EP1203027A1 - Enhanced stability and performance of cells and cell components - Google Patents

Enhanced stability and performance of cells and cell components

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Publication number
EP1203027A1
EP1203027A1 EP00942871A EP00942871A EP1203027A1 EP 1203027 A1 EP1203027 A1 EP 1203027A1 EP 00942871 A EP00942871 A EP 00942871A EP 00942871 A EP00942871 A EP 00942871A EP 1203027 A1 EP1203027 A1 EP 1203027A1
Authority
EP
European Patent Office
Prior art keywords
cells
group
prokaryotic
human
components
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP00942871A
Other languages
German (de)
French (fr)
Other versions
EP1203027A4 (en
Inventor
Malcolm Potts
Richard Helm
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Virginia Tech Intellectual Properties Inc
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Virginia Tech Intellectual Properties Inc
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Filing date
Publication date
Application filed by Virginia Tech Intellectual Properties Inc filed Critical Virginia Tech Intellectual Properties Inc
Publication of EP1203027A1 publication Critical patent/EP1203027A1/en
Publication of EP1203027A4 publication Critical patent/EP1203027A4/en
Withdrawn legal-status Critical Current

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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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof

Definitions

  • the invention generally relates to the stabilization of cells and cell components for long-term storage at room temperature.
  • the invention provides an extracellular polysaccharide (EPS) from the terrestrial cyanobacterium Nostoc commune, and a method of using the EPS to provide desiccation tolerance for mammalian cells and cellular components for long-term stable preservation.
  • EPS extracellular polysaccharide
  • Mammalian cells do not survive common storage procedures such as lyophilization.
  • the only currently available option for long term storage of mammalian cells is storage at -170 °C in a cryoprotective medium.
  • This method has several drawbacks, including the necessity of providing a continual supply of liquid nitrogen in which to store the cells, and the attendant lack of "portability" of the preserved cells. Transportation of and accessibility of cells stored in this manner is not at all convenient.
  • the present invention provides a composition of matter comprising a substantially purified extracellular polysaccharide (EPS) from the cyanobacterium Nostoc commune, having the predominant repeat unit depicted in Formula 1 :
  • EPS extracellular polysaccharide
  • the present invention further provides a product for use in the long-term stabilization of labile material made by the process of obtaining a cell-free supernatant fraction from a culture of a form species of Nostoc commune, isolating a subfraction of the concentrated, cell-free supernatant fraction, converting the subfraction to a cation-free subfraction, and autoclaving the cation-free subfraction.
  • the present invention also provides compositions for desiccation and stable preservation of labile material.
  • the compositions include the EPS and the labile material, and may further include trehalose, sucrose, or other saccharides.
  • the present invention also provides a method for the stable preservation of labile material which includes the steps of mixing the labile material with the Nostoc EPS (and optionally trehalose or other saccharides), desiccating the mixture, and storing the desiccated mixture.
  • the step of desiccation may be carried out by a variety of means, such as lyophilization, spray drying, drying under vacuum, and drying in a stream of air or inert gas.
  • a suggested amount of EPS to be utilized is at least 20 grams liter "1 of the mixture, prior to desiccation. Storage of the material may be at room temperature, or at any other suitable temperature.
  • Examples of labile material which may be preserved by the methods and compositions of the present invention include prokaryotic cells and prokaryotic cellular components and products, eukaryotic cells and eukaryotic cellular components and products, and mammalian tissue (including human cells and tissue).
  • the present invention provides a new composition of matter comprising a novel extracellular polysaccharide (EPS) derived from the terrestrial cyanobacterium Nostoc commune.
  • EPS extracellular polysaccharide
  • the present invention provides a method of use of the Nostoc EPS for the stabilization of labile material, especially biological material.
  • Nostoc commune is known to have a marked capacity for desiccation tolerance and can survive storage at -400 MPa (0% R.H.) for centuries (Potts, 1994).
  • the cells produce large amounts (more than 60% of the dry weight of Nostoc ses colonies) of an unusual excreted polysaccharide that contributes in several ways to the marked stabilization of cells during prolonged storage in the air-dry state, at low or high temperatures.
  • the EPS provides a structural/molecular scaffold with rheological properties which can accommodate the rapid biophysical and physiological changes in the community upon rehydration and during recovery from desiccation.
  • the glycan swells from brittle dried crusts to cartilaginous structures within minutes of rehydration.
  • epiphytes colonize the surfaces of Nostoc colonies, there is no penetration of the glycan due in part to a silicon/calcium-rich pellicle and the inherent resistance of the glycan to enzymatic breakdown.
  • the EPS acts as an immobilization matrix for a range of secreted enzymes which remain fully active after long-term air-dried storage (Hill et al., 1997; Scherer and Potts, 1989; Hill et al., 1994; Shirkey et al., 2000).
  • the EPS material is provided in the free carboxylic acid form and without traces of phenol.
  • the absence of phenol is important to the practice of the present invention since phenol is known to be highly toxic.
  • Provision of the EPS in free carboxylic acid form is an advantage because a more consistent final product can be prepared from different batch fermentation runs.
  • the molecular structure of the material provided by the present invention is different from that described by Hill et al.
  • the material described by Hill et al. was not autoclaved.
  • the preparation of the EPS of the present invention includes a step of autoclaving the material. It has been determined that autoclaving removes a ribose group from the polysaccharide, thus generating a significantly different molecular structure.
  • the EPS has now been purified and characterized and a method of use of the EPS for the desiccation and stable preservation of labile material has been invented.
  • stable preservation we mean that the labile material, by being mixed with the EPS, is able to retain characteristic features or abilities which would otherwise be lost upon desiccation. For example, cells which are stabilized by the methods of the present invention are able to survive desiccation, and to retain the ability to grow and undergo cell division upon rehydration.
  • a composition of matter comprising a substantially purified EPS having the predominant repeat unit depicted in Formula 1 above is provided.
  • "predominant repeat unit” we mean that the polysaccharide produced by Nostoc commune is a xylogalactoglucan with a pendant uronic acid group in an approximate molar ratio of xylose:galactose:glucose:uronic acid of 1 :1 :2:1, respectively.
  • a product for use in the long-term storage of labile material made according to a process outlined herein is provided.
  • the EPS of the present invention may be made using strain DRH1 of a form species of terrestrial cyanobacterium Nostoc commune.
