WO2005003287A1 - Procedes de culture cellulaire - Google Patents

Procedes de culture cellulaire Download PDF

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Publication number
WO2005003287A1
WO2005003287A1 PCT/GB2004/002822 GB2004002822W WO2005003287A1 WO 2005003287 A1 WO2005003287 A1 WO 2005003287A1 GB 2004002822 W GB2004002822 W GB 2004002822W WO 2005003287 A1 WO2005003287 A1 WO 2005003287A1
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WO
WIPO (PCT)
Prior art keywords
volume
foam
cell
cells
culture medium
Prior art date
Application number
PCT/GB2004/002822
Other languages
English (en)
Inventor
Adrian Parton
Original Assignee
Cytrix Technologies Limited
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 Cytrix Technologies Limited filed Critical Cytrix Technologies Limited
Publication of WO2005003287A1 publication Critical patent/WO2005003287A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • the present invention relates to methods of cell culture in which an open cell foam is used as a cell culture support, the volume of the foam being varied by compression during the phases of cell culture leading to a cell concentration and/or metabolite production step performed in the presence of a reduced volume of culture medium, so enabling the production of a higher concentration of metabolite.
  • Traditional cell culture methods have relied on the use of continuous solid surfaces and in the laboratory this has typically been confined to Petri dishes, flat sided flasks or roller bottles. Cells coming in contact with these solid phase surfaces will typically grow until contiguous, confluent monolayers are reached at which point cell growth ceases. This is characteristic of non-cancerous cells.
  • the present invention now provides a method of cell culture comprising a cell seeding step and a cell concentration and/or metabolite production step, wherein:
  • cells to be cultured are seeded onto a culture support comprising a compressible open cell foam in a first volume of a fluid culture medium, and in said cell concentration and/or metabolite production step, the volume of culture medium is reduced, the foam is compressed and the cells are further cultured to produce a cell metabolite in the culture medium,
  • the compressible foam during at least 50% of the cell concentration and/or metabolite production step is compressed to a production volume that is no more than 60% of the free volume of the foam, the volume of culture medium during the cell concentration and/or metabolite production step is no more than 120% of the production volume of the foam, and during the cell seeding step, the volume of the culture medium is at least 150% of the volume of the culture medium during the cell concentration and/or metabolite production step and the foam is at a volume which is at least 150% of said production volume during at least a part of the cell seeding step.
  • the volume of the foam is varied by expansion toward or to the said free volume and re-compression to distribute seeding cells within the foam.
  • said compression of the foam may preferably reduce its volume to a minimum volume which is no more than 75%, more preferably no more than 50%, still more preferably no more than 25%, of said free volume.
  • a minimum volume which is no more than 75%, more preferably no more than 50%, still more preferably no more than 25%, of said free volume.
  • the trigger point may be a variable that is characteristic of individual cell lines.
  • An example of a trigger point for the 1H8 hybridoma cell line is in the range pH 7.0 to pH 6.8, but more preferably pH 7.0.
  • This compression of the foam may again reduce its volume to a minimum volume which is no more than 75% of said free volume, or more preferably the lesser figures given above for compression in the seeding phase.
  • the volume of the foam is progressively expanded to a maximum culturing volume, which is suitably the free volume of the foam.
  • said distribution of growing cells within the foam by expansion and re-compression of the foam is repeated at intervals, preferably at least twice, more preferably at least 5 times .
  • the desired metabolite is secreted into the medi ⁇ m in the metabolite production step, it may be harvested by removal of the medium and subsequent purification.
  • the cells themselves may be harvested and the reduction of the volume of the medium during the cell concentration or metabolite production phase will have the benefit of reducing the cost of the medium consumed and of reducing the volume from which the cells need to be harvested.
  • the cells themselves, rather than any specific metabolite, may be the desired end product.
  • the foam is re-expanded and further cell culture medium is added whereby the volume of the cell culture medium is increased to at least 150% of its volume during the cell concentration and/or metabolite production step and after a further period of culture a second cell concentration and/or metabolite production step is commenced in which the compressible foam during at least 50% of the cell concentration and/or metabolite production step is compressed to a production volume that is no more than 60% of the free volume of the foam.
  • the foams can be compressed and the amount of media reduced to provide an effective way of concentrating the cells for production purposes.
  • the timing of the different phases of the culture process can be predetermined in advance to suit the cells being cultured or can be determined interactively in response to parameters measured during cell culture. These may be parameters that are traditionally monitored in cell culture processes in order to be informed as to when growth of the cells, i.e. increase in cell number, has commenced or to be informed as to when the cells in the production phase are reaching a crisis point and need to be rescued if they are to survive to produce more metabolite in a future production phase .
  • Cells which may be cultured according to the invention include a wide variety of cell types including bacterial cells, fungi such as yeast, insect cells, mammalian (adherent and non-adherent) e.g. hybridoma and CHO cells etc .
  • Metabolites that may be produced by the cells may include antibodies, e.g. monoclonal antibodies, proteins of all kinds (whether secreted into the medium or not) , hormones, glycoproteins, lectins, and more generally organic molecules/compounds such as alcohols, fatty acids or sugars .
  • FIG. 1 is a schematic view of apparatus for use in practising the methods of the invention
  • Figure 2 is a vertical cross section through a cell culture bioreactor for use in the apparatus of Figure 1.
