WO1993005144A1 - Procede de culture de cellules de mammiferes dans un reacteur a lit fluidise - Google Patents

Procede de culture de cellules de mammiferes dans un reacteur a lit fluidise Download PDF

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Publication number
WO1993005144A1
WO1993005144A1 PCT/EP1992/001918 EP9201918W WO9305144A1 WO 1993005144 A1 WO1993005144 A1 WO 1993005144A1 EP 9201918 W EP9201918 W EP 9201918W WO 9305144 A1 WO9305144 A1 WO 9305144A1
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WO
WIPO (PCT)
Prior art keywords
cells
cell
bed reactor
mammalian cells
cultivation
Prior art date
Application number
PCT/EP1992/001918
Other languages
German (de)
English (en)
Inventor
Peter Reuschenbach
Original Assignee
Basf Aktiengesellschaft
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 Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Publication of WO1993005144A1 publication Critical patent/WO1993005144A1/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
    • C12N5/0075—General culture methods using substrates using microcarriers
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation
    • C12M25/20—Fluidized bed
    • 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
    • C12N2533/10—Mineral substrates
    • C12N2533/12—Glass
    • 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
    • C12N2533/70—Polysaccharides

Definitions

  • the cultivation of mammalian cells is the basis for a large number of biotechnological production processes, in particular the production of pharmaceutical proteins.
  • Carrier bodies made of glass are reusable and autoclavable, however, the cell yield is with these
  • EP 303 262 discloses a support body made of glass or ceramic with a content-containing surface layer; however, the use of these carrier bodies for mammalian cell culture is not described.
  • the object of the present invention was to provide a method for the cultivation of mammalian cells immobilized on support bodies, in which the support bodies ensure a high cell yield, are autoclavable, easy to clean and reusable.
  • the method for cultivating mammalian cells in a fluidized bed reactor is particularly advantageous if the mammalian cells are immobilized on porous glass support bodies, the surface layer of which contains amino groups bonded to a support. It has also been found that a surface layer containing diethylaminoethyl dextran (DEAE dextran) is particularly suitable. Furthermore, it was found that the mouse fibroblast cell line C-127 (ATCC CRL 1616) can be cultivated particularly well with this method.
  • the porous glass support body can be in any shape; they are preferably used in a spherical shape.
  • the size of the carrier body is expediently from 0.4 to 5 mm, preferably 1 to 2 mm.
  • the pore size of the porous support body is generally 20 to 500 ⁇ m, preferably 60 to 300 ⁇ m.
  • micropores of 1 to 10 ⁇ m can also be present in the carrier bodies.
  • Open-pore sintered glass carrier bodies e.g. of the Siran® brand (Schott, Mainz).
  • the carrier bodies can consist of soda-lime glass or borosilicate glass.
  • Sintered glass carrier bodies made of borosilicate glass are particularly preferred.
  • Suitable amino groups are dialkylamino groups, in particular dialkylaminoalkyl groups, the alkyl groups advantageously having 1 to 4 carbon atoms.
  • Amino groups bound to a carrier include amino groups which are linked to a polymer, e.g. are covalently linked to a polysaccharide. Amino groups which are bonded to glucans, in particular dextran, are preferred. Diethyla inoethyl-dextran (DEAE-dextran) is particularly preferably used.
  • the glass support bodies containing amino groups bonded to the surface of the support are commercially available (Schott, Mainz, Product Information No. 6196d).
  • Suitable mammalian cells for the method according to the invention are both adherent and suspension cells growing in suspension. Permanent cell lines or primary cultures can be used. Genetically modified (recombinant) cell lines can also be used. Hybridoma cells and fibroblasts, in particular the mouse fibroblast cell line C-127 (ATCC CRL 1616) are preferably used. The cell line C-127 secreting the tissue plasminogen activator (t-PA), which transforms with a bovine papomomirus expression vector, is particularly preferred had been used. The production of this cell line is by Reddy et al. (J. Cell. Biochem., Supplement 10 D, 154, 1986).
  • tissue plasminogen activator t-PA
  • bioreactors are suitable as fluidized bed reactors for the process according to the invention, such as those e.g. by Lobby and Griffiths (Cytotechnology 1, 339-346, 1988; Advances in Ani.mal Cell Biology and Technology for Bioprocesses, Eds. Spier et al., Butterworths, Guilford, 336-344, 1989).