  • form species we mean: a strain that has the classic morphology of Nostoc commune Vaucher sensu Geitler and which contains a defined tRNA Le 7 UAA group I intron with no hypervariable region (Potts, unpublished).
  • strains of Nostoc commune may exist from which equivalent EPSs may be made. All such types of EPS (i.e. those which are useful for the stable preservation of desiccated labile material) made from any appropriate strain of form species Nostoc commune, and methods for their use, are encompassed by the present invention.
  • the EPS of the present invention may be produced by organisms as well.
  • the gene(s) encoding production of the EPS may be cloned into any of a variety of appropriate expression vectors and expressed in any of a variety of suitable host cells (e.g. bacteria such as E. coli, yeast, plant, insect or mammalian cells).
  • suitable host cells e.g. bacteria such as E. coli, yeast, plant, insect or mammalian cells.
  • the vectors which are used to carry out such expression may be modified by the addition of various control elements (for example, promoters) or the addition of sequences designed to direct the secretion of the EPS, or, in the case of multiple genes, the genes may be rearranged on the vector or on multiple vectors.
  • the EPS of the present invention may be produced by any cloned configuration of genetic material in any genetically engineered organism so long as the EPS of the present invention is produced and is capable of being used in the practice of the methods of the present invention.
  • labile material we mean any material which is susceptible to degradation (e.g. discoloration, oxidation, etc.) or any other type of vitiation, upon storage. Examples of such material include but are not limited to biological material, comestible items, chemicals, pharmaceuticals, cosmetics, and the like. Any material which it is desirable to store for prolonged periods of time but which is prone to degradation upon storage, may be stored by the practice of the methods of the present invention.
  • the labile material which is stored by the methods of the present invention is biological in nature.
  • biological materials which may be stored by the methods of the present invention include but are not limited to: viruses; prokaryotic cells and components of prokaryotic cells (e.g. cellular membranes, DNA, RNA, proteins, lipids, and the like); eukaryotic cells and components of eukaryotic cells (e.g. subcellular organelles, DNA, RNA, proteins, lipids, and the like); materials derived from ( i.e. products of) cells, such as vaccine preparations, enzymes and the like; and blood and various components of blood.
  • the present invention encompasses the controlled release of pesticides, various agrochemicals, and the like.
  • the labile material which is stored by the methods of the present invention comprises mammalian cells.
  • types of cells which may be stored by the methods of the present invention include but are not limited to: cells derived directly from a mammal, embryonic stem cells for the purpose of transplant or other purposes, cells which have been passaged in cell culture, red blood cells, platelets, and the like.
  • the labile material which is stored by the methods of the present invention comprises human cells, human tissues, and human cell products and/or components.
  • types of human cells which may be stored by the methods of the present invention include but are not limited to: cells derived directly from a human, human embryonic stem cells for the purpose of transplant or other purposes, human cells which have been passaged in cell culture, human red blood cells, human platelets, and the like.
  • human cell products and/or components include but are not limited to subcellular organelles, DNA, RNA, proteins, lipids, and the like; and materials derived from cells, such as vaccine preparations, enzymes and the like.
  • the EPS, the compositions, and the methods of the present invention are designed to promote the stability of labile material upon desiccation.
  • desiccation we mean the removal of water (i.e. the dehydration) of the labile material.
  • the amount of water which is removed from the labile material in the practice of the present invention may vary from labile material to labile material, but will generally be in the range of 90-100%.
  • the EPS of the present invention may be made by a process which comprises the following general steps: obtaining a cell-free supernatant fraction from a culture of an appropriate strain of form species Nostoc commune, subfractionating the cell-free supernatant fraction, converting the subfraction to a cation-free subfraction, and autoclaving the cation- free subfraction.
  • the cell-free supernatant fraction may be subfractionated in order to further purify or concentrate the EPS of the present invention.
  • a subfraction comprising the EPS of the present invention may be precipitated from the cell-free supernatant fraction using an agent such as aqueous ethanol.
  • an agent such as aqueous ethanol.
  • aqueous ethanol e.g. aqueous methanol, aqueous acetone, or a cationic lipid
  • ethanol precipitation may be carried out once or several times, and the precipitate may be washed with various appropriate solvents.
  • the supernatant fraction may be subfractionated by size (e.g. with a sizing column) or by charge using various types of chromatography, or by affinity fractionation, and the like.
  • the EPS of the present invention may be chemically modified, or cleaved by enzymatic digestion (e.g. with glycosidases) or by chemical means (e.g. via acid hydrolysis). Such reactions may be carried out at any appropriate stage of the production process. All such modifications of the EPS of the present invention are meant to be encompassed by the present invention, so long as the resulting EPS is useful for enhancing the stability of labile material upon desiccation.
  • the subfraction is further converted to a cation-free subfraction by any of several means which are well-known to those of skill in the art.
  • One means exposure to a cation- exchange resin is described in the Methods section of the Examples below.
  • the cation-free subfraction is lyophilized, rehydrated, and autoclaved to ensure sterility.
  • EPS of the present invention may be autoclaved prior to lyophilization and rehydration, or may be relyophilized after autoclaving, etc. All such superficial alterations in the process are encompassed by the present invention, so long as the resulting EPS is useful for enhancing the stability of labile material upon desiccation.
  • the EPS is provided as a lyophilized powder.
  • the EPS may also be provided in a hydrated form, for example as a gel or slurry in water or in cell culture medium or other suitable hydrating agent.
  • These forms of the EPS may optionally contain other materials which enhance the appearance, transport and handling of the EPS.
  • Any form of the EPS which allows the EPS to be combined with labile material and provide stability to the material during desiccation may be used in the practice of the present invention.
  • the present invention also provides compositions which are mixtures of the EPS of the present invention and the labile material which is to be desiccated.
  • the mixture of labile material and the EPS may be obtained by rehydrating the dried EPS with a suitable liquid, for example cell culture medium, and mixing the labile material (for example, cultured cells in liquid media) with the rehydrated EPS.
  • a suitable liquid for example cell culture medium
  • the labile material for example, cultured cells in liquid media
  • a useful amount of EPS is likely to be less than about 5% w/v of the final volume of the labile material, or of a solution which contains labile material.
  • such mixtures may optionally include other components which may be beneficial for the storage process.
  • the mixture may also include culture medium components, serum, polyols (e.g. non-reducing saccharides, cyclitols and alditols), DMSO, buffering agents, minerals, salts, growth factors, antioxidants, and the like.
  • the cells themselves may contain additional materials which promote cell stability.
