RU2270245C1 - Apparatus for suspension cultivation of tissue and microorganism cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: claimed apparatus contains fermentation vessel with closure and nipples for aerating gas feeding and gaseous medium discharge, respectively; and devise for cultural medium aeration and agitation. Said devise contains aerating gas circuit comprising pipeline and screen installed inside of vessel above outlet port of pipeline. Screen is placed horizontally and with gap to vessel walls to form gas cushion under thereof. Device also contains attachment for forming gas cushion vibration to agitate the cultural medium and gas flow controller, interconnected with abovementioned pipeline. Apparatus may be equipped by one or more said devices for cultural medium aeration and agitation. Screen has quarrel cross-section having longitudinal slit in bottom part for reciprocal movement of cultural medium flow. Attachment for forming gas cushion vibration contains pulsator with mobile membrane to produce reciprocal movement of gas flow in gas cushion under screen.

EFFECT: increased mass exchange effectiveness in cell cultivation process due to enhanced gas-liquid boundary surface; apparatus of increased productivity.

4 cl, 4 dwg

Description

The invention relates to biotechnology, and more specifically to apparatus for suspension cultivation of tissue cells and microorganisms, such as, for example, mammalian cells, mycelial fungi, etc., including those whose morphological structure is sensitive to the physical effects of culture suspensions, and can be used in microbiological, medical, food industry and research practice.

A known apparatus for suspension cultivation of cells and microorganisms containing a fermentation tank with a lid and nozzles for supplying aerating gas and venting a gaseous medium and a device for mixing a suspension of cells, which is used as a lattice located in the lower part of the tank. Air is supplied through the grate to the container to create hydrostatic pressure, which ensures the mixing process and prevents the sedimentation of cells from the suspension (US No. 4259449, C 12 M 1/04, 1981).

However, such an apparatus has insufficient efficiency of the mass transfer process, which is due to the need to reduce the intensity of the aeration gas supply in order to avoid injury to cells.

The closest analogue of the proposed apparatus is an apparatus for suspension cultivation of tissue cells and microorganisms, containing a fermentation vessel, a lid and nozzles, respectively, for supplying aerating gas and venting a gaseous medium, and a device for aeration and mixing of the culture medium, including a horizontal impeller mounted on a vertical floor shaft and placed under the cover of the tank (RU, No. 2135579, C 12 M 1/04, 09/27/1999).

The impeller is mounted above the surface of the suspension and its rotation speed forms air vortices that can cause the suspension to move in the cavity of the apparatus along a predetermined path, which creates the conditions for gas and mass transfer of the culture suspension.

The device for aeration and mixing of the culture medium used in the apparatus does not cause mechanical destruction of cultured cells.

The disadvantages of the apparatus are as follows.

The apparatus is effective only with small volumes of the fermentation tank, since it does not provide effective mass transfer over the entire height of the culture volume of the suspension with significant volumes of the fermentation tank.

The active zone of the air vortex extends to the surface layer of the working suspension and damps in the direction of the bottom of the tank, which reduces the productivity of the device.

Mass transfer in the apparatus depends on the height of the suspension column and the centrifugal speed of the vortex formed by the impeller, this dependence significantly limits the scope of use of the apparatus and complicates the optimization of gas and mass transfer conditions, thereby reducing the productivity of the apparatus.

A centrifugal impeller is located in the gas cavity of the tank, which is additionally blown with gases of a given composition, and the exhaust gases are discharged into the atmosphere through bacterial filters. With this design, droplets of cell suspension will be captured in the gas vortex, which will not only contaminate the pipelines for venting gases, but also the bacterial filter of exhaust gases, which can lead to a violation of the aseptic conditions of cell culture and termination of the process.

Clogging of the centrifugal impeller’s cavity by biomass, nutrient medium, and microbial synthesis products, which are captured by an air vortex from the surface of the suspension and are thrown onto the wall of the container, which leads to unpredictable processes of destruction of cellular biomass, contamination of the suspension with the products of cell decomposition and, as a result, reduced productivity and quality of the products obtained.

The technical result of the invention is to increase the efficiency of mass transfer during the cultivation of cells and microorganisms by increasing the surfaces of the gas-liquid phase section and increasing the productivity of the apparatus.

