Apparatus for cultivating tissue cells and microorganisms in...

Chemistry: molecular biology and microbiology – Apparatus – Bioreactor

Reexamination Certificate

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Details

C435S295100, C435S302100, C435S818000

Reexamination Certificate

active

06632657

ABSTRACT:

TECHNICAL FIELD
The present invention relates to biotechnology and more particularly to an apparatus for cultivating tissue cells or microorganisms in suspension.
BACKGROUND ART
An apparatus for cultivating tissue cells and microorganisms in suspension is known in the art (SU, A1, No. 1331888, 1987); which comprises a closed reservoir to the bottom of which branch pipes are connected for feeding an aerating gas. One group of the branch pipes is arranged tangentially, and the other parallel to the cylindrical wall of the reservoir. In the process of cultivating, the aerating gas at once involves the suspension in a vortex motion with simultaneous circulation in the form of ascending and descending streams along the axis of the apparatus.
However, such an apparatus cannot be used for cultivating cells of animal and human tissues: these cells can be easily traumatized, since in the process of cultivating in suspension a large number of gas bubbles is formed, and destruction of these bubbles will cause death of many cells. Aeration by gas blowing leads to the formation of foam which also results in loss of a part of the cells. Foam suppression will require introducing costly non-toxic chemical defoaming agents into the nutrient medium, the technological process of cultivation will be complicated, and the use of defoaming agents will deteriorate the properties of the culture medium.
An apparatus for cultivating tissue cells and microorganisms in suspension is known in the art (U.S. Pat. No. 4,259,449, 1981), which comprises a cylindrical reservoir with a cover and branch pipes for feeding an aerating gas and removing gaseous medium, and a device for stirring the cell suspension, said device being made as a grid disposed in the bottom part of the reservoir. Air is supplied through the grid to the reservoir for creating hydrostatic pressure which prevents precipitation of cells from the suspension.
However, the productivity of such an apparatus in the process of cultivating cells is low owing to worsening of the mass transfer characteristics which, in turn, are impaired, insofar as the intensity of feeding the aerating gas is reduced in order to obviate traumatizing the cells. Even in this case traumatizing the cells cannot be ruled out completely, and intensive foam formation takes place.
Also known in the art is an apparatus for cultivating tissue cells and microorganisms in suspension (WO 92/05245, A1, Feb. 4, 1992), comprising a cylindrical reservoir with a cover and branch pipes for gas feeding and removal, a device for aerating and stirring the suspension. The device for the aeration and stirring comprises a horizontal blade wheel secured on a vertical power shaft and arranged in the top part of the reservoir directly under the cover, and an annular plate placed thereunder, provided with a central opening for the removal of gas, attached over the periphery thereof to the wall of the reservoir, forming an annular space around the blade wheel for gas feeding and removal. Slot openings are provided in the annular partition for the passage of gas, said slots being distributed uniformly along the circumference at an angle to the horizontal plane. The branch pipe for feeding the gas is installed in the cover coaxially with the blade wheel, and the branch pipe for the removal of gas is coupled to said annular space and disposed on the edge of the cover.
The disadvantage of such an apparatus is that formation of vortex motion of liquid (potential vortex with axial reverse flow) therein can be achieved at high gas flow velocities (greater than 15-18 m/s) above the surface of this liquid, i.e., said vortex formation involves considerable power inputs. At the same time, there occurs entrainment of liquid drops from the surface of the suspension with subsequent ejection of these drops onto the wall of the reservoir. Cells in the suspension drops become traumatized by the impact against the wall of the apparatus, i.e., mass death of the cells takes place. With a decrease in the gas flow velocity (6-8 m/s), an unstable flow of the liquid above the surface of the cell suspension is observed, i.e., the axial-symmetric vortex motion of the liquid is periodically changed for auto-oscillations of the liquid, in which mode there originates a wave travelling along the wall of the reservoir. The surface of the liquid becomes curved and represents an asymmetric paraboloid of rotation. All the liquid in the apparatus oscillates as a single whole, the entire apparatus starts rocking, and this produces an unfavorable effect on the cell cultivation process. Besides, the design of the apparatus allows cultivating cells with the reservoir being filled to a height equal to or less than one diameter of this reservoir. If the reservoir is filled with the cell suspension to a height greater than one diameter thereof, a stagnant zone will be formed at the bottom of the reservoir. During cultivation cells will inevitably settle in this zone and perish because of deficiency in oxygen.
An apparatus for cultivating tissue cells or microorganisms in suspension is known, which comprises a cylindrical reservoir with a cover and branch pipes, accordingly, for feeding an aerating gas and removing gaseous medium, and a device for aerating and stirring the suspension, comprising a horizontal blade wheel secured on a vertical hollow shalt and arranged in the top part of the reservoir directly under the cover (WO 93/21301, A1, Oct. 28, 1993—the first embodiment of the apparatus). The device for aerating and stirring the medium is provided with an annular partition installed in the cylindrical reservoir coaxially with the blade wheel, a clearance being formed between the cylindrical wall of the reservoir and the annular partition; the device is also provided with a mechanism for stabilizing the position of the annular partition with respect to the surface of the cell suspension. In accordance with this embodiment of the invention, the mechanism for stabilizing the position of the annular partition with respect to the surface of the cell suspension is made in the form of racks attached to the cover of the reservoir and to the annular partition with help of latches with a possibility of varying the position of the annular partition in relation to the height of the reservoir. The annular partition should be immersed to a depth H≧0.02(D
1
-D
2
), where
D
1
is the diameter of the annular partition;
D
2
is the diameter of the axial opening in the annular partition.
A disadvantage of this embodiment of the apparatus is as follows. In many cases the process of cultivating cells and tissues is accompanied by changes in the level of the initial filling of the bioreactor (for example, owing to periodic sampling or the cultivation in the initial period being carried out with small volumes of the nutrient medium, whereas the final stage of cultivating is carried out with maximum filling of the reservoir of the reactor). As a result, the depth (H) of immersing the annular partition changes, i.e., the condition H≧≧0.02(D
1
-D
2
) is disturbed. A decrease of the value (H) results in “locking” of the liquid drain through the axial opening in the annular partition, and this leads to deterioration of the process of stirring and aerating the cells being cultivated. Should the annular partition hang stationary above the surface of the cell suspension, the mass-transfer parameters will worsen still further. If the depth of immersing (H) of the annular partition is appreciably greater than H=0.02(D
1
-D
2
), a travelling wave will be formed above the surface of the suspension, which gradually results in rocking the whole mass of the cell suspension, bringing it to the state of unstable stirring with lowered mass-transfer parameters. Therefore, under the conditions of periodic variations in the level of filling the reservoir of the apparatus in the process of cultivating, this device will be either almost inoperable, or introducing an additional mechanism will be required for automatic setting of the annular partition to the opti

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