  • the volume of the support body in the unexpanded state is usually 10 to 80%, preferably 20 to 60% of the reactor volume.
  • All conventional cell culture media can be used as nutrient media for cultivating the mammalian cells.
  • Serum-free culture media can also be used. Whether serum needs to be added depends on the mammalian cell used.
  • the immobilization of the mammalian cells on the carrier bodies expediently takes place in that the mammalian cells are pre-cultivated by conventional cell culture techniques and then introduced into the fluidized bed reactor containing the carrier bodies, so that a cell density in the reactor of 5-10 '+ to 1-106, preferably from 1 to 8-105 cells / ml.
  • the cultivation of the mammalian cells according to the method according to the invention can be carried out continuously or batchwise. However, work is preferably carried out continuously.
  • a large number of cells is expediently generated by cell multiplication and then in a second phase (the production phase) these cells form the valuable substance.
  • the method according to the invention makes it possible to cultivate mammalian cells to a high density on porous support bodies.
  • the carrier bodies have excellent mechanical properties, so that in the fluidized bed reactor e.g. there is no abrasion which reduces the cell yield.
  • these carrier bodies can be autoclaved by superheated steam; you can e.g. be sterilized in the reactor before cultivation begins. This also makes it possible to reuse the carrier bodies after cleaning and sterilization.
  • the mammalian cells were first grown in commercially available cell culture bottles. For this purpose, cell culture bottles were inoculated with 0 r 5 - 1 r 0 x 105 cells.
  • DMEM medium 4.5 g glucose per liter, 584 mg glutamine per liter
  • fetal calf serum 10%)
  • polyalkylene glycol were used as growth medium
  • the fluidized bed reactor consisted of a 3.2 cm x 28 cm long glass cylinder provided with a double jacket and conically converging at the lower end and a 2.8 x 13 cm conditioning vessel also provided with a double jacket.
  • the total volume of the unit including the connecting hoses was 380 ml.
  • the medium was conveyed with the help of a peristaltic pump between the conditioning vessel and the fluidized bed reactor at a speed of 20 to 60 cm per minute.
  • Fresh medium was fed into the conditioning vessel via a peristaltic pump and used medium was removed from the conditioning vessel via an overflow.
  • the media exchange was started on the 3rd day with an exchange rate of 0.5 reactor volume per day and gradually increased in the production phase to an average value of 4.3 reactor volume per day.
  • the system was tempered via the double jackets of the reactor and the conditioning vessel using a water bath.
  • the incubation temperature was 37 ° C in the first 6 days, then 35 ° C.
  • the oxygen partial pressure was set to 30% (air saturation). This partial pressure was maintained with the aid of a measuring and control unit by automatically introducing air into the head space or oxygen into the liquid of the conditioning vessel.
  • the pH setpoint was regulated by automatic metering of C0 2 into the head space of the conditioning vessel between pH 6.9 and 7.2.
  • porous sintered glass carrier bodies with a DEAE dextran surface layer and having a diameter of 1 to 2 mm and a pore size between 60 and 300 ⁇ m served as carrier material for the C-127 cells.
  • the reactor was inoculated with 7 x 105 cells / ml.
  • DMEM medium 4.5 g glucose per liter, 584 mg glutamine per liter
  • fetal calf serum 10%
  • Pluronic F-68® (0.157o) were used as culture medium in the first 6 days.
  • a medium consisting of cell culture medium (IGl®, Genzyme), glutamine (292 mg / 1), insulin (5 mg / 1), polyalkylene glycol (Pluronic F-68®) (0.15%), bovine serum albumin (150 mg / l) and fetal calf serum.
  • the serum concentration in the medium was gradually reduced from 10% at the beginning of the fermentation to 0% on the 16th day.
  • the amidolytic activity of the secreted protein was determined using the COA set (S 2251, Kabi vitrum).
  • the cell number was derived from the oxygen consumption.
  • Table I Cultivation of C-127 cells on Siran® carrier bodies containing DEAE dextran surface layer. The data were determined in the production phase (day 16-106, serum-free culture medium).
  • the production phase lasted 49 days.
  • Table 2 Cultivation of C-127 cells on collagen carriers (Verax VX-100®). The data were determined in the production phase (16th-65th day, serum-free culture medium)

