WO1991011508A1 - Procede de culture de cellules - Google Patents

Procede de culture de cellules Download PDF

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Publication number
WO1991011508A1
WO1991011508A1 PCT/EP1991/000001 EP9100001W WO9111508A1 WO 1991011508 A1 WO1991011508 A1 WO 1991011508A1 EP 9100001 W EP9100001 W EP 9100001W WO 9111508 A1 WO9111508 A1 WO 9111508A1
Authority
WO
WIPO (PCT)
Prior art keywords
bubbles
medium
cells
mol
microbubbles
Prior art date
Application number
PCT/EP1991/000001
Other languages
English (en)
Inventor
Isabella Maria Leonarda JÖBSES
Gerardus Cornelis Hoogendoorn
Johannes Margaretha Maria Oomens
Original Assignee
Akzo N.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Akzo N.V. filed Critical Akzo N.V.
Priority to KR1019920701825A priority Critical patent/KR920703788A/ko
Priority to AU70787/91A priority patent/AU651080B2/en
Publication of WO1991011508A1 publication Critical patent/WO1991011508A1/fr
Priority to FI922409A priority patent/FI922409A/fi

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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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/02—Tissue, human, animal or plant cell, or virus culture apparatus with means providing suspensions

Definitions

  • the present invention relates to a method and apparatus for supplying cultured cells with gaseous nutrients, wherein the gaseous nutrients are delivered in the form of bubbles to a reactor comprising culture medium and cells in or on micro-carriers or hollow fibers, or in suspension.
  • Important biochemicals include, among others, monoclonal antibodies, recombinant proteins, cytokines etc.
  • Vaccins, artificial pancreas and artificial livers may be examples of cells useful as endproducts.
  • mammalian and other sensitive cells usually the methods described for micro organisms are used in large scale cell cultures. But, unlike yeasts and bacteria, mammalian cells and plant cells are much more sensitive to mechanical forces such as shear, which occur upon stirring of the culture medium and upon supplying the culture medium with gaseous nutrients. Also, though anchorage dependent cells themselves may be cultured more or less protected from shear forces, their products, which may be sensitive to shear as well, are not. Moreover, microcarriers or anchorage dependent cells growing on microcarriers are often also sensitive to shear. Therefore the large scale cell culture of mammalian or plant cells, as well as the large scale culture of cells producing shear-sensitive biochemicals or culture methods using microcarriers, have so far achieved limited success at best.
  • This invention relates to the supply of gaseous nutrients to culture media comprising cells or cells on microcarriers in suspension.
  • Usual methods of supplying gaseous nutrients to such media are culturing in aerated stirred tankreactors and culturing in reactors comprising columns of bubbles of gaseous nutrients.
  • suspension cells are very sensitive and which are caused by the relativily high liquid velocities which occur when using these methods. 2) A very large fraction of the volume of the gaseous nutrients escapes from the liquid surface, which leads to the formation of foam. The foam layer will entrain the cells or microcarriers, resulting in cell death, therefore anti-foam agents have to be added to the medium.
  • anti-foam agents may lead to decreased cell growth as well as to a decreased production of biochemicals and "may hamper down-stream processing.
  • the present invention provides a method and an apparatus for supplying culture media with gaseous nutrients which do not have the aforementioned disadvantages.
  • the invention relates to a method of providing a bioreactor comprising a culture medium and cells (optionally on/in microcarriers) with bubbles of gaseous nutrients wherein substantially all of the bubbles are dissolved and most of the gaseous nutrients consumed before penetrating the fluid surface.
  • the bubbles should have an initial diameter equal to or smaller than about 200 ⁇ m.
  • the bubbles according to the invention are further referred to as microbubbles.
  • the microbubbles Apart from rising to the surface according to Stokes law the microbubbles are subject to forces which move them in other directions.
  • the moving liquid will entrain the microbubbles if its mean velocity is higher than the rising velocity of the microbubbles. This is the case for all practical applicable bioreactors.
  • the effect of stirring is that bubbles have a longer residence time in the medium. This can be explained by several observations. First, the bubbles reaching the surface may become entrained in a downward liquid flow and therefore not break through the surface. Secondly, the bubbles which do reach the surface need some time to break through that surface. They may even need to associate with other bubbles before they lead to foam. During this time that they are "hanging" below the surface they may also become entrained in a downward liquid flow.
  • stirring generally enlarges the mean residence time of microbubbles in a certain bioreactor compared to the residence time of microbubbles is a non-stirred bioreactor, the given formula for non- stirred reactors gives an estimation for a "safe" size of microbubbles which will not lead to foam in stirred reactors with the same column height of medium.