  • dentain we mean that the material is physically associated with the cell. Such an association may be, for example, with the cellular membrane (either externally or within the membrane), or intracellular, or with a particular subcellular site, or at more than one of these locations.
  • association may be of any type (e.g. covalent, non-covalent, ionic, and the like).
  • additional materials which promote cell stability include but are not limited to polyols, e.g.: non-reducing saccharides such as sucrose, trehalose (including ⁇ , ⁇ ; ⁇ , ⁇ ; and 0C, ⁇ isomers), raffinose, melezitose, planteose, stachyose, and the like; cyclitols such as inositol and quercitol; and alditols such as mannitol and glucitol; and derivatives (e.g. phosphate derivatives) of polyols.
  • non-reducing saccharides such as sucrose, trehalose (including ⁇ , ⁇ ; ⁇ , ⁇ ; and 0C, ⁇ isomers), raffinose, melezitose, planteose, stachyose, and the like
  • cyclitols
  • Such additional materials which promote cell stability may be introduced prior to, after, or concomitant with combining the cells with EPS. Further, the cells may contain only one or more than one type of such material.
  • saccharides may be introduced into the cell as a result of genetic engineering, e.g. a cell which does not normally produce a saccharide may be genetically engineered to do so, either directly, or by genetically engineering into the cell an enzyme or enzymes which function to produce a precursor of the saccharide (see, for example, Billi et al., 2000).
  • the cells may be exposed to such materials in a manner which allows the uptake of the materials by the cells.
  • such materials may be introduced into cells by passive diffusion (e.g.
  • the amount of time needed for exposure of the labile material to the EPS prior to desiccation is not crucial. In general, an exposure time of from about 1 to 24 hours may be utilized in the practice of the present invention. However, this may vary somewhat from material to material. Those of skill in the art will recognize that any exposure time which results in stabilization of the labile material may be used in the practice of the present invention.
  • the mixture of EPS and labile material is then desiccated.
  • desiccation Several means of desiccation are well-known to those of skill in the art.
  • the mixture may be rapidly frozen and lyophilized, dried under vacuum in a desiccator chamber, dried in a stream of sterile air or inert gas such as N 2 , dried using a rapid spray dryer, or by regulation of matric water potential (i.e. where the water content of the air which the cells are exposed to is regulated by concentrated water solutions, glycerol, and the like).
  • the optimal means for desiccation of the stored material may vary depending on the exact nature of the labile material. Any suitable means of desiccation may be used in the practice of the present invention.
  • the temperature of storage of the mixture of labile material and EPS is room temperature.
  • the optimum temperature for storage may vary from material to material.
  • some labile material may preferably be stored at colder temperatures.
  • the desiccated mixture may be stored at any temperature at which stability of the labile material is achieved.
  • the EPS of the present invention may also be utilized to encapsulate substances for "timed-release delivery".
  • medicaments coated with the EPS may be administered to a patient in a "dry", protected state but would be released gradually upon exposure to the internal environment of the patient as the EPS is broken down.
  • Other biomedical and pharmaceutical products, such as liposomes which contain other substances may also be encapsulated for timed release delivery.
  • EPS of the present invention may also have other diverse applications, including use in microelectronic circuitry where resilient components are required, use as a molecular scaffold for covalent attachment of compounds such as UV-absorbing compounds, the protection of biomedical products during long-term space flight, and as a substrate for the identification of novel EPS modifying enzymes.
  • Rehydration of EPS-treated labile material after storage may be accomplished in any of a variety of ways which are appropriate for the particular labile material which has been stored.
  • the material need only be rehydrated by the addition of an appropriate quantity of suitable medium such as water, cell culture medium, saline, and the like, in a manner that allows adequate mixing of the desiccated labile material and the rehydrating agent.
  • suitable medium such as water, cell culture medium, saline, and the like
  • the amount of time needed for rehydration will vary from material to material. However, for other purposes, such as the timed-release delivery of agrochemicals, no rehydration is necessary. Instead, the lyophilized material may be administered directly to target areas (for example, to the soil around a crop) and the product or EPS will be rehydrated and broken down gradually by the action of water, enzymes, or mechanical wear.
  • Nostoc commune DRH-1 Cultures were grown in either an air-lift fermentor (2 L) or a 15-liter Bellco reactor, at 25 °C, in BG 11 0 medium (Rippka et al., 1979). When the 2 liter fermentor was used, the fermentor and growth medium were autoclaved and subsequently inoculated with N. ses DRH-1 (250 ml) taken from a smaller culture. The cells were grown under an incident photon flux density for two weeks, during which time the culture was sparged with air, after which time the culture was harvested by a combination of centrifugation and filtration.
  • a photon flux density of up to between about 500 to about 1750 ⁇ mol photons m ' V should be maintained throughout the growth period.
  • a heavy duty motor provides agitation with a paddle and the culture is sparged with sterile compressed air.
  • a bank of radial lights provides a photon flux of up to about 1750 ⁇ mol photons m ' V throughout the growth period.
  • the material was then dissolved in water (200 ml) and passed through a cation-exchange resin (Dowex, H + -form) to generate the cation- free polysaccharide, which was obtained by freeze-drying as a white mass with the consistency of cotton (300-600 mg). Percent recoveries, based on the mass obtained after the tangential flow filtration, ranged from 30-50%.
  • the freeze-dried EPS Prior to use as a preservative, the freeze-dried EPS is dissolved in water and autoclaved to ensure sterility. After cooling, the sterile solution of EPS may be combined with the material which is to be preserved.
  • other sterile components e.g. buffering agents, salts, sucrose or trehalose, etc.
  • buffering agents e.g. buffering agents, salts, sucrose or trehalose, etc.
  • EXAMPLE 1 Stabilization of Attachment Independent Mammalian Cell Lines with Nostoc EPS.
  • L5.1 cells were grown in 12-well microtiter plates. A stock solution (5% w/v of EPS) was layered onto the top of the cells in the plate at an approximately 1 :1 dilution of EPS stock to cell medium. The cells were then desiccated by fast drying in a sterile air stream for 9 hours. Cells were subsequently rehydrated by the addition of growth medium. The results showed that the presence of the glycan allowed the L5.1 cells to maintain active metabolism, as defined by Alamar blue reduction, trypan blue exclusion and cell growth.
  • EXAMPLE 2 Stabilization of Attachment Dependent Mammalian Cell Lines with Nostoc EPS.
