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WO1997031100A1 - Procede pour la croissance de cellules de plantes dans des cultures en suspension liquides, et agents chimiotherapeutiques derives de plantes - Google Patents

Procede pour la croissance de cellules de plantes dans des cultures en suspension liquides, et agents chimiotherapeutiques derives de plantes Download PDF

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Publication number
WO1997031100A1
WO1997031100A1 PCT/US1997/002935 US9702935W WO9731100A1 WO 1997031100 A1 WO1997031100 A1 WO 1997031100A1 US 9702935 W US9702935 W US 9702935W WO 9731100 A1 WO9731100 A1 WO 9731100A1
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WO
WIPO (PCT)
Prior art keywords
plant cells
growth
cells produce
culture
plant
Prior art date
Application number
PCT/US1997/002935
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English (en)
Inventor
Patrick Soon-Shiong
Original Assignee
Vivorx Pharmaceuticals, 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
Application filed by Vivorx Pharmaceuticals, Inc. filed Critical Vivorx Pharmaceuticals, Inc.
Priority to AU19744/97A priority Critical patent/AU1974497A/en
Publication of WO1997031100A1 publication Critical patent/WO1997031100A1/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
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms

Definitions

  • the present invention relates to methods and compositions for the growth of plant cells and the production of naturally occurring therapeutic agents
  • 5 including taxol, taxanes, homoharringtonine, antibiotics, immunosuppressives, antifungals, and antivirals.
  • Plant-derived pharmaceuticals are usually obtained by extraction of harvested plant materials or by chemical synthesis. Production of plant-derived
  • Cell culture ensures a limitless and continuous source of product and is not subject to weather or blight. Cell cultures can be induced to overproduce the product of
  • Free-living microorganisms e.g., bacteria or fungi
  • Microorganisms synthesize nearly all of the biomolecules essential to life.
  • cells from higher organisms including plants
  • Large-scale processes are better developed and less difficult for culturing bacteria than for culturing plant cells.
  • Bacterial cells can be grown under vigorous agitation in large volumes of simple liquid medium.
  • plant cells are more difficult to grow as they are easily damaged by the shear stresses of turbulent fluid flow.
  • plant cells require complex nutrient media to support cell growth.
  • Plant cell cultures are established by isolating from the intact plant a living explant tissue, usually in the form of a callus (which is an unorganized proliferating mass of undifferentiated plant cells) . Induction of the callus requires an environment allowing some of the cells to divide and proliferate, provided by a special nutrient medium supplemented with plant growth regulators. Two media (Gamborg B5 or Murashige & Skoog) have been widely used in a number of plant culture systems, although Gamborg
  • Plant cell suspension cultures are induced from established callus cultures by transferring callus into a type of liquid medium which favors suspension-culture growth. Plant cells typically grow as aggregates because of the failure of dividing cells to separate completely. Suspension cultures are advantageous because they are easily scaled up for large scale (75,000 liter) mass production. Suspension cultures are the method of choice when designing bioreactor systems for production of desirable plant-derived products, since they are relatively homogeneous and carefully controlled.
  • Taxol is a diterpenoid alkaloid originally isolated from the bark of the pacific yew, Taxus brevifolia.
  • taxol is extremely effective against various cancers (including ovarian and breast) .
  • the supply of natural taxol is severely limited; several pacific yew trees are required to obtain enough of the drug to treat one patient, and the yew has become nearly extinct due to logging.
  • cultures of herbaceous plants have been relatively easy to grow, cultures of woody plants or conifers have usually been more difficult.
  • the invention employs a microgravity culture vessel which provides a quiescent low-shear environment for the maintenance of plant cells in the form of high-fidelity three-dimensional structures.
  • the invention utilizes processes for growing plant cells under optimized conditions which lead to the production of large amounts of useful therapeutic agents.
  • the invention encompasses the construction and use of improved or altered plant cells which are especially suited to growth and production in suspension culture, including cells obtained by selective breeding or recombinant DNA methods.
  • the invention extends to all therapeutic agents produced or manufactured by this process, including natural products as well as recombinant products.
  • the invention employs a complex cell-culture medium containing various nutrients and growth factors which are necessary or sufficient to promote long-term cell growth or multiplication and to avoid senescence or loss of biological function.
  • the invention involves the use of appropriate plant growth regulating factors to maintain plant cells in liquid suspension culture.
  • methods for growing plant cells comprising cultivating, in nutrient medium, plant cells derived from callus culture or suspension culture or both.
  • callus is an unorganized proliferating mass of undifferentiated plant cells.
  • suspension culture means structurally undifferentiated cells in various stages of aggregation dispersed throughout a liquid nutrient medium.
  • nutrient medium refers to a medium suitable for cultivation of plant cell callus and suspension cultures, and may be either a growth medium or a production medium.
  • growth medium refers to a nutrient medium which promotes rapid growth of cultured cells.
  • production medium refers to a nutrient medium which promotes biosynthesis of a desired product (e.g., taxol) in cultured cells.
  • taxol refers to a diterpenoid alkaloid originally isolated from the bark of the pacific yew, Taxus brevifolia, and it possesses antitumor activity.
  • taxanes refers to chemical compounds structurally related to taxol.