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

Abstract

L'invention concerne un nouveau procédé de culture de cellules de mammifères dans un réacteur à lit fluidisé.
PCT/EP1992/001918 1991-08-30 1992-08-21 Procede de culture de cellules de mammiferes dans un reacteur a lit fluidise WO1993005144A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE4128953A DE4128953A1 (de) 1991-08-30 1991-08-30 Verfahren zur kultivierung von saeugerzellen im fliessbettreaktor
DEP4128953.6 1991-08-30

Publications (1)

Publication Number Publication Date
WO1993005144A1 true WO1993005144A1 (fr) 1993-03-18

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1992/001918 WO1993005144A1 (fr) 1991-08-30 1992-08-21 Procede de culture de cellules de mammiferes dans un reacteur a lit fluidise

Country Status (3)

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DE (1) DE4128953A1 (fr)
MX (1) MX9204984A (fr)
WO (1) WO1993005144A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE332364T1 (de) 1999-02-22 2006-07-15 Transgene Sa Verfahren zur gewinnung von purifizierter virenzuammensetzung
DE10048822A1 (de) * 2000-09-29 2002-04-18 Nimbus Biotechnologie Gmbh Verfahren zur Immobilisierung von Lipidschichten

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4224413A (en) * 1975-05-21 1980-09-23 Beecham Group Limited Cell culture method
FR2510604A1 (fr) * 1981-07-15 1983-02-04 Yeda Res & Dev Production d'activateur de plasminogene
EP0303262A2 (fr) * 1987-08-13 1989-02-15 Forschungszentrum Jülich Gmbh Support inorganique avec une couche superficielle contenant des amines pour l'immobilisation de micro-organismes ou de cellules, procédé d'obtention et application
EP0338716A2 (fr) * 1988-04-21 1989-10-25 Berlex Laboratories, Inc. Méthode et appareillage pour la production de protéines non dégradées par des cellules de mammifères

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4224413A (en) * 1975-05-21 1980-09-23 Beecham Group Limited Cell culture method
FR2510604A1 (fr) * 1981-07-15 1983-02-04 Yeda Res & Dev Production d'activateur de plasminogene
EP0303262A2 (fr) * 1987-08-13 1989-02-15 Forschungszentrum Jülich Gmbh Support inorganique avec une couche superficielle contenant des amines pour l'immobilisation de micro-organismes ou de cellules, procédé d'obtention et application
EP0338716A2 (fr) * 1988-04-21 1989-10-25 Berlex Laboratories, Inc. Méthode et appareillage pour la production de protéines non dégradées par des cellules de mammifères

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOTECH 83. 1983, ONLINE PUBLICATIONS,NORTHWOOD, UK. Seiten 853 - 862 CLARK, J. & HIRTENSTEIN, M. 'Recent developments in microcarrier cell culture.' *
Journal of Cellular Biochemistry, Supplement 10d, 1986. Abstracts. P. 154, abstract no. 0218. Reddy, V.B. et al. in der Anmeldung erwähnt "Construction and analysis of BPV vectors for the expression of t-PA in mouse cells". *
TIBTECH. Bd. 8, August 1990, ELSEVIER SCIENCE PUBLISHERS, UK. Seiten 204 - 209 LOOBY, D. & GRIFFITHS,B. 'Immobilization of animal cells in porous carrier culture.' in der Anmeldung erwähnt *

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Publication number Publication date
MX9204984A (es) 1993-04-01
DE4128953A1 (de) 1993-03-04

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