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WO2010111255A1 - Substances biologiques desséchées et procédés de préparation - Google Patents

Substances biologiques desséchées et procédés de préparation Download PDF

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Publication number
WO2010111255A1
WO2010111255A1 PCT/US2010/028296 US2010028296W WO2010111255A1 WO 2010111255 A1 WO2010111255 A1 WO 2010111255A1 US 2010028296 W US2010028296 W US 2010028296W WO 2010111255 A1 WO2010111255 A1 WO 2010111255A1
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Prior art keywords
cells
biologic
factor
protein
cell
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English (en)
Inventor
David H. Ho
Stephen P. Bruttig
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HeMemics Biotechnologies Inc
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HeMemics Biotechnologies Inc
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Publication of WO2010111255A1 publication Critical patent/WO2010111255A1/fr
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    • 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
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media

Definitions

  • the present invention is directed, in part, to compositions comprising desiccated biologies and to methods of preparing the same.
  • biologies such as cells and biomolecules
  • anucleated cells such as platelets
  • nucleated cells such as reproductive cells (Dinnyes et al, Reprod. Fertil. Dev., 2007, 19, 719-31), stem cells (De Sousa et al, Reproduction, 2006, 132, 681-9) and hepatocytes (Bakala et al., Pol. J. Vet. ScL, 2007, 10, 11-8) must be maintained in expensive storage devices and possess limited shelf-life at room temperature.
  • Freezing cells can promote ice crystal formation as well as osmotic changes during the process and result in disruption of intracellular organelles and membranes, resulting in loss of cells (i.e., transient warming effect) or loss or significant diminution of cell functions. Further, freeze-drying can, and often does, result in generating microparticles that are apparently formed from the cellular debris.
  • freezing protocols for hepatocyte suspensions mostly devastating results such as low recovery and severe loss of functions occurred (Koebe et al., Chem. Biol. Interact., 1999, 121, 99-115).
  • experiments showed that a mechanical interaction between ice crystals and red blood cell membrane induced mechanical damage to the membrane (Ishiguro et al, Cryobiology, 1994, 31, 483-500).
  • the current protocols for preserving and/or storing biologies are not sufficient to dry cells and to recover desired functions upon reconstitution.
  • the present invention provides methods of preserving and/or storing biologies to preserve cell structures and functions in the dried or semi-dried states. These processes can result in cells that will recover full or partial function upon reconstitution and rehydration.
  • compositions comprising: one or more biologies; one or more membrane penetrable sugars; and one or more membrane impenetrable sugars; wherein the moisture content of the composition is from about 5% to about 95%.
  • the biologic is a cell.
  • the cell is anucleated.
  • the anucleated cell is a platelet or red blood cell.
  • the cell is nucleated.
  • the nucleated cell is a white blood cell, a stem cell, a stem cell progenitor cell, a gamete, a gamete progenitor cell, a hepatocyte, a muscle cell, an endothelial cell, an epithelial cell, an erythroblast, a leukoblast, a chondroblast, or a pancreatic cell or other nucleated cell.
  • the biologic is a virus, protein, nucleic acid, carbohydrate, or lipid.
  • the membrane penetrable sugar is trehalose. In some embodiments, the membrane impenetrable sugar is dextran. In some embodiments, the membrane impenetrable sugar is a combination of more than one sugar (e.g., a mixture of dextran and other sugars with a molecular weight of 50,000 Daltons or more).
  • the moisture content is from about 15% to about 40%. In some embodiments, the moisture content is from about 20% to about 25%. In some embodiments, the moisture content is from about 55% to about 60%. In some embodiments, the moisture content is from about 60% to about 95%.
  • the biologic is a platelet, the membrane penetrable sugar is trehalose, the membrane impenetrable sugar is dextran, and the moisture content is about 15%.
  • the biologic is a red blood cell, the membrane penetrable sugar is trehalose, the membrane impenetrable sugar is dextran, and the moisture content is about 25%.
  • the biologic is a white blood cell, the membrane penetrable sugar is trehalose, the membrane impenetrable sugar is dextran, and the moisture content is about 50%.
  • the membrane penetrable sugar is trehalose
  • the membrane impenetrable sugar is dextran alone or in combination with another sugar(s) with a molecular weight of 50,000 Daltons or more
  • the moisture content is from about 15% to about 90%.