  • growth of plant cells occurs within a vessel generating a quiescent microgravity environment to diminish shear stresses and to maintain three-dimensional cell-aggregate structures.
  • Products contemplated for production according to the present invention include naturally occurring or genetically recombinant products useful as chemotherapeutic agents.
  • Exemplary products include taxol, taxane or other compounds structurally similar to taxol, homoharringtonine, antibiotic compounds, immunosuppressive compounds, antifungal compounds, antiviral compounds, and the like.
  • Taxol is present in many parts of the Taxus plant, but is most highly concentrated in its bark and new- growth needles. Explants from the bark or needles of T. brevifolia or T. chinensis have been successfully cultured to produce taxol and related taxanes from both callus and suspension cultures. As used herein, "explants" are isolated samples of living plant tissue which have been separated from the intact plant.
  • Taxus cell lines For the preparation of Taxus cell lines, needles and meristematic regions of the Taxus plant are removed and surface sterilized (e.g., using chlorine bleach) prior to introduction into the culture medium.
  • Antimicrobial agents e.g. , phosphomycin, gentamycin sulfate, ampicillin, cefoxitin, or benlate, cloxacillin
  • media appropriate to the individual cell line are used for induction and proliferation of the callus.
  • proliferation means an increase in total cell number.
  • Gamborg B5 medium at pH 5.5 between 3.0 and 7.0, but preferably between 4.0 and 6.0
  • casamino acids and 1-2 mg/L 2,4-dichlorophenoxyacetic acid is optimal for growth and subculture of callus.
  • This medium contains an effective cell-growth-promo ing concentration of water, sodium (Na*) ions, potassium (K*) ions, calcium (Ca**) ions, magnesium (Mg* + ) ions, zinc (Zn++) ions, chloride (Cl " ) ions, sulfate (S04 “ ) ions, all essential amino acids, water-soluble vitamins, coenzymes, and glucose.
  • Antibiotics such as benlate or ceftoxin
  • Elevated levels of carbohydrate e.g.
  • elicitors are compounds added to the culture medium which stimulate secondary metabolite production. They can be abiotic
  • Air is introduced into liquid cultures by means of tubing into the culture vessels.
  • the temperature is typically maintained between 20°C and 26°C.
  • cultures may be grown under any conditions of illumination, when taxol-producing cells are being cultured, total taxol production is highest in cultures exposed to light (between 100 and 3000 foot candles) .
  • the culture medium is prepared using a standard medium as a basis.
  • Gamborg B5 medium at pH 5.5, supplemented with 0.2% casamino acids and 1-2 mg/L 2,4-dichlorophenoxyacetic acid is optimal for growth and subculture of callus.
  • This medium contains effective cell- growth-promoting concentrations of water, calcium ions, sodium ions, all essential amino acids, water-soluble vitamins, coenzymes, and glucose.
  • Antibiotics (such as benlate or ceftoxin) are added to the medium to reduce explant losses due to microbial contamination.
  • Elevated levels of carbohydrate e.g., 7% to 10% sucrose have been employed to raise osmotic pressure and inhibit cell growth in a mature culture, thereby stimulating the accumulation of secondary metabolites such as taxol.
  • Addition of other elicitors such as inexpensive salts (e.g., 20 to 50 mM sodium chloride or potassium chloride) can likewise elicit secondary metabolite production
  • the suspension cultures are maintained for 1 to 8 weeks after subcultu ing, and are then harvested by removal and filtration of the growth medium employing standard techniques. After the culture has been weighed, lyophilized, and ground to a fine powder, the desired product (e.g., taxol) can be recovered by suitable means, e.g., by extraction using conventional solvent extraction methods.
  • suitable means e.g., by extraction using conventional solvent extraction methods.
  • the microgravitational process employed in the practice of the present invention greatly enhances the ability to form and maintain three-dimensional living tissues. It simultaneously minimizes the fluid shear stress, provides three-dimensional freedom for cell and substrate spatial orientation, and increases localization of cells, tissues, and substrates in a similar spatial region for significant periods during the cell culture.
  • cells, tissues, and substrates rotate about an axis nearly perpendicular to gravity. Particles of greatly different sedimentation rates orbit in particular paths and remain spatially localized for many minutes or hours. This allows particles sufficient interaction time to form multicellular structures and to associate with each other.
  • Suitable vessel diameters are chosen based on the volume required for the intended quantity of cultured material, and which will allow a sufficient seeding density of cells, tissues, and substrates. As understood by those of skill in the art, the outward particle drift due to centrifugal force is exaggerated at higher vessel radii and for rapidly sedimenting particles. Selected levels of shear stress may be introduced into the culture environment by differential rotation of the vessel components, as a means for controlling the rate and size of tissue formation and for maintaining optimal particle sizes and associated sedimentation rates.
  • plant cells, cultures, and cell lines developed specifically for growth and production using the cell culture methods described herein.
  • plant cells, cultures, and cell lines produced using the cell culture methods described herein.
  • therapeutic agents both naturally occurring and genetically recombinant, which are produced using the cell culture methods described herein.
  • Taxol-producing plant cells when cultured under microgravity conditions, are stabilized with respect to the formation and maintenance of three-dimensional aggregates.
  • callus was introduced into a microgravity vessel containing culture medium and growth factors.
  • Simulated microgravity was created (in ordinary unit gravity) by modulating the horizontal rotation of a culture vessel completely filled with culture medium containing the matrix. These conditions cause cells to collocate in one spatial region and encourage the maintenance of aggregates because shear stresses arising from the relative motion of the medium with respect to the walls of the vessel are minimized.