  • the bioreactor apparatus of Figure 1 comprises a bioreactor cylinder (1) with an outlet (2) at the base which allows liquid to be removed, an inlet (3) with a central porous plunger (4) which is used to generate the compression/decompression cycles.
  • Open cell reticulated foam discs (5) are held between the base of the cylinder and the plunger.
  • An optional motor 6 can be used to drive the plunger according to pre-programmed schedules.
  • a handle may be provided for manual compression and decompression of the foam.
  • the use of a peristaltic pump (7) can be incorporated to provide mixing of the sample.
  • Sensors 8 can be provided to monitor the condition of the culture medium and a gas exchange valve 9 can be used to introduce gas such as air into the culture medium.
  • a glass closed bottomed cylinder 21 has a screw threaded top receiving a threaded cap 30 which has a top plate pierced by four ports (two visible) 32,34 disposed at the corners of a square.
  • Respective tubes 36 pass through each of these ports, in sealing engagement with bungs 35 and extend to close to the bottom of the cylinder 21.
  • a further port 38 at the centre of the top plate of the cap 30 receives a plug 40 which has a threaded through bore in screw engagement with which is a threaded shaft 42 having at its upper end a plain (unthreaded portion) terminating in a T handle 44 and at its lower end a circular disc 46 acting as a piston displaceable up and down the cylinder by the rotation of handle 44.
  • Discs of open cell reticulated foam (not shown) are positioned below the disc 46.
  • the plain portion of shaft 42 is surrounded by an elongate collar48 which can be filled with liquid to maintain a sterile seal of the cylinder.
  • Two of the tubes 36 are connected via a pump (not shown) as inlet and outlet respectively for the circulation of culture medium (as in Figure 1) . Another of said tubes 36 is used for introducing and/or removing culture medium, and the last of them is used for introducing gas to the medium. Further similar tubes may be provided for other purposes as desired.
  • CD14 Cells in 2 ml RPMI 1640 medium containing 10% foetal calf serum were either cultured alone or in the presence of 0.5 cm diameter sterile foam (60 ppi (24 pores per cm)) disks. Cells were introduced into the foam and bubbles removed by gentle compression/decompression. Microscopic examination showed that the cells collect in foam pockets at different planes throughout the culture. Supernatants were harvested over 4 days at 37°C/5% C0 2 and comparisons were made between wells containing cells alone and wells containing foam compressed to half its free volume. The volume of culture medium used during the production phase of the production cycles in the presence of the foam was half that used in its absence.
  • Standard ELISA was used to assay mouse IgG in the supernatants. Results were 6.4 ⁇ g/ml monoclonal antibody in the absence of foam compared with 19.8 ⁇ g/ml in foam cultures. Thus, in this example, a 3-fold increase in antibody productivity was obtained.
  • Step 1 Using the same cells as were used in Example 1, a similar comparison between culture in open wells and in compressed foam was conducted. With ⁇ 2ml medium in each well after cell seeding, an intitial ⁇ 2ml free volume of the foam was compressed to ⁇ 0.5ml after distribution of cells and bubble removal by cycles of compression/de-compression cycling between 2 ml and 0.5 ml.
  • Step 2 After 24 hours during which time cell growth had started (known for this hybridoma - but could be readily monitored by standard techniques of cell sampling, pH, [glucose] etc) , decompression/compression cycles were carried out to distribute cells and the foam was expanded to ⁇ lml, thus allowing a further phase of growth in a larger volume of foam.
  • Step 3 After a further 24 hours, step 2 was repeated leaving the foam fully decompressed at 2ml.
  • Step 4 After a further 24hrs of growth, the foam was compressed to 1ml to commence the cell concentration and/or metabolite production step and 1ml of supernatant cell culture medium was removed. From this a first quantity of antibody was recovered (Harvest 1) .
  • Step 5 The production phase (in 1ml medium) was allowed to proceed for 24hours after which time the foam was fully compressed and supernatant harvested. From this a second quantity of antibody was recovered (Harvest 2) .
  • Step 6 Monitoring of the biomass during concentrated production cycles using standard parameters - e.g. cell sampling, pH, [glucose] would allow steps to be taken (e.g. medium change, reduction of biomass) before crisis such that further production cycles could proceed (although in this example, additional production cycles were not carried out.
  • standard parameters e.g. cell sampling, pH, [glucose]
  • the culture was incubated at 37°C in an atmosphere of 5% C0 2 in air. After 3 days, 100 ml of culture was removed and replaced with 100 ml fresh medium. Thereafter, medium was replenished every 1-2 days with a feed volume of 75-100 ml.
  • Example 4 Gradual seeding of foam bioreactor with hybridoma cells and subsequent monoclonal antibody production
  • Phase 1 2xl0 5 viable cells/ml set up in 75 ml RPMI containing 10% FCS in bioreactor apparatus as described in Figure 1 fitted with a motor-driven plunger.
  • Foam cylinder (diameter 50mm, height 55mm) of 60ppi (24 pores per cm) open reticulated polyester foam was used. Plunging action was used (1) to expel air bubbles from the foam and (2) to distribute cells throughout the foam pockets as follows: • 3 cycles of rapid compression/decompression (motor speed 100 rpm on screwed plunger) from maximum to 50% compression with 1-second intervals between cycles; • followed by 12 cycles of slower compression/decompression at motor speed 50 rpm from 50% compression to 0% with 15 minutes between cycles;
  • the initial culture foam volume for the first 3 days was 50% of maximum.
  • the culture was incubated at 37°C in an atmosphere of 5% C0 2 in air.
  • Phase 2 After 3 days, 37.5 ml of culture medium was added and the foam decompressed to 75% of its full volume and cycles of compression/decompression set as follows:
  • Phase 3 At the end of day 5, 37.5 ml of culture medium was added and the foam decompressed to its full volume and cycles of compression/decompression set as follows:
  • Antibody productivity was compared with a culture seeded fully at ' 150 ml with 2xl0 5 viable cells/ml in full foam volume, ie without gradual release of the foam. This culture is shown as Control Culture in the Results table.