  • the effect of Nostoc EPS on the stability of the attachment-dependent D3 ES cells was also analyzed. The studies were carried out as described in Example 1 with glycan alone, and also with added trehalose (from a stock solution of glycamtrehalose, 1:1). Results showed that the attachment-dependent cells survived desiccation, could be transferred, and that differentiation was easily induced in the transferred cells. Use of the Nostoc glycan appeared to be essential for cell survival. Further, the results showed that the glycan alone worked as well to stabilize the desiccated cells as did the glycan plus trehalose.
  • EXAMPLE 3 Effect on Cell Stability of 1) Time of Addition of the Nostoc EPS and 2) Length of Exposure to the Nostoc EPS
  • Example 1 Further investigations were carried out as described in Example 1 in which the stability of desiccated D3 ES cells was correlated with 1) the dependence on time of addition of the Nostoc EPS to the cells, and 2) the length of exposure of the cells to the glycan prior to dessication. Results indicated that the time of addition of the EPS is critical with this cell line. Highest stability was achieved when the EPS was added at day 2 after cell transfer. However, the length of time the D3 cells were incubated with glycan prior to dessication (from 3-24 hours) was not critical. The cells recovered at between 4-7 days after rehydration.

Abstract

The present invention provides a composition of matter comprising a substantially purified extracellular polysaccharide (EPS) from the terrestrial cyanobacterium Nostoc commune. When mixed with labile material (such as cells or cellular components) the EPS affords protection to the cells during desiccation and long-term storage. The invention thus provides an improved method for room temperature long-term storage of labile material.

Description

ENHANCED STABILITY AND PERFORMANCE OF CELLS AND CELL
COMPONENTS
DESCRIPTION
BACKGROUND OF THE INVENTION
Field of the Invention
The invention generally relates to the stabilization of cells and cell components for long-term storage at room temperature. In particular, the invention provides an extracellular polysaccharide (EPS) from the terrestrial cyanobacterium Nostoc commune, and a method of using the EPS to provide desiccation tolerance for mammalian cells and cellular components for long-term stable preservation.
Background of the Invention
The ability to effect the stable, long-term storage of cells, cellular components, and cellular products is a desideratum with many potential applications related to, for example, pharmaceutical preparations, medical procedures, and laboratory research endeavors. In particular, the facile, long term, room temperature storage of viable cells and cell components, especially those of mammals, is highly desirable.
Mammalian cells do not survive common storage procedures such as lyophilization. The only currently available option for long term storage of mammalian cells is storage at -170 °C in a cryoprotective medium. This method has several drawbacks, including the necessity of providing a continual supply of liquid nitrogen in which to store the cells, and the attendant lack of "portability" of the preserved cells. Transportation of and accessibility of cells stored in this manner is not at all convenient.
It would be a boon to researchers and clinicians if there were available a straightforward means of storing cells, and cellular components and products, that provided ease of handling and transport, together with maintaining high levels of cellular integrity and viability during the storage period.
SUMMARY OF THE INVENTION
The present invention provides a composition of matter comprising a substantially purified extracellular polysaccharide (EPS) from the cyanobacterium Nostoc commune, having the predominant repeat unit depicted in Formula 1 :
NosAβ l y
6 ► 4Glc/>β l - ► 4Gajpαl 4Glcpβ l - ■ 4XyIpβ l ->
FORMULA 1.
The present invention further provides a product for use in the long-term stabilization of labile material made by the process of obtaining a cell-free supernatant fraction from a culture of a form species of Nostoc commune, isolating a subfraction of the concentrated, cell-free supernatant fraction, converting the subfraction to a cation-free subfraction, and autoclaving the cation-free subfraction.
The present invention also provides compositions for desiccation and stable preservation of labile material. The compositions include the EPS and the labile material, and may further include trehalose, sucrose, or other saccharides.
The present invention also provides a method for the stable preservation of labile material which includes the steps of mixing the labile material with the Nostoc EPS (and optionally trehalose or other saccharides), desiccating the mixture, and storing the desiccated mixture. The step of desiccation may be carried out by a variety of means, such as lyophilization, spray drying, drying under vacuum, and drying in a stream of air or inert gas. A suggested amount of EPS to be utilized is at least 20 grams liter"1 of the mixture, prior to desiccation. Storage of the material may be at room temperature, or at any other suitable temperature. Examples of labile material which may be preserved by the methods and compositions of the present invention include prokaryotic cells and prokaryotic cellular components and products, eukaryotic cells and eukaryotic cellular components and products, and mammalian tissue (including human cells and tissue).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
The present invention provides a new composition of matter comprising a novel extracellular polysaccharide (EPS) derived from the terrestrial cyanobacterium Nostoc commune. In addition, the present invention provides a method of use of the Nostoc EPS for the stabilization of labile material, especially biological material. Nostoc commune is known to have a marked capacity for desiccation tolerance and can survive storage at -400 MPa (0% R.H.) for centuries (Potts, 1994). The cells produce large amounts (more than 60% of the dry weight of Nostoc commune colonies) of an unusual excreted polysaccharide that contributes in several ways to the marked stabilization of cells during prolonged storage in the air-dry state, at low or high temperatures. For example, the EPS provides a structural/molecular scaffold with rheological properties which can accommodate the rapid biophysical and physiological changes in the community upon rehydration and during recovery from desiccation. The glycan swells from brittle dried crusts to cartilaginous structures within minutes of rehydration. And, although epiphytes colonize the surfaces of Nostoc colonies, there is no penetration of the glycan due in part to a silicon/calcium-rich pellicle and the inherent resistance of the glycan to enzymatic breakdown.
Further, the EPS acts as an immobilization matrix for a range of secreted enzymes which remain fully active after long-term air-dried storage (Hill et al., 1997; Scherer and Potts, 1989; Hill et al., 1994; Shirkey et al., 2000).
An extracelluar polysaccharide from Nostoc commune has previously been identified (Hill et al., 1997). However, the EPS which was described at that time was obtained by methods which resulted in a product that is dissimilar to the EPS of the present invention in composition. Specifically, in the isolation procedure described by Hill et al., the supernatant obtained after centrifugation of the Nostoc commune culture was extracted with phenol hloro form and the EPS was recovered by precipitation in 95% ethanol at -70°C. The material was then washed with 70% ethanol and air dried. This method provided a material with trace levels of phenol as well as the EPS in carboxylate form. In contrast, in the procedure of the present invention which is outlined below, the EPS material is provided in the free carboxylic acid form and without traces of phenol. The absence of phenol is important to the practice of the present invention since phenol is known to be highly toxic. Provision of the EPS in free carboxylic acid form is an advantage because a more consistent final product can be prepared from different batch fermentation runs. Further, the molecular structure of the material provided by the present invention is different from that described by Hill et al. The material described by Hill et al. was not autoclaved. The preparation of the EPS of the present invention includes a step of autoclaving the material. It has been determined that autoclaving removes a ribose group from the polysaccharide, thus generating a significantly different molecular structure.
