WO1995023212A1 - Procede pour cultiver des cellules de mammiferes et d'insectes dans un milieu contenant du serum de poissons - Google Patents

Procede pour cultiver des cellules de mammiferes et d'insectes dans un milieu contenant du serum de poissons Download PDF

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Publication number
WO1995023212A1
WO1995023212A1 PCT/US1994/014221 US9414221W WO9523212A1 WO 1995023212 A1 WO1995023212 A1 WO 1995023212A1 US 9414221 W US9414221 W US 9414221W WO 9523212 A1 WO9523212 A1 WO 9523212A1
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WIPO (PCT)
Prior art keywords
cells
serum
fish
culture medium
defined culture
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Application number
PCT/US1994/014221
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English (en)
Inventor
Evelyn S. Sawyer
Philip J. Sawyer
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Sea Run Holdings, Inc.
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
Priority claimed from US08/200,639 external-priority patent/US5401653A/en
Priority claimed from US08/263,250 external-priority patent/US5426045A/en
Application filed by Sea Run Holdings, Inc. filed Critical Sea Run Holdings, Inc.
Priority to AU15511/95A priority Critical patent/AU1551195A/en
Publication of WO1995023212A1 publication Critical patent/WO1995023212A1/fr

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    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
    • 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
    • C12N5/0018—Culture media for cell or tissue culture
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals

