Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Reexamination Certificate
2002-12-30
2004-09-21
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
C435S296100, C435S299100, C435S299200
Reexamination Certificate
active
06794183
ABSTRACT:
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.
BACKGROUND OF THE INVENTION
The culturing of cells for the production of cell material and especially 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 occurring 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, cell suspension cultures which consist of undifferentiated individual cells or 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 (somaclonal 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, epigenetic factors play a quite important influence for the biosynthesis.
Plant organ cultures and here, above all, the commercially interesting transfonned 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 regulators. By way of example, reference is made to the synthesis of taxoids, podophyllotoxins or rosmaric acids which 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 with 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 is 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 in 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 234 868 and U.S. Pat No. 5,585,266). For the fermentation of so-called “hairy root cultures”, the use of fermenters which contain a system of taut wires 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 85/10958).
The fermentation vessels of the prior art usually consist, for reasons of stability, of metal, for example steel or aluminum sheet, or, because of transparency, of glass or acrylic glass and preponderantly have a cubic shape which makes possible a space-saving arrangement of several parallel 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 stable materials 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 a simple handling and servicing.
In particu
Ripplinger Peter
Wildi Eckhart
Wildi Reinhart
Foley & Lardner LLP
Redding David A.
Rootec Gesellschaft Für Bioaktive Wirkstoffe mbH
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