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

Abstract

Selon l'invention, les rendements de métabolites cellulaires désirés peuvent être améliorés et la consommation du milieu de culture cellulaire peut être réduite par un procédé consistant à cultiver des cellules dans un support de culture en mousse réticulée, laquelle est comprimée de façon à présenter un volume réduit lors d'une étape de production de métabolites par rapport à un volume utilisé lors de l'ensemencement des cellules de sorte que son volume de production n'excède pas 60 % de son volume libre. Par ailleurs, le volume de milieu de culture cellulaire utilisé n'excède pas 120 % du volume de production de la mousse.
PCT/GB2004/002822 2003-06-30 2004-06-29 Procedes de culture cellulaire WO2005003287A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0315262A GB0315262D0 (en) 2003-06-30 2003-06-30 Methods of cell culture
GB0315262.6 2003-06-30

Publications (1)

Publication Number Publication Date
WO2005003287A1 true WO2005003287A1 (fr) 2005-01-13

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PCT/GB2004/002822 WO2005003287A1 (fr) 2003-06-30 2004-06-29 Procedes de culture cellulaire

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GB (1) GB0315262D0 (fr)
WO (1) WO2005003287A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102006058730A1 (de) * 2006-12-13 2008-06-19 Dot Gmbh Zellkulturträger
CN101113432B (zh) * 2007-06-25 2011-04-27 南华大学 一种无血清或低血清浓度的细胞培养方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3027305A (en) * 1959-01-16 1962-03-27 Robert R Freeman Apparatus for the cultivation of microorganisms
DE3530332A1 (de) * 1985-08-24 1986-05-15 Heinz Prof. Dr.-Ing. 1000 Berlin Brauer Puls-bioreaktor mit schwammelementen fuer die fixierung von mikroorganismen oder enzymen
JPS6336783A (ja) * 1986-07-31 1988-02-17 Ishikawa Seisakusho:Kk 多孔性素材を用いる化学反応方法及び装置
US5266476A (en) * 1985-06-18 1993-11-30 Yeda Research & Development Co., Ltd. Fibrous matrix for in vitro cell cultivation
US6329196B1 (en) * 1996-11-27 2001-12-11 William Nevil Heaton Johnson Methods and apparatus for enhancement of mass transfer of a fluid in a porous matrix system containing biomass
WO2003020871A2 (fr) * 2001-08-30 2003-03-13 Arbomedics Gmbh Procede et dispositif de culture cellulaire in vitro

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3027305A (en) * 1959-01-16 1962-03-27 Robert R Freeman Apparatus for the cultivation of microorganisms
US5266476A (en) * 1985-06-18 1993-11-30 Yeda Research & Development Co., Ltd. Fibrous matrix for in vitro cell cultivation
DE3530332A1 (de) * 1985-08-24 1986-05-15 Heinz Prof. Dr.-Ing. 1000 Berlin Brauer Puls-bioreaktor mit schwammelementen fuer die fixierung von mikroorganismen oder enzymen
JPS6336783A (ja) * 1986-07-31 1988-02-17 Ishikawa Seisakusho:Kk 多孔性素材を用いる化学反応方法及び装置
US6329196B1 (en) * 1996-11-27 2001-12-11 William Nevil Heaton Johnson Methods and apparatus for enhancement of mass transfer of a fluid in a porous matrix system containing biomass
WO2003020871A2 (fr) * 2001-08-30 2003-03-13 Arbomedics Gmbh Procede et dispositif de culture cellulaire in vitro

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN vol. 0122, no. 48 (C - 511) 13 July 1988 (1988-07-13) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102006058730A1 (de) * 2006-12-13 2008-06-19 Dot Gmbh Zellkulturträger
CN101113432B (zh) * 2007-06-25 2011-04-27 南华大学 一种无血清或低血清浓度的细胞培养方法

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