This result is achieved by the fact that the apparatus for suspension cultivation of tissue cells and microorganisms includes a fermentation tank with a lid and nozzles, respectively, for supplying aerating gas and venting a gaseous medium and a device for aeration and mixing of the culture medium, the latter contains an aeration gas circuit consisting of a pipe and placed above its outlet inside the screen, located horizontally to form a gas cushion beneath it and installed with a gap to enkam container, and means for creating a gas cushion vibrations for stirring the culture medium and a gas flow controller in communication with said conduit.

The apparatus may have at least one more device for aeration and mixing of the culture medium.

The screen in cross section may be in the form of a rhombus with a longitudinal slit in its lower part for the reciprocal flow of the culture medium.

The means for creating oscillations of the gas cushion comprises a pulsator with a movable diaphragm for creating a reciprocating movement of the gas flow into the gas cushion under the screen.

The invention is illustrated in the drawing, in which:

figure 1 - schematically shows the apparatus in longitudinal section;

figure 2 is a section along aa of figure 1;

figure 3 - apparatus with two devices for aeration and mixing of the culture medium;

figure 4 is a variant of the apparatus with a vertical arrangement of the fermentation tank;

figure 5 is a screen in cross section having the shape of a rhombus with a longitudinal slit.

The apparatus for suspension cultivation of cells and microorganisms (see figure 1) contains a fermentation tank 1, covers 2, covering the tank 1 on both sides, a pipe 3 (see figure 2) for loading and a pipe 4 for unloading the tank 1, pipe 5 and 6, respectively, for supplying aerating gas and discharging a gaseous medium and a device for aeration and mixing of the culture medium. The nozzles 5 and 6 are diagonally located in the upper parts of different covers 2, covering the tank 1 on both sides.

A device for aeration and mixing of the culture medium includes an aeration gas circuit and means for generating vibrations of the gas pad for mixing the culture medium.

The aerating gas circuit contains a screen 7 mounted using a screen holder 8 inside the fermentation tank 1. The screen 7 is mounted horizontally with a gap to the walls of the tank 1 above the outlet of the pipe 9 for supplying aerating gas under the screen to form a gas cushion underneath.

Means for creating oscillations of the gas cushion is communicated with the pipeline 9 and contains a pulsator 10 with a movable diaphragm 11 to create a reciprocating movement of gas into the cushion. The movement of the diaphragm 11 is provided by the actuator 12. As the actuator 12, for example, a motor mechanically coupled to the diaphragm 11 and providing its reciprocating movement, or a pulse generator of compressed air forcing the diaphragm 11. to move, can be used. The other end of the pipe 9 through the valve 14 and regulator 11 are in communication with a bacterial filter 16 through which aerating gas is supplied.

The filter 16 is also communicated through a gas flow regulator 15 with a nozzle 5 for purging the gas cavity of the apparatus with aerating gas through a nozzle 6 and a bacterial filter 17. The apparatus for suspension cultivation of cells and microorganisms is equipped with a control device 18.

The screen 7 has the form of, for example, an umbrella or a gable surface, is fixed inside the fermentation tank by any known method, for example, by means of a holder 8 on the pipe 9. To intensify the gas exchange of the culture medium, the screen 7 can be made of a gas-permeable hydrophobic material, for example, tiflon. To intensify the process of mixing (mass transfer) of the culture suspension, the screen 7 in cross section may be in the form of a rhombus with a longitudinal slot for the reciprocal flow of the culture medium (see Fig. 5).

As gas flow regulators 14 and 15 can be used rotameters, peristaltic pumps or other similar devices.

To ensure optimal conditions of gas and mass transfer during the cultivation of various microorganisms, the apparatus may contain several devices for aeration and mixing of the culture medium, while the required number of screens 7 at different heights can be installed in the fermentation tank 1 (see figure 3).

The apparatus for suspension cultivation of cells and microorganisms may contain a fermentation tank 1 located both horizontally and vertically (see figure 4). With a vertical arrangement of the container 1, the nozzles 5 and 6 are diagonally located in the upper cover 2 that covers the container 1. The fermentation tank 1 may have a different shape, but the cylindrical shape of the container is preferable. Capacity 1, if necessary, can be made of transparent heat-resistant glass.

