expenditure as was previously usual when the different cultures were cultured in separate fermenters.
The nutrient solution is continuously circulated between fermenter and a nutrient solution tank. Interposed absorption columns in which the metabolites of the fermen-ation are absorbed on a suitable column material, filters for solid material floated out from the fermenter, inlets for nutrient material or devices or the removal of spent solution are provided as in case of known fermenters.
In an especially simple and economic embodimental form, the nutrient solution tank also consists of a simple plastic sack which, for reasons of stability, lies in a metal or synthetic material trough and is connected via a valve connection, for example lock closures, with the supply pipes, A. magnetic stirrer device provides for a contamination-free mixing up of the sack content. In the case of the culture change, this economic tank can be disposed of directly and thus saves the usual laborious cleaning. By means of the arrangement in each case of one or more fermenters to a nutrient tank in a transportable stand, one obtains compact, mobile units.
In the following Figures, the invention is explained in more detail without, however, limiting it thereto,
Fig; 1 shows a fermenter I with external plastic tube 2 and lid part 5 which firmly holds the tube 2 via a squeeze closure 4. The lid 3 contains the central supply pipe 5 on which the carrier plates 6 are fixed in the form of a cross,ii) the supply pipe 5 with the tube sections 5a and intermediate adapters 5b are integrated spray nozzles 7 for the nutrient solution.. In the periphery of the lid 5 are provided a sucking-off pipe 8 for the spent nutrient solution and inlet and removal connections 9/10 for gases. In the illustrated case, two plate modules 6 are provided over one another.
Fig, 2 shows a vertical section through another variant in which the supply and removal pipes 5, 8, 9, 10 are fixed in an outer, ring 3a and run downwardly in the periphery of the fermenter l,,On the middle lid top 3b are fixed the carrier plates 6 via a central holding 11„ The pipe 2 is firmly held between lid ring 3a and tension connection 4.
Fig. 3 shows the supplying of the nutrient solution in a horizontal sectional view according to Fig 2 via the spray nozzles 7 which are assembled in the ring pipe 5c. Plastic pipe 2 and carrier plates correspond to Fig, 2,.
Fig.4 shows a group of fermenters 1 according to Fig, 1 which are transportable fixed via common holding device 14 to a rail system 12. Nutrient solution supply devices 5 and removal devices 8 are assembled and are connected via a not-shown pump device to the central nutrient solution tank 13-
Fig. 5 shows a preferred construction of the lid ring 3a according to Fig, 2 in which all supply and removal pipes for liquids and gases 5, 8, 9 and 10 are mounted on one side and, via ring p-ipes 5c, 8c, 9c, 10c, a distribution over the circumference of the reactor takes place and from which go the passing over into the reactor. This construction has the advantage that, in the case of the opening of the reactor, sterile air can be blown in from the side facing away from the pipes which protects the reactor content from germ contamination (indicated by arrows on the right side),. The ring pipes 5c, 8c, 9d, 10a preferably conseist of a synthetic material tube which is guided in a recesS or groove of the lid ring 5a end possesses T-shaped connection pieces for the pipes into the reactor,For reasons of illustration, only one connection 8a is shown,
Fig, 6 shows; a preferred construction of the spray nozzles 7 for the nutrient solution in which gas supply pipes 8b are guided by holding means 15 parallel with the nutrient solution inlet 5b, At the height of the carrier plates 6, all ,three pipes 5a, 8a are provided with 0.1 -1. mm sized bores 7 so that the nutrient solution stream emerging under pressure in operation from the middle pipe 5a is whirled by the gas stream emerging from the gas supply pipes 8a and divided up into fine droplets. Bore diameter and pressure, as well as the angle under which the gas streams impinge on the liquid, permit the Spray character to be optimally adjusted,
Fig. 6a shows the arrangement of the spray nozzle bores 7.
Fig. bb shows a holding means 15 for the pipes 5a, 8a, 8a , as well as adjustment devices 16, 17 with which the bores 7 can be aligned.
Fig. 7 shows a further construction of the carrier plate 6 in the form of preforated steel or: aluminium sheets or synthetic resin plates which are attached on both sides to a central holding means 19 via not illustrated tilting hinges. The tilting hinges enable the tilting angle of the plates 18 (0 - 90o) and thus the run-off speed of the nutrient solution over the plates to be adjusted. Furthermore, it is possible to apply the plates 18 wholly on the holding means, whereby the device takes up less room, which, in the case of the cleaning and sterilising of the device, requires smaller cleaning containers (cf. Fig.. 7b).
Fig. 7B shows schematically a perforated plate 18, as well as, in section, the central holding means 19 which is also preferably constructed as perforated plate or grid plate in order to make possible a gas and liquid passage. The plaes 18 are arranged, over on another at distances" of 5 - 25 cm, preferably 10 cm, so that, depending upon the reactor height, there is given a number of 4 - 10..