  • microbubbles may be chosen larger than calculated according to that formula.
  • a suitable method of producing these microbubbles can be arrived at by using a saturator.
  • a saturator is an external column separated from the cell column reactor comprising culture medium which is practically free of cells.
  • the culture medium is saturated with gaseous nutrients at a pressure greater than atmospheric pressure (for instance 5 bar).
  • the saturated medium is transfered to the bottom of the cell culture reactor where it releases the gaseous nutrients as microbubbles, because of oversaturation at this lower pressure.
  • the formation of microbubbles from an oversaturated liquid has been described in Perry R.H., Green D., 1988, Perry's Chemical Engineers' Handbook, 6 th edition, McGraw-Hill Bookco.
  • the microbubbles were so far only produced for studying flotation behavior of these bubbles, which means they have to rise to the surface.
  • gaseous nutrient delivered to the medium as microbubbles is pure oxygen.
  • the above described method of oxygenation has an advantage over normal external oxygenation, because at an over pressure of for instance 5 bar, 6 times as much oxygen can be dissolved in the medium.
  • the oxygen supply can be provided within the fresh medium supply, or, depending on the rate of consumption, in a recirculation loop, with a flow at least six times lower than with known methods.
  • the methods according to the invention are also very suitable for supplying cultures of anchorage dependent cells with gaseous nutrients. Especially when the anchorage dependent cells are grown attached to carriers which are kept in suspension, such as cells attached to microcarriers.
  • the invention also relates to an apparatus for carrying out the methods according to the invention.
  • the apparatus usually comprises a reaction vessel, provided with a means for supplying gaseous nutrients, such as a saturator. Although other methods of providing microbubbles are also included.
  • the reaction vessel will also be provided with an outlet for culture medium and cells which will lead to a separator where the medium and the cells are separated from the waste products and possibly the end product and recirculated together with new medium into the reaction vessel.
  • the medium may also be separated from the cells and be lead through the saturator to be supplied with gaseous nutrients.
  • the reaction vessel may be supplied with a stirring device although this is not absolutely necessary. Of course, in continuous processes new medium may also be provided separately from the recirculated medium.
  • An apparatus according to the invention is represented in fig. 1.
  • a reaction vessel (4) is represented, provided with an inlet (3) for medium saturated with gaseous nutrients through a saturator (1).
  • the reaction vessel is also provided with an outlet (5) for cells and medium from which in a first separator (6) the cells are separated and lead back to the vessel (7).
  • a part of the medium is then recirculated (9) to the saturator and mixed with fresh medium (10) just before entering the saturator.
  • "old" medium is withdrawn.
  • CHO cells Choinese Hamster Ovary cells; 1.8x10 10 cells/l are cultured on microcarriers (Cytodex 3).
  • the apparatus is provided with a stirring device which stirs at low shear rates (100 rpm).
  • the concentration of oxygen is measured with an oxygen electrode and recorded.
  • the oxygen is provided by a saturator at a pressure of 5 bar. Regulation of the amount of oxygen delivered to the desired value is carried out by adjusting the flow through the saturator.
  • the microcarriers were separated from the flow through the saturator by filtration with a screen filter with pores of 44 ⁇ m diameter.
  • the culture medium comprises DMEM/F12 with 5% FCS (Fetal Calf Serum). The reactor is kept at a temperature of 37 °C.
  • the peffusion rate was 40 liter/day and the medium recirculation rate for oxygenation was 4 liter/hr.
  • a steady state cell density of 3.10 10 cells/liter was maintained during 2.5 months.
  • microbubbles and possible "shear" effects caused by cell-microbubble collision the long term cultivation of cells in the presence of microbubbles appeared to be harmless with regard to cell growth and product formation rate.
  • a microcarrier settler was placed inside the fermentor to obtain a microcarrier-free medium for the
  • the static zone in the settler was 12 cm high.
  • microbubbles are dissolved before they reach the fluid surface.
  • microbubbles were small enough to be kept in suspension by stirring until they were
  • the bubbles are dissolved at 80% of the height of the liquid column 5)
  • the bubbles rise as rigid spheres at a velocity calculated according to Stokes' law.
  • ⁇ C driving force for mass transfer, i.e.
  • A bubble area [m 2 ]
  • V b bubble volume [m 3 ]
  • the mean velocity is defined as: (3)
  • the mean velocity is therefore:
  • the bubbles have to be dissolved before reaching the surface so t dis ⁇ t r
  • V b .d b 3
  • the mean velocity is defined as: v(t)dt (7)
  • V (t) V (t)
  • the initial bubble diameter can be calculated from
  • the maximal initial bubble diameter is calculated to be
  • the residence time of the bubbles must be equal to the total dissolution time:

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

Abstract

L'invention concerne un procédé et un appareil permettant d'apporter des nutriments gazeux à des cellules de culture, ces nutriments ayant la forme de micro-bulles et une taille choisie de façon que la plupart des bulles soient dissoutes et consommées avant d'atteindre la surface du milieu de culture. Les micro-bulles sont produites de préférence avec un saturateur.
PCT/EP1991/000001 1990-02-01 1991-01-29 Procede de culture de cellules WO1991011508A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
KR1019920701825A KR920703788A (ko) 1990-02-01 1991-01-29 세포 배양 방법
AU70787/91A AU651080B2 (en) 1990-02-01 1991-01-29 Method for culturing cells
FI922409A FI922409A (fi) 1990-02-01 1992-05-26 Foerfarande foer cellodling.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP90200243 1990-02-01
EP90200243.5 1990-02-01

Publications (1)

Publication Number Publication Date
WO1991011508A1 true WO1991011508A1 (fr) 1991-08-08

Family

ID=8204928

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1991/000001 WO1991011508A1 (fr) 1990-02-01 1991-01-29 Procede de culture de cellules

Country Status (7)

Country Link
EP (1) EP0513057A1 (fr)
JP (1) JPH05503848A (fr)
KR (1) KR920703788A (fr)
AU (1) AU651080B2 (fr)
CA (1) CA2075059A1 (fr)
FI (1) FI922409A (fr)
WO (1) WO1991011508A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USH1532H (en) * 1993-11-03 1996-05-07 Genetics Institute, Inc. Adaption of mammalian cell lines to high cell densities
US6949244B1 (en) 1995-12-20 2005-09-27 The Board Of Trustees Of The University Of Kentucky Murine monoclonal anti-idiotype antibody 11D10 and methods of use thereof
WO2010069492A1 (fr) * 2008-12-20 2010-06-24 Bayer Technology Services Gmbh Bioréacteur
WO2020051042A1 (fr) * 2018-09-06 2020-03-12 Momenta Pharmaceuticals, Inc. Procédés de culture cellulaire continue
US11719704B2 (en) 2015-12-30 2023-08-08 Momenta Pharmaceuticals, Inc. Methods related to biologics
CN116606713A (zh) * 2023-07-12 2023-08-18 无锡海拓环保装备科技有限公司 一种菌群培养装置