Definitions

  • the present invention relates generally to cell culture, and more specifically to the culture of mammalian cells and insect cells using a serum derived from fish.
  • the technique has significant advantages over the more commonly used technique of using blood serum derived from fetal calves or other mammals as more fully set forth below.
  • Animal cell culture is a basic technique in the fields of biology and medicine. The production of living cells in vitro, in the laboratory, permits numerous applications that would be difficult or impossible in vivo, in the living animal.
  • the culture of animal cells requires a defined medium containing specific quantities of certain chemicals, and in addition for most cells, up to 15% of an undefined nutrient medium usually fetal bovine serum (FBS). Serum from newborn calves and other mammals is also used, but FBS is preferred because of its high level of growth factors and low cross- reactivity with other animal cells. FBS or other mammalian products are also used to coat the surface of culture-ware to promote cell attachment.
  • FBS fetal bovine serum
  • FBS FBS
  • the production of FBS in this country is an estimated 700,000 liters annually worth $300 to $400 million.
  • the industry obtains fetal calves for bleeding from slaughter houses, or in some cases, rears herds of cattle for this purpose. These herds are held in as isolated a situation as possible in order to prevent disease.
  • Whole blood is obtained aseptically (by syringe) from an animal, centrifuged to separate cells from serum, and the serum filtered to 0.22 microns to remove most bacteria. Often, serum is heated to 56°C to inactivate the complement system, a group of immune proteins.
  • Insect cell culture has also been conducted for many years to develop control methods for this important animal group, and has been used as a model for biological processes in humans and higher animals. More recently, insect cell culture and a virus vector have become valuable tools for the expression of foreign genes. This technique is superior to the production of foreign proteins by bacteria as higher yields, better "copies", and more complex eukaryotic proteins can be obtained (Smith et al., 1983). Some examples of the recombinant proteins produced by insect cell expression systems are human interferon and interleukin-2, substances that are injected as therapy in human subjects.
  • An example of the current method of protein production includes the following steps: 1) culture of an insect cell line (Spodoptera frugiperda); 2) insertion of the desired foreign gene in the baculovirus, Autographa Californica Polyhedral Virus (Ac NPV); 3) infection of the insect cell line with this virus; and 4) extraction of the resulting foreign protein from the infected insect cells.
  • an insect cell line Spodoptera frugiperda
  • Ac NPV Autographa Californica Polyhedral Virus
  • BSE Bovine Spongifor Encephalopathy
  • U.S. Patent No. 4,449,480 to Isom et al. discloses freshwater mussels in an artificial habitat utilizing growth media.
  • the Isom et al. invention is directed to larviculture, that is, the provision of food and habitat for the larval stage of young animals such as aquatic invertebrates.
  • Isom et al. are not concerned with cell culture.
  • Larviculture is an interim technique, having the goal of keeping the larval animal alive until it can progress to the next stage, or in this particular case, until it can transform from a parasitic stage and feed independently on its own (Pennack 1953).
  • the Isom et al. invention is not applicable to the instant problem, as cell lines do not grow and transform to become independent of their culture medium and learn to feed on their own.
  • Isom et al. can only operate on the parasitic stage of whole, multicellular animals (Barnes, 1963), and is not applicable to cell lines derived from the organs and tissues of a whole animal.
  • a fundamental purpose of cell culture is to permit processes and experimentation in cells that would not be possible to perform on a whole animal.
  • Isom et al. borrow certain techniques from cell culture (for example, a modified solution or medium of salts and proteins) and from bacteriology (for example, antibiotics). The use of these tools, however, does not render the Isom et al. method a cell culture or bacteriology process, and the disclosed method cannot function as such. Thus, the Isom et al. method cannot provide a solution to the instant problem.
  • the method of the present invention is especially timely as BSE has recently been found in Canadian cattle (Campbell, 1993) and is strongly implicated as the cause of death in Wisconsin mink which were fed protein meal made from dairy cow carcasses (Marsh, 1993). In Europe there is evidence of the disease in humans receiving contaminated human growth hormone (Knauer, 1993). Summary of the Invention
  • the present invention is a method of using fish serum instead of bovine or other mammalian sera for mammalian cell culture and for insect cell culture. This method would appear contradictory to those skilled in the art of cell culture. Because of the great phylogenetic differences between fish and mammals, and between fish and insects, there is a reasonable expectation of failure. Searches of the non-patent literature show no prior use of fish serum for mammalian cell culture or for insect cell culture.
  • the mammalian cell lines used in the method of the present invention are commonly used for a number of experimental and commercial applications. These applications include toxicology, biochemistry, cancer research, and the production of recombinant proteins for therapy in humans and domestic animals. There is growing concern that these therapeutants could carry the infectious organisms found in bovine serum (Anon. 1994). Therefore, the use of fish serum instead of mammalian serum in the culture of these cells provides important safety advantages for the recipients of these recombinant products.
  • Fish serum can be used for cell culture applications that now employ bovine or other mammalian sera, and for applications where these sera are ineffective or unsafe.
