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WO1997031100A1 - Method for growing plant cells in liquid suspension cultures, and chemotherapeutic agents derived from plants - Google Patents

Method for growing plant cells in liquid suspension cultures, and chemotherapeutic agents derived from plants 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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Prior art keywords
plant cells
growth
cells produce
culture
plant
Prior art date
Application number
PCT/US1997/002935
Other languages
French (fr)
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/en

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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
    • C12N5/04—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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  • General Engineering & Computer Science (AREA)
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Abstract

A microgravity liquid-suspension culture method has been developed for growing aggregates of plant cells which produce naturally occurring chemical products for use as therapeutic agents, including chemotherapy for cancer and other diseases. The invention method involves the use of a complex cell-culture medium containing necessary nutrients and growth factors, a microgravity culture vessel for promoting three-dimensional growth, and a set of growth-promoting plant growth factors.

Description

METHOD FOR GROWING PLANT CELLS IN LIQUID SUSPENSION CULTURES. AND CHEMOTHERAPEUTICAGENTSDERIVEDFROMPLANTS
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
Higher plants are important sources of pharmaceuticals and specialty chemicals. Many of these
10 chemicals are secondary metabolites (not essential for plant growth) which protect the plant from various pathogens. Plant-derived pharmaceuticals are usually obtained by extraction of harvested plant materials or by chemical synthesis. Production of plant-derived
15 pharmaceuticals from liquid suspension culture is an enabling technology which could greatly increase the supply of chemotherapeutic chemicals such as taxol. Cell culture ultimately offers reliable year-round production of these valuable chemicals under controlled conditions. In
20 addition, it is expected that these methods can be continually improved in light of advances in technology and genetics. 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
25 interest, thereby simplifying the process of separation and purification.
Free-living microorganisms (e.g., bacteria or fungi) possess tough cell walls for resistance to most environmental stresses. Microorganisms synthesize nearly all of the biomolecules essential to life. However, cells from higher organisms (including plants) are structurally delicate and require constant supplements of specific nutrients to maintain viability. 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.
In contrast, however, plant cells are more difficult to grow as they are easily damaged by the shear stresses of turbulent fluid flow. In addition, 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
B5 (with supplements) has been most effective in taxol- producing cultures.
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. The National Cancer Institute has shown that crude bark extracts exhibit anti-tumor activities, and clinical trials have confirmed that taxol is extremely effective against various cancers (including ovarian and breast) . Unfortunately, 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. Although cultures of herbaceous plants have been relatively easy to grow, cultures of woody plants or conifers have usually been more difficult.
SUMMARY OF THE INVENTION
In accordance with the present invention, methods have been developed for the rapid and efficient initiation of cultures from different plants, e.g., different Taxus species, for their successful growth on artificial nutrient media, and for the production of high levels of useful plant derived products, e.g., taxol and taxanes, in a short period of time.
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.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there are provided methods for growing plant cells, said methods comprising cultivating, in nutrient medium, plant cells derived from callus culture or suspension culture or both.
As employed herein, "callus" is an unorganized proliferating mass of undifferentiated plant cells.
As employed herein "suspension culture" means structurally undifferentiated cells in various stages of aggregation dispersed throughout a liquid nutrient medium.
As employed herein "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.
As employed herein, "growth medium" refers to a nutrient medium which promotes rapid growth of cultured cells.
As employed herein, "production medium" refers to a nutrient medium which promotes biosynthesis of a desired product (e.g., taxol) in cultured cells. As employed herein, "taxol" refers to a diterpenoid alkaloid originally isolated from the bark of the pacific yew, Taxus brevifolia, and it possesses antitumor activity. As employed herein, "taxanes" refers to chemical compounds structurally related to taxol.
In a preferred aspect of the invention, 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.
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) may also be employed for surface sterilization of plant material.
For callus and suspension growth according to the invention, media appropriate to the individual cell line are used for induction and proliferation of the callus. As used herein, "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), 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 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) 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. As used herein, "elicitors" are compounds added to the culture medium which stimulate secondary metabolite production. They can be abiotic
(e.g., salts and sugars and metal ions) or biotic (e.g., autoclaved fungi and bacteria) .
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. Although 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) .
Nutrients and factors supplemented into growth medium are critical for the long-term viability and health of plant cells in culture. 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.
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. Thus, in the microgravity vessel employed in the practice of the present invention, 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.
In accordance with another aspect of the present invention, there are provided plant cells, cultures, and cell lines developed specifically for growth and production using the cell culture methods described herein.
In accordance with yet another aspect of the present invention, there are provided plant cells, cultures, and cell lines produced using the cell culture methods described herein.
In accordance with yet another aspect of the present invention, there are provided therapeutic agents, both naturally occurring and genetically recombinant, which are produced using the cell culture methods described herein.
The invention will now be described in greater detail by reference to the following non-limiting example. EXAMPLE
Taxol-producing plant cells, when cultured under microgravity conditions, are stabilized with respect to the formation and maintenance of three-dimensional aggregates. Thus, 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.
While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

Claims

That which is claimed is:
1. A method for growing plant cells, said method comprising cultivating, in nutrient medium, plant cells derived from callus culture or suspension culture or both.
2. A method according to claim 1 wherein growth of said plant cells occurs within a vessel generating a quiescent microgravity environment to diminish shear stresses and to maintain three-dimensional cell-aggregate structures.
3. A method according to claim 1 wherein said plant cells produce a naturally occurring or genetically recombinant product for use as a chemotherapeutic agent.
4. A method according to claim 1 wherein said plant cells produce taxol.
5. A method according to claim 1 wherein said plant cells produce any taxane or other compound structurally similar to taxol.
6. A method according to claim 1 wherein said plant cells produce homoharringtonine.
7. A method according to claim 1 wherein said plant cells produce an antibiotic compound.
8. A method according to claim 1 wherein said plant cells produce an immunosuppressive compound.
9. A method according to claim 1 wherein said plant cells produce an antifungal compound.
10. A method according to claim 1 wherein said plant cells produce an antiviral compound.
11. Plant cells, cultures, and cell lines developed specifically for growth and production using the method of claim 1.
12. Plant cells, cultures, and cell lines produced by the method of claim 1.
13. Therapeutic agents, both naturally occurring and genetically recombinant, which are produced using the method of claim 1.
PCT/US1997/002935 1996-02-22 1997-02-20 Method for growing plant cells in liquid suspension cultures, and chemotherapeutic agents derived from plants WO1997031100A1 (en)

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US60/012,071 1996-02-22

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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

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