The EPS has now been purified and characterized and a method of use of the EPS for the desiccation and stable preservation of labile material has been invented. By "stable preservation" we mean that the labile material, by being mixed with the EPS, is able to retain characteristic features or abilities which would otherwise be lost upon desiccation. For example, cells which are stabilized by the methods of the present invention are able to survive desiccation, and to retain the ability to grow and undergo cell division upon rehydration.
In a preferred embodiment of the present invention, a composition of matter comprising a substantially purified EPS having the predominant repeat unit depicted in Formula 1 above is provided. By "predominant repeat unit" we mean that the polysaccharide produced by Nostoc commune is a xylogalactoglucan with a pendant uronic acid group in an approximate molar ratio of xylose:galactose:glucose:uronic acid of 1 :1 :2:1, respectively. In another preferred embodiment of the present invention, a product for use in the long-term storage of labile material made according to a process outlined herein is provided. In one embodiment of the present invention, the EPS of the present invention may be made using strain DRH1 of a form species of terrestrial cyanobacterium Nostoc commune. By "form species" we mean: a strain that has the classic morphology of Nostoc commune Vaucher sensu Geitler and which contains a defined tRNA Le7UAA group I intron with no hypervariable region (Potts, unpublished). However, those of skill in the art will recognize that many strains of Nostoc commune may exist from which equivalent EPSs may be made. All such types of EPS (i.e. those which are useful for the stable preservation of desiccated labile material) made from any appropriate strain of form species Nostoc commune, and methods for their use, are encompassed by the present invention.
Further, those of skill in the art will recognize that the EPS of the present invention may be produced by organisms as well. For example, the gene(s) encoding production of the EPS may be cloned into any of a variety of appropriate expression vectors and expressed in any of a variety of suitable host cells (e.g. bacteria such as E. coli, yeast, plant, insect or mammalian cells). The vectors which are used to carry out such expression may be modified by the addition of various control elements (for example, promoters) or the addition of sequences designed to direct the secretion of the EPS, or, in the case of multiple genes, the genes may be rearranged on the vector or on multiple vectors. For example, interveneing sequences may be removed, genes which in nature are located at distal sites may be placed close together, etc. The EPS of the present invention may be produced by any cloned configuration of genetic material in any genetically engineered organism so long as the EPS of the present invention is produced and is capable of being used in the practice of the methods of the present invention. By "labile material" we mean any material which is susceptible to degradation (e.g. discoloration, oxidation, etc.) or any other type of vitiation, upon storage. Examples of such material include but are not limited to biological material, comestible items, chemicals, pharmaceuticals, cosmetics, and the like. Any material which it is desirable to store for prolonged periods of time but which is prone to degradation upon storage, may be stored by the practice of the methods of the present invention.
In a preferred embodiment, the labile material which is stored by the methods of the present invention is biological in nature. Examples of biological materials which may be stored by the methods of the present invention include but are not limited to: viruses; prokaryotic cells and components of prokaryotic cells (e.g. cellular membranes, DNA, RNA, proteins, lipids, and the like); eukaryotic cells and components of eukaryotic cells (e.g. subcellular organelles, DNA, RNA, proteins, lipids, and the like); materials derived from ( i.e. products of) cells, such as vaccine preparations, enzymes and the like; and blood and various components of blood. In yet another preferred embodiment, the present invention encompasses the controlled release of pesticides, various agrochemicals, and the like.
In a preferred embodiment of the present invention, the labile material which is stored by the methods of the present invention comprises mammalian cells. Examples of types of cells which may be stored by the methods of the present invention include but are not limited to: cells derived directly from a mammal, embryonic stem cells for the purpose of transplant or other purposes, cells which have been passaged in cell culture, red blood cells, platelets, and the like.
In a preferred embodiment of the present invention, the labile material which is stored by the methods of the present invention comprises human cells, human tissues, and human cell products and/or components. Examples of types of human cells, which may be stored by the methods of the present invention include but are not limited to: cells derived directly from a human, human embryonic stem cells for the purpose of transplant or other purposes, human cells which have been passaged in cell culture, human red blood cells, human platelets, and the like. Examples of human cell products and/or components include but are not limited to subcellular organelles, DNA, RNA, proteins, lipids, and the like; and materials derived from cells, such as vaccine preparations, enzymes and the like.
The EPS, the compositions, and the methods of the present invention are designed to promote the stability of labile material upon desiccation. By "desiccation" we mean the removal of water (i.e. the dehydration) of the labile material. The amount of water which is removed from the labile material in the practice of the present invention may vary from labile material to labile material, but will generally be in the range of 90-100%.
The EPS of the present invention may be made by a process which comprises the following general steps: obtaining a cell-free supernatant fraction from a culture of an appropriate strain of form species Nostoc commune, subfractionating the cell-free supernatant fraction, converting the subfraction to a cation-free subfraction, and autoclaving the cation- free subfraction.
One example of a specific means of culturing the cyanobacterium and obtaining the cell-free supernatant fraction is given in the Methods section of the Examples below. However, those of skill in the art will recognize that there are many modifications which may be made to such a procedure, such as varying the volume of the culture, the amount of aeration, the photon flux density, the media components, the length of time of growth, and the like. Further, those of skill in the art will recognize that several appropriate alternatives exist for obtaining and handling the cell-free supernatant from such a culture. For example, the culture may be centrifuged or filtered, and the supernatant may be concentrated by any appropriate available means, e.g. via tangential flow filtration, lyophilization, and the like. Any suitable means for obtaining an appropriate cell-free supernatant fraction may be used in the practice of the present invention.