  • the ideal serum for cell culture would provide the nutrients and growth factors that maintain cells and support their growth.
  • the serum should be (A) consistent in quality, (B) have serum immune proteins (immunoglobulins) , either at very low levels (as in FSB) or unlike those of mammals and insects for low cross-reactivity, and (C) be free of mammalian infectious agents that would contaminate cell lines or cell culture products, or endanger researchers.
  • serum from fishes is effective for mammalian cell culture and for insect cell culture, and meets the characteristics of the ideal serum with respect to consistency, low cross-reactivity, safety, and control of content.
  • sera from several species of aquacultured fishes can be used to grow insect cells. Research was conducted with four species of aquacultured fish, representing three families of teleosts; two salmonids, the rainbow trout and its seawater form the steelhead (Oncorhynchus mykiss); the Atlantic Salmon (Sal o salar); one cyclopterid, the lumpfish (Cyclopterus lumpus); and one ictalurid, the channel catfish (Ictalurus punctatus). Serum from these fishes is effective for insect cell culture and meets the characteristics of the ideal serum including consistency, low cross-reactivity, safety, and control of content.
  • the preferred specific rearing conditions for the donor fish are disclosed below. However, other conditions are desirable to alter a component in fish serum for culture of specific mammalian cells and insect cells, as described below.
  • the fishes are a distinct evolutionary group from the mammals and from insects. As such, many of their enzyme and immune system proteins, especially immunoglobulins, differ from those of mammals and insects. For example, most fishes are cold-blooded vertebrates having a body temperature that approximates that of the waters where they live. Cold water fishes normally live in water with a temperature range of 0°C to 18°C, and do not survive long above 20°C. Therefore, their serum proteins, including antibody proteins, function within a range far below the 37°C and higher body temperatures characteristic of the warm-blooded vertebrates such as humans and other mammals. This functional temperature difference strongly implies a difference in protein structure.
  • a major advantage of fish serum for cell culture is safety. Serum from fishes is unlikely to contain infectious agents harmful to mammals, including humans. Fish are cold-blooded animals with body temperatures that approximate the waters where they live. Therefore their pathogens, especially those of cold-water fishes, prefer temperatures well below the body temperatures of most mammals.
  • viruses of cold-water fishes are often controlled by raising water temperature (and therefore the fishes' body temperature) above 18°C (Wolf, 1988). Therefore, infection of mammalian cell cultures or of researchers by an agent in fish serum is highly unlikely.
  • D. Control of Content An additional advantage of using fish serum for cell- culture applications is control of serum content. Levels of certain substances in fish serum can be controlled by procedures that would be impossible with mammals for biological or regulatory reasons. For example, mammalian genetic triploids are not viable, but in salmonids, triploids live and grow normally and serum from the female triploid contains no sex steroids (Schreck and Moyle, 1990). Conversely, the sexual maturity of donor fish can be induced by light or hormone injections if high sex steroid serum is desired. Also, fish can be held under conditions unacceptable for mammals, such as total darkness, to increase certain hormones such as melatonin in serum. Detailed Description of the Present Invention
  • the mammalian cell lines used were Chinese hamster ovary cells (CHOs), supplied by the ATCC (#CCL-61), and monkey kidney cells (VEROs), also supplied by the ATCC #CCL-81. Spodoptera cells and Drosophila sp cells are used as the insect cell lines.
  • the fish serum used was taken from two species of salmonids, the rainbow trout and the steelhead (Oncorhynchus mykiss), and the Atlantic salmon (Salmo salar), the lumpfish ( Cyclopterus lumpus), and the channel catfish ( Ictalurus punctatus). These species were used because consistent and reproducible methods for their production are well established, large numbers of these species are reared in commercial aquaculture and therefore large amounts of serum can be obtained, and individual fish are large enough (over two pounds) so that blood can be drawn easily. Other species of aquacultured fish fit these criteria, particularly the sturgeon and the striped bass.
  • the process begins with the consistent and reproducible conditions in which donor fish are reared. All fish used as serum sources are 1) progeny of domesticated broodstock, 2) inspected for disease according to the American Fisheries Society Blue Book standards, 3) sexually immature, 4) in the log phase of growth, 5) larger than two pounds (range from 2- 12 pounds), 6) reared by standard husbandry methods appropriate to the species as described in Piper (1988), and 7) fed commercially manufactured pelleted feed of a composition consistent with that recommended by Halver (1972) and commonly used for each species. Rainbow trout and catfish are reared in freshwater; steelhead, salmon and lumpfish are reared in seawater.
  • Water temperature at the time of bleeding is normally 8° to 12°C, but water temperatures of 4° to 14°C are suitable for rainbow trout, salmon, steelhead, and lumpfish, and temperatures of 4° to 30°C for catfish; the objective being to avoid handling stress in the donor fish. Handling stress is further reduced by starving fish for 48 hours before bleeding.
  • Each fish is stunned by administering a sharp blow to the fish's head, by immersion in ice-water, or by immersion in water containing C0 2 or other fish anesthetic, the objective being to stun the fish to a level of loss of reflex activity (loss of consciousness) as defined by Schreck and Moyle (1990).
  • Serum is removed from the collection tubes and sterilized by passing first through a .45 ⁇ filter, and then through a .22 ⁇ filter.