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

Abstract

L'invention concerne un procédé de culture en suspension liquide par microgravité destiné à assurer la croissance d'agrégats de cellules de plantes qui produisent des produits chimiques naturels pouvant être utilisés comme agents thérapeutiques, y compris dans la chimiothérapie pour le cancer et d'autres maladies. Le procédé selon l'invention consiste à utiliser un milieu de culture de cellules complexe contenant les substances nutritives et les facteurs de croissance nécessaires, une cuve de culture par microgravité pour favoriser la croissance tridimensionnelle et un ensemble de facteurs de croissance de plantes stimulant la croissance.
PCT/US1997/002935 1996-02-22 1997-02-20 Procede pour la croissance de cellules de plantes dans des cultures en suspension liquides, et agents chimiotherapeutiques derives de plantes WO1997031100A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU19744/97A AU1974497A (en) 1996-02-22 1997-02-20 Method for growing plant cells in liquid suspension cultures, and chemotherapeutic agents derived from plants

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1207196P 1996-02-22 1996-02-22
US60/012,071 1996-02-22

Publications (1)

Publication Number Publication Date
WO1997031100A1 true WO1997031100A1 (fr) 1997-08-28

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Application Number Title Priority Date Filing Date
PCT/US1997/002935 WO1997031100A1 (fr) 1996-02-22 1997-02-20 Procede pour la croissance de cellules de plantes dans des cultures en suspension liquides, et agents chimiotherapeutiques derives de plantes

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AU (1) AU1974497A (fr)
WO (1) WO1997031100A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2347410A (en) * 1999-01-21 2000-09-06 Tony Norman Marsh Plant cell growth promoting material
US7264951B1 (en) 1992-02-20 2007-09-04 Phyton, Inc. Enhanced production of taxol and taxanes by cell cultures of Taxus species

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5030573A (en) * 1980-10-22 1991-07-09 Nestec S.A. Process for cultivating plant cells in vitro
US5312740A (en) * 1991-02-12 1994-05-17 Nippon Steel Corporation Process for producing taxol by cell culture of taxus species
US5409828A (en) * 1985-12-18 1995-04-25 Rutgers, The State University Of New Jersey Method for stimulating cell multiplication, differentiation, embroyogenesis and respiration in plant cell tissue culture by the addition of a glycoprotein extensin to culture medium

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5030573A (en) * 1980-10-22 1991-07-09 Nestec S.A. Process for cultivating plant cells in vitro
US5409828A (en) * 1985-12-18 1995-04-25 Rutgers, The State University Of New Jersey Method for stimulating cell multiplication, differentiation, embroyogenesis and respiration in plant cell tissue culture by the addition of a glycoprotein extensin to culture medium
US5312740A (en) * 1991-02-12 1994-05-17 Nippon Steel Corporation Process for producing taxol by cell culture of taxus species

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOSYS ABSTRACT, Abstract Number 80:185463, BAGRATHISHVILI et al., "Production of a Tea Plant Suspension Cell Culture", 1979; & FIZIOL. RAST., (MOSC), Vol. 26, No. 2, pages 449-451. *
BIOSYS ABSTRACT, Abstract Number 87:341533, 1987, SCHNEIDER et al., "Metabolism of 2-2 4 Dichlorophenoxyisobutyric Acid and its Glucosyl Ester in Plants and Suspension Cultured Cells of Agrostemma-Githago"; & J. PLANT PHYSIOL., Vol. 127, No. 1-2, pages 147-152. *
BIOSYS ABSTRACT, Abstract Number 96:467066, PESTCHANKER et al., "Kinetics of Taxol Production and Nutrient use in Suspension Cultures of Taxus Cuspidata in Shake Flasks and a Wilson-Type Bioreactor"; & ENZYME AND MICROBIAL TECHNOL., 1996, Vol. 19, No. 4, pages 256-260. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7264951B1 (en) 1992-02-20 2007-09-04 Phyton, Inc. Enhanced production of taxol and taxanes by cell cultures of Taxus species
GB2347410A (en) * 1999-01-21 2000-09-06 Tony Norman Marsh Plant cell growth promoting material
GB2347410B (en) * 1999-01-21 2003-08-06 Tony Norman Marsh Plant cell growth promotion material

Also Published As

Publication number Publication date
AU1974497A (en) 1997-09-10

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