The cell-free supernatant fraction may be subfractionated in order to further purify or concentrate the EPS of the present invention. For example, a subfraction comprising the EPS of the present invention may be precipitated from the cell-free supernatant fraction using an agent such as aqueous ethanol. The details of one such procedure are given in the Methods section of the Examples below. However, those of skill in the art will recognize that many variations for purifying and/or concentrating exist. For example, other precipitating agents may be utilized (e.g. aqueous methanol, aqueous acetone, or a cationic lipid) either instead of or in addition to ethanol precipitation. Also, ethanol precipitation may be carried out once or several times, and the precipitate may be washed with various appropriate solvents. The supernatant fraction may be subfractionated by size (e.g. with a sizing column) or by charge using various types of chromatography, or by affinity fractionation, and the like. Those of skill in the art will recognize that many means of further purifying and/or concentrating the supernatant fraction are available and all such means may be utilized in the practice of the present invention, providing the resulting product or EPS is useful for effecting the stability of labile material for desiccation.
In addition, those of skill in the art will be familiar with other means of potentially modifying the the EPS of the present invention in such a way as to enhance their properties, for example, to promote their efficacy with respect to stabilizing labile material for desiccation, or to promote ease of handling, dissolution and/or rehydration of the desiccated material, or to isolate highly active subfractions, and the like. For example, the EPS of the present invention may be chemically modified, or cleaved by enzymatic digestion (e.g. with glycosidases) or by chemical means (e.g. via acid hydrolysis). Such reactions may be carried out at any appropriate stage of the production process. All such modifications of the EPS of the present invention are meant to be encompassed by the present invention, so long as the resulting EPS is useful for enhancing the stability of labile material upon desiccation.
The subfraction is further converted to a cation-free subfraction by any of several means which are well-known to those of skill in the art. One means (exposure to a cation- exchange resin) is described in the Methods section of the Examples below.
In the process which is outlined in the Methods section of the Examples below, the cation-free subfraction is lyophilized, rehydrated, and autoclaved to ensure sterility. Those of skill in the art will recognize that the exact sequence of these steps is not crucial to the practice of the present invention and may be altered. For example, the EPS of the present invention may be autoclaved prior to lyophilization and rehydration, or may be relyophilized after autoclaving, etc. All such superficial alterations in the process are encompassed by the present invention, so long as the resulting EPS is useful for enhancing the stability of labile material upon desiccation.
In a preferred embodiment of the present invention, the EPS is provided as a lyophilized powder. However, those of skill in the art will recognize that the EPS may also be provided in a hydrated form, for example as a gel or slurry in water or in cell culture medium or other suitable hydrating agent. These forms of the EPS may optionally contain other materials which enhance the appearance, transport and handling of the EPS. Any form of the EPS which allows the EPS to be combined with labile material and provide stability to the material during desiccation may be used in the practice of the present invention.
The present invention also provides compositions which are mixtures of the EPS of the present invention and the labile material which is to be desiccated. The mixture of labile material and the EPS may be obtained by rehydrating the dried EPS with a suitable liquid, for example cell culture medium, and mixing the labile material (for example, cultured cells in liquid media) with the rehydrated EPS. Those of skill in the art will recognize that the exact amount of EPS necessary to promote the stability of the labile material upon desiccation will vary from material to material. However, a useful amount of EPS is likely to be less than about 5% w/v of the final volume of the labile material, or of a solution which contains labile material. In addition, such mixtures may optionally include other components which may be beneficial for the storage process. For example, the mixture may also include culture medium components, serum, polyols (e.g. non-reducing saccharides, cyclitols and alditols), DMSO, buffering agents, minerals, salts, growth factors, antioxidants, and the like. In addition, in the case of the preservation of prokaryotic and eukaryotic cells, the cells themselves may contain additional materials which promote cell stability. By "contain" we mean that the material is physically associated with the cell. Such an association may be, for example, with the cellular membrane (either externally or within the membrane), or intracellular, or with a particular subcellular site, or at more than one of these locations. Likewise, the association may be of any type (e.g. covalent, non-covalent, ionic, and the like). Examples of additional materials which promote cell stability include but are not limited to polyols, e.g.: non-reducing saccharides such as sucrose, trehalose (including α,α; β,β; and 0C,β isomers), raffinose, melezitose, planteose, stachyose, and the like; cyclitols such as inositol and quercitol; and alditols such as mannitol and glucitol; and derivatives (e.g. phosphate derivatives) of polyols.
Such additional materials which promote cell stability may be introduced prior to, after, or concomitant with combining the cells with EPS. Further, the cells may contain only one or more than one type of such material.
Such additional materials may be present as a result of a variety of procedures which are well-known to those of skill in the art. For example, saccharides may be introduced into the cell as a result of genetic engineering, e.g. a cell which does not normally produce a saccharide may be genetically engineered to do so, either directly, or by genetically engineering into the cell an enzyme or enzymes which function to produce a precursor of the saccharide (see, for example, Billi et al., 2000). Alternatively, the cells may be exposed to such materials in a manner which allows the uptake of the materials by the cells. For example, such materials may be introduced into cells by passive diffusion (e.g. by exposing the cells to high concentrations of a saccharide); or by some means which promotes the passage of the material across the cell membrane, e.g. via chemical or physical modification of membrane permeability; or by temperature regulation. Those of skill in the art will recognize that many means of promoting the uptake of such material into a cell exist, and that all such means may be utilized in the practice of the present invention. Further, the term "uptake" is meant to include all such means.
The amount of time needed for exposure of the labile material to the EPS prior to desiccation is not crucial. In general, an exposure time of from about 1 to 24 hours may be utilized in the practice of the present invention. However, this may vary somewhat from material to material. Those of skill in the art will recognize that any exposure time which results in stabilization of the labile material may be used in the practice of the present invention.
In a preferred embodiment of the present invention, the mixture of EPS and labile material is then desiccated. Several means of desiccation are well-known to those of skill in the art. For example, the mixture may be rapidly frozen and lyophilized, dried under vacuum in a desiccator chamber, dried in a stream of sterile air or inert gas such as N2, dried using a rapid spray dryer, or by regulation of matric water potential (i.e. where the water content of the air which the cells are exposed to is regulated by concentrated water solutions, glycerol, and the like). The optimal means for desiccation of the stored material may vary depending on the exact nature of the labile material. Any suitable means of desiccation may be used in the practice of the present invention.