  • Serum from 6 or more fish of the same species is combined as a numbered lot, and frozen at -70°C. No heat treatment such as that commonly used for bovine serum to denature complement immune system proteins is needed.
  • CHO or VERO cells shipped frozen from the American Type Culture Collection are thawed, counted, and seeded immediately in 25 cm sterile tissue culture flasks containing 5.0 ml of medium.
  • the medium is RPMI-1640, a widely-used defined medium (which may be purchased from Sigma Chemical Co.), plus 10% (v/v) FBS.
  • This flask containing cells and medium is placed in an incubator at 37°C and 5% CO .
  • the insect cells are grown in flasks or other vessels in Grace's medium supplemented with TC Yeastolate and Lactalbumin Hydrolysate and 10% FBS at 27°C in a closed atmosphere.
  • Mammalian cells are allowed to grow and increase in numbers over a period of one week. During this time, the cells are "split" or subcultured every 48 hours as follows. After the first 48 hours, cells from the original flask are detached using the standard trypsinization technique described by Pollard and Walker (1984) and are transferred to two new 25 cm 2 tissue culture flasks. This procedure is repeated every 48 hours until a total of eight flasks have been seeded.
  • the process of weaning to lower FBS levels begins.
  • the RPMI-1640 plus 10% FBS is removed by aspiration or pipette from each flask, and replaced with RPMI-1640 plus 7.5% FBS.
  • These flasks are placed in the incubator and the cells are allowed to grow and increase in number for 48 hours.
  • the RPMI-1640 with 7.5% FBS is removed from the eight flasks and replaced with RPMI-1640 plus 5% FBS.
  • the process may be repeated to lower FBS levels to 2.5% or 1%.
  • the weaning process takes up to 10 days to acclimate cells to the lower concentrations of FBS.
  • Cells growing in the flasks containing RPMI-1640 plus 5% or 2.5% or 1% FBS are then harvested using standard trypsinization techniques, washed with serum-free RPMI-1640, centrifuged at 300g for 3.5 minutes at room temperature, counted in a hemocytometer, and resuspended in new 25 cm 2 tissue culture flasks containing serum-free RPMI-1640. Aliquots from these flasks containing 5 x 10 4 cells/ml (by calculation) are then seeded in new flasks containing 5 ml of RPMI-1640 plus thawed fish serum as described above, at a concentration of 2.5% or 5% (v/v). Flasks containing the cells and media are then incubated at 37°C in 5% C0 2 /95% air.
  • Insect cell culture follows the same procedure.
  • the process of weaning to lower FBS levels begins.
  • the medium plus 10% FBS is removed by aspiration or pipette from each flask, and replaced with medium plus 7.5% FBS.
  • These flasks are placed in an incubator and the cells are allowed to grow and increase in number for 48 hours.
  • the medium with 7.5% FBS is removed from the flasks and replaced with medium plus 5% FBS.
  • the process may be repeated to lower FBS levels to 2.5% or 1%.
  • the weaning process takes up to 10 days to acclimate cells to the lower concentrations of FBS.
  • Cells growing in the flasks containing medium plus 5% or 2.5% or 1% FBS are then harvested using standard trypsinization techniques, washed with serum-free medium, centrifuged at 300g for 3.5 minutes at room temperature, counted in a hemocytometer, and resuspended in new 25 cm 2 tissue culture flasks containing serum-free medium. Aliquots from these flasks containing 5 x 10 4 cells/ml (by calculation) are then seeded in new flasks containing 5 ml of medium plus thawed fish serum as described above, at a concentration of 2.5% or 5% (v/v).
  • Flasks containing the cells and media are then incubated at 37°C in 5% C0 2 /95% air.
  • mammalian cells will grow normally with an approximate doubling time of 24 hours, the same as would be expected if they were cultured in FBS.
  • those cultured with 1% or 2.5% fish serum are normal and qualitatively similar to those cultured in media containing FBS.
  • Cells cultured at 5% fish serum appear normal except for small lipid-filled vacuoles in the cytoplasm.
  • Results show that insect cells survive and grow in sera from all species of fishes tested. Insect cells in lumpfish serum survived and did not attach for 48 hours but attached normally when FBS was then added. Compared to the FBS control, insect cells grown in fish serum showed no obvious difference in appearance, and for lumpfish and steelhead serum, cell growth was similar to growth in FBS. Compared to cells in FBS, final cell numbers were lower and attachment less for cells grown in sera from the other fish species tested.
  • mammalian cells and insect cells can be subcultured for experimental or commercial purposes using fish serum as a replacement for FBS, in either flasks or suspension culture.
  • Serum lipid in the fish species tested was high. Typical triglycerides were greater than 400 mg/dL and cholesterol was greater than 450 mg/dL, ten times or more higher than those of FBS. High serum lipoproteins are potentially growth inhibitory (Ito et al., 1982), therefore it is inferred that for some insect species lower lipid content in fish serum would improve cell growth.
  • the species of fish used as a source of serum may vary depending on what mammalian cells are to be cultured. However, the reproducibility of fish species and strain used and of fish culture conditions required for consistent serum quality limits donor species to those grown in aquaculture. Content of serum is influenced by such factors as husbandry, feed, reproductive status, genetics, age, and handling. Also, some fishes containing trimethyl amine oxide or high levels of other non-protein nitrogen (ammonia or amines) in their serum may be unsuitable as donors.
  • the method of obtaining the serum is not critical as long as the fish are treated in a humane manner and serum is obtained and processed (centrifuged, filtered, packaged, etc.) aseptically. Although the method is described using CHOs and VEROs, later experimentation has shown that normal diploid fibroblasts can be cultured using the fish serum method.
  • the defined medium may be replaced with other media selected from the group consisting of Eagles MEM, BME, Medium 199, or McCoys, all widely-used defined media.