List of references
fermenter vessel plastic pipe lid
outer lid ring middle ring cap
tension connections (squeeze connections) supply pipe tube piece
intermediate adapter ring pipes carrier plates spray nozzles sucking-Off pipe tube piece
intermediate adapter ring pipe
gas inlet and outlet connections ring pipe central holding means rail system
nutrient solution tank holding device adjustment device perforated plates central holding means
Amended patent claims filed at the European Patent Office
on the 7th June, 2002
1. Device for the culturing of plant and animal tissue, comprising a fermenter vessel (l), inlet for liquid nutrients and gases, as well as devices for removal of spent nutrient liquids and gases (8) and solid carrier plates (6)for the tissue which are permeable for the said liquid nutrients and gases and are fixedly arranged within the fermenter vessel (1), characterised in that the fermenter vessel (l) consists of a form-stable lid part (3) which carries the various inlet and outlet pipea (5, 8, 9, 10), as well as the carrier plates (5) for the cells and consists of a cup- or bag-shaped plastic tube (2), the opening of which is sealed off and fixed against the lid (5).
2. Devices according to claim 1, characterised in that
3-8 carrier plates (6) are radially fixed on a central
holding means (11) and the supply of the nutrient solution
takes place via 3-8 supply pipes (5) present in the
outer region between the plates (6).
3, Device according to claim 1,, characterised in that
the supply pipes (5a) contain as nozzles bores (7) for
the outlet of the nutrient solution and gas inlet
pipes (9) are guided parallel to the pipe (5a) which, in
appropriate places, also contain bores (7) from which
4. Device according to claim 1 - 5. characterised in that in the outer region between the carrier plates (6) are arranged one or more sucking-off pipes (8/10) for spent nutrient liquid and spent gas which on the end contain a sucking-off nozzle,
5. Device according to claim 1,, charaoterised in that the inlet pipe represents a central pipe (5) which consists of two or more tube pieces combined via intermediate adapters (.5b), whereby the adapter (5b) contains
nozzles- for the atomisBtion of the nutrient solution and the tube pieces (59) carry 3-8 radially projecting lattice plates (18) as carrier plates (6) for the cells, whereby the length of the assembled supply pipe (5) corresponds to the length of the plastic tube (2) of the fermenter vessel (1) and the cross-section through the carrier lattice (18) to the opening of the plastic tube (2:) or to the size of the lid (3).
6, Device according to claim 5, characterised in that, in
the outer region between the carrier plates (5), one or
more sucking-off pipes (8/10) for spent nutrient liquid and spent gases are arranged which also consist of tube pieces (8a)which correspond in length to the tube pieces (5a) of the supply pipe (5) and contain adapter piec'es (8b) for the connecting of the tube pieces (8a), as well as a sucking-off nozzle on the end, whereby the length of this pipe (8) corresponds to the length of the plastic pipe (2).
7. Device according to claim 1, characterised in that the
carrier plates (18) are arranged transversely to the
longitudinal axis of the reactor,
8.. Device according to claim 7, characterised in that the
carrier plates (18) can be adjusted on a central holding
means (19) via tilt devices at an angle of 0 - 90o to the
9. Device according to any of claims 1-8, characterised
in that lid (3) and plastic pipe (2) are connected via
seals and tension connections (4)-
10,. Device, according to one ©f claims 1-8, characterised
in that the lid part (3) is connected via a screw and
flange connection with the tension closure (4) for the
fixing of the plastic pipe (2).
11,. Device according to one of claims 1 - 10, characterised
in that one or more such devices (l) are connected via
appropriate pipe connections (5,8) andholding devices
(14) to a central supply system (15).
12. Device according to one of claims 1 - 11, characterised
in that the lid part (5) consists of an outer lid ring
(3a) and a middle lid (3b), whereby all nutrient solution
and gas supply and removsl pipes (5, 8, 9, 10) are
provided in the ring (3a) and on the middle lid (5b) are
fixed the carrier plates (6) via holding means (11) and
the supply and removal pipes are guided downwardly in the peripheral region of the fermenter (1) and the tensiotD connection (4) firmly, holds the plastic pipe (2) against the lid ring (39).
13. Device according to claim 12, characterised in that
all supply and removal pipes (5, 8, 9, 10)are provided
on one side of the lid ring (33) and are guided over
ring pipes (5c, 8c, 9c, 10c) in the lid ring (33) over
14. Device according to one of claims 1 - 13, characterised in that the nutrient solution is circulated via a nutrient solution tank (13) in the form of plastic sack.
15. Device for the culturing of plant and animal tissue substantially as herein described with reference to the accompanying drawings.
Dated this 3 day of October 2002
|Indian Patent Application Number||IN/PCT/2002/1606/CHE|
|PG Journal Number||26/2007|
|Date of Filing||03-Oct-2002|
|Name of Patentee||M/S. ROOTEC GESELLSCHAFT FÜR BIOAKTIVE WIRKSTOFFE MBH|
|Applicant Address||Rischer Strasse 12 69123 Heidelberg|
|PCT International Classification Number||C12M1/26|
|PCT International Application Number||PCT/EP2001/003716|
|PCT International Filing date||2001-04-02|