In a preferred embodiment of the present invention, the temperature of storage of the mixture of labile material and EPS is room temperature. However, those of skill in the art will recognize that the optimum temperature for storage may vary from material to material. For example, some labile material may preferably be stored at colder temperatures. The desiccated mixture may be stored at any temperature at which stability of the labile material is achieved. In addition, due to its unusual rheological properties, the EPS of the present invention may also be utilized to encapsulate substances for "timed-release delivery". For example, medicaments coated with the EPS may be administered to a patient in a "dry", protected state but would be released gradually upon exposure to the internal environment of the patient as the EPS is broken down. Other biomedical and pharmaceutical products, such as liposomes which contain other substances, may also be encapsulated for timed release delivery.
Additionally, agricultural chemicals (e.g. herbicides, fungicides, pesticides and the like) may be encapsulated with the Nostoc EPS for slow release after delivery. Any suitable substance for which timed-release delivery is desirable may be encapsulated with the EPS of the present invention, either in its purified form or in combination with other appropriate substances. The EPS of the present invention may also have other diverse applications, including use in microelectronic circuitry where resilient components are required, use as a molecular scaffold for covalent attachment of compounds such as UV-absorbing compounds, the protection of biomedical products during long-term space flight, and as a substrate for the identification of novel EPS modifying enzymes. Rehydration of EPS-treated labile material after storage may be accomplished in any of a variety of ways which are appropriate for the particular labile material which has been stored. In general, the material need only be rehydrated by the addition of an appropriate quantity of suitable medium such as water, cell culture medium, saline, and the like, in a manner that allows adequate mixing of the desiccated labile material and the rehydrating agent. The amount of time needed for rehydration will vary from material to material. However, for other purposes, such as the timed-release delivery of agrochemicals, no rehydration is necessary. Instead, the lyophilized material may be administered directly to target areas (for example, to the soil around a crop) and the product or EPS will be rehydrated and broken down gradually by the action of water, enzymes, or mechanical wear.
The following Examples are intended to illustrate various embodiments of the present invention but should not be construed as limiting the scope of the invention in any way.
EXAMPLES
Methods Growth of Nostoc commune DRH-1 —Cultures were grown in either an air-lift fermentor (2 L) or a 15-liter Bellco reactor, at 25 °C, in BG 110 medium (Rippka et al., 1979). When the 2 liter fermentor was used, the fermentor and growth medium were autoclaved and subsequently inoculated with N. commune DRH-1 (250 ml) taken from a smaller culture. The cells were grown under an incident photon flux density for two weeks, during which time the culture was sparged with air, after which time the culture was harvested by a combination of centrifugation and filtration. When the 2 liter fermentor is used, a photon flux density of up to between about 500 to about 1750 μmol photons m'V should be maintained throughout the growth period. When the 15 liter reactor is utilized, a heavy duty motor provides agitation with a paddle and the culture is sparged with sterile compressed air. A bank of radial lights provides a photon flux of up to about 1750 μmol photons m'V throughout the growth period.
Procedure for Preparing EPS- The cell-free supernatant fraction (ca. 1.5 L from a 2-liter fermentor) was passed through a tangential flow filtration concentrator (10,000 MWCO; Millipore Corp.) which reduced the volume approximately 10-fold. The solution was freeze-dried to provide an amber powder (1-2.5 g) which was dissolved in water (250 ml) and precipitated by pouring into a rapidly stirred solution of ethanol (95%, 750 ml). The insoluble material was recovered by filtration, washed successively with ethanol and acetone, and subsequently dried to provide a straw-colored material (500-750 mg). The material was then dissolved in water (200 ml) and passed through a cation-exchange resin (Dowex, H+-form) to generate the cation- free polysaccharide, which was obtained by freeze-drying as a white mass with the consistency of cotton (300-600 mg). Percent recoveries, based on the mass obtained after the tangential flow filtration, ranged from 30-50%. Prior to use as a preservative, the freeze-dried EPS is dissolved in water and autoclaved to ensure sterility. After cooling, the sterile solution of EPS may be combined with the material which is to be preserved. In addition, other sterile components (e.g. buffering agents, salts, sucrose or trehalose, etc.) which may be beneficial for the preservation process may also be added.
Cell lines:
The following cell lines were used to assess the effect of the Nostoc EPS on cell stability during desiccation: attachment-independent mouse hybridoma cell line L5.1 , and attachment- dependent Embryonic Stem (ES) cell line D3.
EXAMPLE 1. Stabilization of Attachment Independent Mammalian Cell Lines with Nostoc EPS.
The effect of Nostoc EPS on the stability of the attachment-independent hybridoma cell line L5.1 was analyzed. L5.1 cells were grown in 12-well microtiter plates. A stock solution (5% w/v of EPS) was layered onto the top of the cells in the plate at an approximately 1 :1 dilution of EPS stock to cell medium. The cells were then desiccated by fast drying in a sterile air stream for 9 hours. Cells were subsequently rehydrated by the addition of growth medium. The results showed that the presence of the glycan allowed the L5.1 cells to maintain active metabolism, as defined by Alamar blue reduction, trypan blue exclusion and cell growth.
EXAMPLE 2. Stabilization of Attachment Dependent Mammalian Cell Lines with Nostoc EPS. The effect of Nostoc EPS on the stability of the attachment-dependent D3 ES cells was also analyzed. The studies were carried out as described in Example 1 with glycan alone, and also with added trehalose (from a stock solution of glycamtrehalose, 1:1). Results showed that the attachment-dependent cells survived desiccation, could be transferred, and that differentiation was easily induced in the transferred cells. Use of the Nostoc glycan appeared to be essential for cell survival. Further, the results showed that the glycan alone worked as well to stabilize the desiccated cells as did the glycan plus trehalose.
EXAMPLE 3. Effect on Cell Stability of 1) Time of Addition of the Nostoc EPS and 2) Length of Exposure to the Nostoc EPS Further investigations were carried out as described in Example 1 in which the stability of desiccated D3 ES cells was correlated with 1) the dependence on time of addition of the Nostoc EPS to the cells, and 2) the length of exposure of the cells to the glycan prior to dessication. Results indicated that the time of addition of the EPS is critical with this cell line. Highest stability was achieved when the EPS was added at day 2 after cell transfer. However, the length of time the D3 cells were incubated with glycan prior to dessication (from 3-24 hours) was not critical. The cells recovered at between 4-7 days after rehydration.
While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims.
REFERENCES
Billi, D., Wright, D.J., Helm, R. F., Prickett, T., Potts, M., and Crowe, J.H. (2000) Appl. Environ. Bacteriol. 66, 1680-1684.