The operation of the apparatus for suspension cultivation of cells and microorganisms (see figure 1) is as follows.

Before starting work, well-known procedures are carried out for sealing and sterilizing fermentation equipment, culture media and obtaining inoculum cells.

In the fermentation tank 1 under aseptic conditions, through the valve and pipe 3 make the nutrient medium and the inoculum of the seed cells. The valve is closed, and on the regulator 15 the flow rate of the aerating gas is blown through the filter 16, the nozzle 5, the upper part of the tank 1, the nozzle 6 and the filter 17. On the regulator 14, the flow rate of the aerating gas is blown through the filter 16 and the device for aeration and mixing of the culture suspensions. The gas flows through the filter 16, the regulator 14 and the valve 10, fills the cavity under the diaphragm 11 of the pulsator 10 and along the aeration gas circuit, enters under the screen 7, displaces the culture medium from under it and forms a gas cushion under the screen. Excessive gas pressure generated under the screen is discharged into the culture medium in the form of gas bubbles and then through the pipe 6 and filter 17 into the atmosphere. Aeration of the culture medium is ensured by gas exchange over the entire surface of the gas cushion located under the screen 7 and in contact with the culture medium and the aeration gas flow above the surface layer of the culture suspension in the tank 1.

On the control device 18, the frequency of operation of the means for generating oscillations of the gas cushion is set, namely, the frequency of operation of the drive 12, which drives the diaphragm 11 of the pulsator 10, is set in reciprocating motion. When moving, the diaphragm 11 displaces the gas from the pulsator 10. Gas under excessive pressure enters through a pipe 9 under the screen 7 into the gas cushion formed underneath. The volume of gas under the screen 7 increases, acts on the culture medium, forcing it to move upward through the gaps between the edges of the screen and the walls of the fermentation tank 1, while the level of culture suspension in the tank 1 rises. When moving the diaphragm 11 in the pulsator 10, which sucks gas through line 9 with the valve 10 closed, from the air cushion located under the screen 7, the gas pressure in the cushion decreases, the volume of the cushion decreases. When this culture medium flows down through the gaps between the wall of the tank 1 and the edges of the screen 7 and the level of the culture medium in the tank 1 is returned to its original position. A cyclic change in the gas pressure in the gas cushion raises and lowers the level of the culture medium in the tank 1, while the latter flows under the conditions of reciprocating movement through the gap formed between the walls of the tank 1 and the edges of the screen 7. The reciprocating flow of the suspension through the specified gaps with a given the frequency provides mixing of the culture medium under conditions that are gentle for cells. The gas cushion located under the screen and immersed in the culture medium simultaneously experiences the pressure of the column of the culture medium, the frequency of the gas pressure from the means for creating oscillations of the gas cushion, and gas displacement in it along the aeration gas circuit from the gas flow regulator 14, which ensures controlled gas exchange .

If there are several devices for aeration and mixing of the culture medium and screens 7 installed at different heights inside the fermentation tank, the device operates similarly to the one described above. The presence of several devices for aeration and mixing of the culture medium and screens provides additional layer-by-layer aeration and mixing of the culture medium and optimal conditions for cultivation in more viscous suspensions.

Claims (4)

1. Apparatus for suspension cultivation of tissue cells and microorganisms, including a fermentation tank with a lid and nozzles, respectively, for supplying aerating gas and venting a gaseous medium and a device for aeration and mixing of the culture medium, characterized in that the latter contains an aeration gas circuit consisting of a pipeline and placed above its outlet in the inside of the screen, located horizontally to form a gas cushion under it and installed with a gap to the walls of the tank spine, as well as a means of creating oscillations of the gas pad for mixing the culture medium and a gas flow regulator in communication with the specified pipeline. 2. The apparatus according to claim 1, characterized in that it is equipped with at least one more device for aeration and mixing of the culture medium. 3. The apparatus according to claim 1, characterized in that the screen in cross section has the shape of a rhombus with a longitudinal slot in its lower part for the reciprocal flow of the culture medium. 4. The apparatus according to claim 1, characterized in that the means for generating oscillations of the gas cushion comprises a pulsator with a movable diaphragm for creating a reciprocating movement of the gas flow into the gas cushion under the screen.

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