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011078371A (ja) * 2009-10-09 2011-04-21 Tomotaka Marui 細胞変化を促進する微小気泡含有組成物、およびその微小気泡含有組成物を製造する装置、ならびに微小気泡含有組成物を用いた細胞変化促進方法。
JP5549209B2 (ja) * 2009-12-11 2014-07-16 株式会社Ｉｈｉ 付着性細胞培養装置
JP2014183742A (ja) * 2013-03-21 2014-10-02 Yamagata Univ ポリ塩化ビフェニル類無害化複合組成物ならびにその製造方法
WO2016079820A1 (fr) * 2014-11-19 2016-05-26 三菱化学エンジニアリング株式会社 Dispositif de réaction biologique, procédé de réaction biologique, structure poreuse porteuse de micro-organisme aérobie à utiliser dans le dispositif de réaction biologique, et procédé de production de structure poreuse
WO2017017830A1 (fr) * 2015-07-30 2017-02-02 三菱化学エンジニアリング株式会社 Bioréacteur utilisant des micro/nano-bulles enrichies en oxygène et procédé de bioréaction utilisant le bioréacteur utilisant des micro/nano-bulles enrichies en oxygène
JP6138390B1 (ja) * 2017-01-25 2017-05-31 三菱化学エンジニアリング株式会社 マイクロナノバブルを用いた生物反応装置およびこの生物反応装置を用いた生物反応方法
KR102476633B1 (ko) 2018-06-27 2022-12-09 후지필름 가부시키가이샤 세포 배양 방법, 생산물의 제조 방법, 및 세포 배양 장치

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3968035A (en) * 1973-04-05 1976-07-06 Eli Lilly And Company Super-oxygenation method
US4654305A (en) * 1981-08-25 1987-03-31 The Board Of Governors For Higher Education State Of Rhode Island And Providence Plantations Multiphase reactor systems based on foams for simultaneous growth and separation of products

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3968035A (en) * 1973-04-05 1976-07-06 Eli Lilly And Company Super-oxygenation method
US4654305A (en) * 1981-08-25 1987-03-31 The Board Of Governors For Higher Education State Of Rhode Island And Providence Plantations Multiphase reactor systems based on foams for simultaneous growth and separation of products

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Patent Abstracts of Japan, vol. 10, no. 219 (C-363)(2275), 31 July 1986; & JP-A-6158582 (ADVANCE RES. & DEV. CO. LTD) 25 March 1986 *
Patent Abstracts of Japan, vol. 12, no. 213 (C-505)(3060), 17 June 1988; & JP-A-6314687 (MARUBISHI BAIOENJI K.K.) 21 January 1988 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USH1532H (en) * 1993-11-03 1996-05-07 Genetics Institute, Inc. Adaption of mammalian cell lines to high cell densities
US7399849B2 (en) 1995-01-29 2008-07-15 University Of Kentucky Research Foundation Murine monoclonal anti-idiotype antibody 11D10 and methods of use thereof
US6949244B1 (en) 1995-12-20 2005-09-27 The Board Of Trustees Of The University Of Kentucky Murine monoclonal anti-idiotype antibody 11D10 and methods of use thereof
WO2010069492A1 (fr) * 2008-12-20 2010-06-24 Bayer Technology Services Gmbh Bioréacteur
US11719704B2 (en) 2015-12-30 2023-08-08 Momenta Pharmaceuticals, Inc. Methods related to biologics
WO2020051042A1 (fr) * 2018-09-06 2020-03-12 Momenta Pharmaceuticals, Inc. Procédés de culture cellulaire continue
CN113286650A (zh) * 2018-09-06 2021-08-20 动量制药公司 连续细胞培养方法
EP3846924A4 (fr) * 2018-09-06 2022-07-06 Momenta Pharmaceuticals, Inc. Procédés de culture cellulaire continue
CN116606713A (zh) * 2023-07-12 2023-08-18 无锡海拓环保装备科技有限公司 一种菌群培养装置
CN116606713B (zh) * 2023-07-12 2024-03-15 无锡海拓环保装备科技有限公司 一种菌群培养装置

Also Published As

Publication number Publication date
EP0513057A1 (fr) 1992-11-19
FI922409A0 (fi) 1992-05-26
JPH05503848A (ja) 1993-06-24
AU7078791A (en) 1991-08-21
KR920703788A (ko) 1992-12-18
CA2075059A1 (fr) 1991-08-02
AU651080B2 (en) 1994-07-14
FI922409A (fi) 1992-05-26

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