Helm, R.F., Huang, Z., Edwards, D., Leeson, H., Peery, W., and Potts, M. (2000) J Bacteriol. 182, 974-982. Hill, D.R., Keenan, T. W., Helm, R.F., Potts, M., Crowe, L.M. and Crowe, J.H. ( 1997) J. Appl. Phycol. 9, 237-248.
Potts, M. (1994) Microbiol. Rev. 58, 755-805.
Rippka, R., Deruelles J., Waterbury, J.B., Herdman, M., Stanier R.Y. (1979) J. Gen. Microbiol. Ill, 1-61.
Scherer, S. and Potts, M. (1989) J. Biol. Chem. 264, 12546-12553.
Shirkey, B., Kovarcik, D.P., Wright, D.J., Wilmoth, G., Prickett, T.F., Helm, R.F., Gregory, E.M., and Potts, M. (2000) J. Bacteriol. 182, 189-197.

Claims

We claim: L A composition of matter, comprising: a substantially purified extracellular polysaccharide having the predominant repeat unit:
NosAβ l
Y
6 ---► 4Glcpβl - 4Galpαl ^ 4Glcpβ l - 4Xylpβ l
2. A composition for desiccation and stable preservation of labile material, comprising: an admixture of said labile material and the extracellular polysaccharide of claim 1.
3. The composition of claim 2 further comprising at least one polyol.
4. The composition of claim 3 wherein said polyol is selected from the group consisting of: non- reducing saccharides, cyclitols, and alditols.
5. The composition of claim 4 wherein said non-reducing saccharide is selected from the group consisting of: trehalose, sucrose, melezitose, planteose, raffinose and stachyose.
6. The composition of claim 2 wherein said labile material is selected from the group consisting of: viruses, prokaryotic cells, components of prokaryotic cells, prokaryotic cell products, eukaryotic cells, components of eukaryotic cells, eukaryotic cell products, mammalian tissue, human cells, human tissues, components of human cells, human cell products, pharmaceutical products, pesticides, and agribiochemicals.
7. A method for the stable preservation of labile material, comprising: forming an admixture comprising said labile material and the extracellular polysaccharide of claim 1, desiccating said admixture, and storing said desiccated admixture.
8. The method of claim 7 wherein said labile material is selected from the group consisting of: viruses, prokaryotic cells, components of prokaryotic cells, prokaryotic cell products, eukaryotic cells, components of eukaryotic cells, eukaryotic cell products, mammalian tissue, human cells, human tissues, components of human cells, human cell products, pharmaceutical products, pesticides, and agribiochemicals.
9. The method of claim 7 wherein said desiccating step is carried out by a means selected from the group consisting of: lyophilization, spray drying, drying under vacuum, drying in a stream of air, and drying in a stream of inert gas.
10. The method of claim 7 wherein said step of storing is carried out at room temperature.
11. The method of claim 7 wherein said admixture further comprises at least one polyol.
12. The method of claim 11 wherein said polyol is selected from the group consisting of: non- reducing saccharides, cyclitols, and alditols.
13. The method of claim 12 wherein said non-reducing saccharide is selected from the group consisting of: trehalose, sucrose, melezitose, planteose, raffinose and stachyose.
14. The method of claim 12 wherein said admixture contains at least 5% w/v of the extracellular polysaccharide of claim 1.
15. The method of claim 12 wherein said prokaryotic cells and said eukaryotic cells contain an intracellular non-reducing saccharide.
16. The method of claim 15 wherein said prokaryotic cells and said eukaryotic cells are genetically engineered to produce said saccharide.
17. The method of claim 15 wherein said prokaryotic cells and said eukaryotic cells contain said intracellular non-reducing saccharide as a result of the uptake of said saccharide by said prokaryotic cells and said eukaryotic cells.
18. A product for use in the desiccation and stable preservation of labile material, said product being made from a process comprising the steps of: obtaining a concentrated, cell-free supernatant fraction from a culture of a form species of Nostoc commune, isolating a subfraction of said concentrated, cell-free supernatant fraction, converting said subfraction to a cation-free subfraction, and autoclaving said cation-free subfraction.
19. A composition for desiccation and stable preservation of labile material, comprising: an admixture of said labile material and the product of claim 18.
20. The composition of claim 19 further comprising at least one polyol.
21. The composition of claim 20 wherein said polyol is selected from the group consisting of: non-reducing saccharides, cyclitols, or alditols.
22. The composition of claim 21 wherein said non-reducing saccharide is selected from the group consisting of: trehalose and sucrose.
23. The composition of claim 19 wherein said labile material is selected from the group consisting of: viruses, prokaryotic cells, components of prokaryotic cells, prokaryotic cell products, eukaryotic cells, components of eukaryotic cells, eukaryotic cell products, mammalian tissue, human cells, human tissues, components of human cells, human cell products, pharmaceutical products, pesticides, and agribiochemicals.
24. A method for the stable preservation of labile material, comprising: forming an admixture comprising said labile material and the product of claim 18, desiccating said admixture, and storing said desiccated admixture.
25. The method of claim 24 wherein said labile material is selected from the group consisting of: viruses, prokaryotic cells, components of prokaryotic cells, prokaryotic cell products, eukaryotic cells, components of eukaryotic cells, eukaryotic cell products, mammalian tissue, human cells, human tissues, components of human cells, human cell products, pharmaceutical products, pesticides, and agribiochemicals.
26. The method of claim 24 wherein said step of desiccating is carried out by a means selected from the group consisting of: lyophilization, spray drying, drying under vacuum, drying in a stream of air, and drying in a stream of inert gas.
27. The method of claim 24 wherein said step of storing is carried out at room temperature.
28. The method of claim 24 wherein said admixture further comprises at least one polyol.
29. The method of claim 28 wherein said polyol is selected from the group consisting of: non- reducing saccharides, cyclitols, and alditols.
30. The method of claim 29 wherein said non-reducing saccharide is selected from the group consisting of: trehalose, sucrose, melezitose, planteose, raffinose and stachyose.
31. The method of claim 24 wherein said admixture contains at least 5% w/v of the product of claim 18.
32. The method of claim 25 wherein said prokaryotic cells and said eukaryotic cells contain an intracellular non-reducing saccharide.
33. The method of claim 32 wherein said prokaryotic cells and said eukaryotic cells are genetically engineered to produce said saccharide.
34. The method of claim 32 wherein said prokaryotic cells and said eukaryotic cells contain said intracellular non-reducing saccharide as a result of the uptake of said non-reducing saccharide by said prokaryotic cells and said eukaryotic cells.
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