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Abstract

L'invention concerne un procédé pour cultiver des cellules de mammifères, utilisant du sérum de poissons. Le procédé fait appel à du sérum extrait du sang de poissons pour cultiver des cellules de mammifères, destinées à différentes utilisations. Le procédé présente les avantages décisifs suivants: 1) absence d'agents infectieux de mammifères qui pourraient contaminer les lignées cellulaires et exposer à des risques les chercheurs ou les receveurs des préparations thérapeutiques dérivées des cultures des cellules de mammifères; 2) une qualité et une composition constantes et reproductibles du sérum; 3) une réactivité croisée faible; et 4) la présence d'éléments nutritifs sériques nécessaires à la croissance des cellules de mammifères. Le sérum de poissons est utilisé en combinaison avec des milieux définis spécifiques, pour assurer la croissance des cellules de mammifères ainsi que la viabilité des populations de ces cellules. Le sérum est obtenu à partir de poissons d'élévage, élevés dans des conditions contrôlées afin que l'alimentation, le milieu d'élévage, les caractéristiques génétiques, le déroulement de l'élévage, et la fertilité restent sensiblement constants et reproductibles.
PCT/US1994/014221 1994-02-23 1994-12-09 Procede pour cultiver des cellules de mammiferes et d'insectes dans un milieu contenant du serum de poissons WO1995023212A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU15511/95A AU1551195A (en) 1994-02-23 1994-12-09 Method for culturing mammalian cells and insect cells in a medium containing fish serum

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US08/200,639 1994-02-23
US08/200,639 US5401653A (en) 1994-02-23 1994-02-23 Method for culturing insect cells in a medium containing fish serum
US08/263,250 1994-06-21
US08/263,250 US5426045A (en) 1993-12-16 1994-06-21 Method for culturing mammalian cells in a medium containing fish serum

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WO1995023212A1 true WO1995023212A1 (fr) 1995-08-31

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CA (1) CA2183980A1 (fr)
WO (1) WO1995023212A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1085083A1 (fr) * 1998-06-01 2001-03-21 Chugai Seiyaku Kabushiki Kaisha Milieu de culture de cellules animales et procede de production de proteines en utilisant ce milieu
WO2006073070A1 (fr) * 2005-01-05 2006-07-13 Chugai Seiyaku Kabushiki Kaisha Méthode de culture cellulaire et emploi de ladite méthode

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5238824A (en) * 1989-11-03 1993-08-24 The United States Of America, As Represented By The Secretary Of Agriculture Hybridomas and monoclonal antibodies therefrom reactive toward antigens from edwardsiella ictaluri

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5238824A (en) * 1989-11-03 1993-08-24 The United States Of America, As Represented By The Secretary Of Agriculture Hybridomas and monoclonal antibodies therefrom reactive toward antigens from edwardsiella ictaluri

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1085083A1 (fr) * 1998-06-01 2001-03-21 Chugai Seiyaku Kabushiki Kaisha Milieu de culture de cellules animales et procede de production de proteines en utilisant ce milieu
EP1085083A4 (fr) * 1998-06-01 2002-11-27 Chugai Pharmaceutical Co Ltd Milieu de culture pour culture de cellules animales et procede de production de proteines en utilisant ce milieu
US6537782B1 (en) 1998-06-01 2003-03-25 Chugai Seiyaku Kabushiki Kaisha Media for culturing animal cells and process for producing protein by using the same
US6962812B2 (en) 1998-06-01 2005-11-08 Chugai Seiyaku Kabushiki Kaisha Culture medium for culture of animal cell and method for producing protein using same
WO2006073070A1 (fr) * 2005-01-05 2006-07-13 Chugai Seiyaku Kabushiki Kaisha Méthode de culture cellulaire et emploi de ladite méthode
EP2843040A1 (fr) * 2005-01-05 2015-03-04 Chugai Seiyaku Kabushiki Kaisha Procédé de culture de cellules et utilisation de celui-ci
US9714410B2 (en) 2005-01-05 2017-07-25 Chugai Seiyaku Kabushiki Kaisha Cell culture method and utilization of the same

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CA2183980A1 (fr) 1995-08-31

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