Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
  • C12M27/02—Stirrer or mobile mixing elements

Definitions

• the present invention relates to a method for culturing cells in a bioreactor.
• Biotechnologically manufactured products from cell cultures are becoming increasingly important, particularly in the medical-pharmaceutical sector.
• the cultivation of tissue cells on an industrial scale has so far been quite cumbersome, in contrast to less demanding bacteria and yeast cell cultures. Problems related to this are mentioned, such as adequate ventilation, sterility of the culture exchange of the medium and in particular the mechanical stress on the tissue cells in order to ensure adequate mixing of the culture medium.
• Fluidized bed or also multi-phase vortex reactors have proven to be quite useful (Large Scale Cell Culture Technology edited by Björn K. Lydersen, Hanser Publishers, Kunststoff, Vienna, New York). These devices are quite complicated in structure and therefore prone to failure and difficult to handle. Fin main problem in the cultivation of tissue cells. • .11: .- are based on their extremely weak stability against mechanical loads, as they inevitably occur in the cultivation methods according to the prior art.
• the object on which the invention is based is to provide a method for the cultivation of wallless cells, in particular strong mechanical stress on the cells being prevented and nevertheless adequate aeration and mixing and mixing of the culture fluid being achieved.
• the reactor can be gassed in the headspace or directly in the medium.
• this object is achieved by a method in which the culture medium is overturned, since part of the medium in the reactor of the submerged culture of wall-less cells is continuously in the reactor with a circulation device, preferably a propeller, at the bottom of the reactor vessel into an open top and brought via the circulating device to the vessel contents in connection with the annular space between the guide body and the vessel wall of the reactor, conveyed upwards and added from above to the main amount of the reactor contents located in the reactor vessel.
• a circulation device preferably a propeller
• the pumping around of the reactor contents is preferably carried out in such a way that the flow conditions occurring in the annular space are turbulent or laminar in nature.
• the hollow antel-like annular space of the bioreactor can also be designed as an arrangement of tubes with different diameters.
• the branched-off part of the medium can be poured onto the main amount of the reactor contents with the formation of a trumpet by suitable process control.
• the reactor space is preferably gassed in the head space of the bioreactor. Fumigation can ensure an adequate supply of the necessary gases, such as oxygen and CO. be guaranteed.
• the pH regulation with carbon dioxide can be done by gassing the reactor in the head space or directly from the medium. Under certain conditions, it is advantageous to match the flow velocity of the medium in the annular space to the number of cells present. This adjustment takes place via the circulation device, for example by the speed of the propeller. All methods suitable for this purpose can be used as the determination method for the cell density, such as, for example, measuring the optical density or the fluorescence. A reliable control of the flow rate depending on the number of cells can also be achieved with the flow injection analysis.
• the medium is preferably circulated at least 0.2 times per minute.
• Be ⁇ Sonders preferably a Ummélzfreguenz is from 0.2 to 50 min.
• the method according to the invention enables the fermentation of wallless cells in a particularly advantageous manner.
• the procedure according to the invention achieves a thorough mixing of the culture medium without mechanically destroying the cells.
• the method according to the invention can be applied to all cultivation methods / thus also -i. -T carriers adsorbed or immobilized cells. A powerful system for the cultivation of wallless cells is thus available.
• the invention is illustrated by the following example.
• Erythrocytes are prepared in 7 liters of fermentation medium (phosphate-buffered saline, pH 7.2). The temperature is about 25 "C. Immediately after inoculating the bioreactor with sheep blood, 2 samples are taken. The first sample is used to determine the hemoglobin content in the supernatant at the time of inoculation second sample is left in a tube for reference for the duration of the entire experiment. After the experiment is completed, the free hemoglobin content is determined. The mechanical destruction of the cells is determined by the release of hemoglobin in the supernatant by measurement at 540 nm. The maximum value of erythrocyte lysis is determined after osmotic disruption of the organelles in water.
• fermentation medium phosphate-buffered saline, pH 7.2
• the samples are centrifuged at 1000 rpm for 10 minutes in a laboratory centrifuge.
• a commercial overturning fermenter (manufacturer, Rosenmund, Liestal Switzerland) is used as the bioreactor.
• the mechanical destruction of the cells is determined depending on the speed of the pump wheel (propeller). The corresponding values at a pump wheel speed of 250 rpm and the associated zero value determination at the time of inoculation are shown in the table.
• the table shows that at a pump wheel speed of 250 rpm, the number of cells in the bioreactor has not changed compared to the reference, taking into account the measurement inaccuracy of the method.

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• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Microbiology (AREA)
• Sustainable Development (AREA)
• Biotechnology (AREA)
• Biomedical Technology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Analytical Chemistry (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)

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Citations (11)

• 1988
  • 1988-12-23 DE DE19883843493 patent/DE3843493C1/de not_active Expired
• 1989
  • 1989-12-19 WO PCT/EP1989/001561 patent/WO1990007570A1/de not_active Ceased

Patent Citations (11)

Non-Patent Citations (1)

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