Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Reexamination Certificate
1999-06-02
2001-04-03
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
C435S304200, C435S304300, C435S308100, C210S296000
Reexamination Certificate
active
06210959
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an apparatus for the cultivation and concentration of non-adherent cells as well as for cocultivation of two different cell species which are adherent or non-adherent.
BACKGROUND OF THE INVENTION
In cell cultivation cells are introduced into culture flasks containing a nutritive medium where they grow, depending on the respective type of cell, either adherently, i.e. on the surface of the culture flask, or non-adherently, i.e. in a suspension in the nutritive medium. For harvesting and the continuing cultivation of non-adherently growing cells it has so far been common to concentrate the cells by centrifuging after the growth phase and before the further processing. There is the problem, however, that centrifuging gives rise to an undesirable aggregation of cells and to the occurrence of damage to the cells. Moreover, the manipulation involves a high risk of contamination by bacteria and fungi.
In the technique of co-culturing two cell species so far common mobile inserts are used which are equipped with a liquid-permeable but not cell-permeable membrane, which are placed into culture dishes for separating them into two cell compartments. The known inserts in culture disks, however, permit only the co-cultivation of two cell species only at a small scale and with different conditions only because one cell species is in the tissue culture dish whilst the other one is placed on the membrane. It is moreover inexpedient that the exchange of the culturing medium, the collection of the excess culture quantity and the repeated adjustment of the cell concentration is not possible or possible only within narrow limits.
From the document WO 96/00780 a cell culture apparatus subdivided into compartments is known wherein the cell compartment is defined by a lower gas-permeable membrane and by an upper membrane permeable to the nutritive medium so as to allow for a better supply of the cells with a simultaneous concentration of the macromolecular cell products in the cell compartment. Even though this cell culture apparatus allows for an exchange of the cell culture medium without centrifugation it does not permit harvesting of the macromolecular cell products without a subsequent separation of the cells by centrifuging. Apart therefrom, a co-culture of two cell species is not possible with such an apparatus.
SUMMARY OF THE INVENTION
The present invention is now based on the problem of providing an apparatus of the general type outlined above, which presents a simple structure and which serves to allow for cell production without centrifuging at a reduced risk of contamination and moreover a simple adjustment of the cell concentration.
In accordance with the present invention this problem is solved by the features defined in Patent claim
1
. Preferred features which ensure-an expedient improvement of the invention are disclosed in the dependent patent claims.
On account of the inventive design of the apparatus with a fixedly mounted partial partitioning wall a partial two-chamber system is made available in an expedient manner, wherein by the time of harvesting the free excess volume of cells may expediently flow through the membrane to the other side of the chamber and the desired quantity of excess cell volume may be removed, whereupon new medium can be supplied. Whilst in culturing operation the partitioning wall is preferably located above the cell suspension as a result of the arrangement of the hollow vessel it is thus possible, without centrifugation after vertical positioning of the hollow vessel, not only to exchange the culture medium but also to adjust the cell concentration and to collect the excess culture volume. With these provisions a cell aggregation and damage to the cells are largely avoided and the adjustment of the cell concentration is facilitated by controlled exchange of the culture medium. Moreover there are less manipulations on the cell culture so that the contamination risk will be reduced.
Owing to its design and structure, the inventive apparatus is suitable for the culture of high cell concentrations with frequent and simple exchange of the medium whilst it enables the co-culture of high numbers of cells of two species under definitely identical culture conditions in the vertical orientation.
Not only the careful treatment of the cells by omission of the forces of gravitation, which are due to the centrifuging operation, but also the fact that a cell-free excess culture volume is easier to harvest than it were with the conventional centrifuging and subsequent filtering operations must be deemed to involve a special advantage of the invention. Apart therefrom, there is no loss of cells or only a loss smaller than with centrifuging and subsequent exhaustion or decanting of the excess culture medium when the inventive apparatus is employed.
It is moreover expedient that the manipulations are comparatively simpler than in prior art. For instance, the exchange of the media by conventional centrifuging takes approximately 12 minutes whereas the inventive apparatus reduces this time to roughly four minutes, especially because the manipulation is restricted to a simple tilting operation for tilting the hollow vessel from the resting surface to the surface on which it stands upright.
Another advantageous aspect is the avoidance or reduction of cell aggregation which may occur in the cell pellet during the centrifuging operation. Moreover, there is only an expediently low risk of contamination due to the reduced scope of manipulations.
Another advantage resides in the fact that due to the use of the apparatus the cluttering of a high cell concentration with frequent and simple exchange of the medium becomes possible. Moreover, there is the opportunity of permanent exchange of the medium, for instance in the production of antibodies or in the large-scale expansion of cells. Furthermore the possibility of co-culturing two different cell species is expedient, e.g. for studies into their humoral interaction.
As far as the design of the inventive apparatus is concerned it is expedient for the function and the manufacture that the passage is closed by a micro-porous membrane or a cell-impermeable screen flush with the partitioning wall, or presents a laterally projecting border or skirt. The size, shape, material, pore or mesh size of the membrane or the screen may be optionally varied in response to the respective requirements within the general framework of the desired purpose, i.e. in terms of the permeability to the medium and the excess cell volume but not to the cells. As an alternative it may also be expedient to design the entire partitioning wall—possible with the exception of a structurally required marginal plastic web—as a micro-porous membrane or cell-impermeable screen, instead of providing the at least one passage, which ensures an expedient acceleration of an exchange of the liquid in the vertical position of the hollow vessel.
The membrane which is permeable to liquids but impermeable to cells, or the screen permeable to liquids but impermeable to cells, respectively, expediently consist of a translucent material but such transparency is not definitely required whereas the hollow vessel is preferably made of a transparent material. These provisions advantageously ensure that the hollow vessel can be visually inspected in a so-called inverting microscope, i.e. with illumination from the top and with positioning of the hollow vessel above the lens so that the culture may be viewed from the bottom of the hollow vessel. The hollow vessel, and preferably the partitioning wall as well, consists of an inert transparent material, particularly even though not exclusively of polystyrene.
The height h of the partitioning wall, seen in the upright position of the hollow vessel, may expediently be so dimensioned that the cell suspension cannot flow beyond the partitioning wall into the chamber receiving the excess cell volume when the hollow vessel is in the upright position.
In cell-culturing operation the partit
Kremer Jean-Pierre
Liu Guoquing
Lodri Antal
Reisbach Gilbert
GSF-Forschungszentrum fur Umwelt und Gesundheit GmbH
Redding David A.
Sughrue Mion Zinn Macpeak & Seas, PLLC
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