  • the present invention also provides methods of preserving a biologic comprising: contacting the biologic with at least one membrane penetrable sugar and at least one membrane impenetrable sugar; optionally, contacting the biologic with a fixative agent; and drying the biologic by vacuum desiccation to a final moisture content of from about 5% to about 95%.
  • the biologic is a cell.
  • the cell is anucleated.
  • the anucleated cell is a platelet or red blood cell.
  • the cell is nucleated.
  • the nucleated cell is a white blood cell, a stem cell, a stem cell progenitor cell, a gamete, a gamete progenitor cell, a hepatocyte, a muscle cell, an endothelial cell, an epithelial cell, an erythroblast, a leukoblast, a chondroblast, or a pancreatic cell, or other nucleated cell.
  • the biologic is a virus, protein, nucleic acid, carbohydrate, or lipid.
  • the membrane penetrable sugar is trehalose. In some embodiments, the membrane impenetrable sugar is dextran. In some embodiments, the moisture content is from about 15% to about 40%. In some embodiments, the moisture content is from about 20% to about 25%.
  • the fixative agent is glutaraldehyde or paraldehyde.
  • the biologic is dried by vacuum desiccation from about 0 0 C to about 40 0 C for about 1 hours to about 24 hours. In some embodiments, the biologic is dried by vacuum desiccation from about 32°C to about 34°C for about 3 hours.
  • the method further comprises storing the biologic in a vacuum sealed container in the presence or absence of a desiccant. In some embodiments, the method further comprises rehydrating the biologic. In some embodiments, the rehydration comprises contacting the biologic with water, followed by saline.
  • the biologic is a platelet, the membrane penetrable sugar is trehalose, the membrane impenetrable sugar is dextran, and the moisture content is about 15%.
  • the biologic is a red blood cell, the membrane penetrable sugar is trehalose, the membrane impenetrable sugar is dextran, and the moisture content is about 25%.
  • the biologic is a white blood cell, the membrane penetrable sugar is trehalose, the membrane impenetrable sugar is dextran, and the moisture content is about 50%.
  • Figure 1 shows a schematic representation of a representative desiccation process.
  • Figure 2 shows a schematic representation of sugar uptake to stabilize the cells in dry format and a hydration process.
  • Figure 3 shows hydration of desiccated red blood cells compared to fresh blood cells where the cells in both panel maintain the bi-concave structures.
  • Figure 4 shows platelet-sizing profile using Freeze-drying (FD Pits) and Desiccation
  • Figure 5 shows nucleated cells maintain cell membrane integrity upon reconstitution as stained with trypan blue.
  • Figure 6 shows osmotic fragility of fresh versus dried RBC wherein the fragility of fresh (diamond symbol) versus dried and reconstituted (square symbol) RBC was compared under various osmotic conditions (data are means + SEM).
  • Figure 7 shows initial determination of RBC elasticity, wherein desiccated and rehydrated human RBCs compare favorably with fresh human blood diluted to the same HCT.
  • Figure 8 depicts the first set of oxygen dissociation curve data from "normal" whole human blood (the curve on the right) and duplicate determinations from desiccated and rehydrated human blood (both curves on the left, desiccated).
  • Figure 9 depicts the first set of oxygen dissociation curve data from "normal" whole human blood (the curve on the right) and duplicate determinations from desiccated and rehydrated human blood (both curves on the left, desiccated 3 days earlier).
  • Figure 10 depicts a representative rotary evaporation/storage flask system.
  • Figure 11 depicts a representative rotary evaporation/storage flask/roller system.
  • Figure 12 depicts a representative blood bag desiccating system.
  • the present invention provides methods of preserving and/or storing biologies, individually, together, or in combination in a dried or semi-dried format.
  • the present invention also provides compositions comprising a biologic in a desiccated state.
  • the term "biologic” means a cell and/or a biomolecule.
  • the term “cell” means nucleated cells (i.e, cells containing one or more nuclei) or anucleated cells (i.e., platelets and red blood cells; cells that have no nucleus).
  • Cells may be in the form of individual cells, tissue(s), and/or organ(s). Cells can be derived from any organ. Different cells can be present in the same sample being desiccated. In addition, cells can be altered by humans such as, for example, cell lines and hybridomas. Cells include animal cells and/or plant cells.
  • biomolecule means any protein, nucleic acid, carbohydrate, lipid, or other such molecule, produced or existing free in other body/biological fluids.
  • Biomolecules can be present alone, or in combination with other biomolecules and/or cells, such as plasma products (i.e., blood cells, biomolecules, and salts), tissue, and/or organs, such as the vasculature bed containing endothelial cells, smooth muscle cells and some combination of other cell types.
  • Biomolecules also include, for example, antibodies and peptides, or compositions of biomolecules such as, for example, the proteins, peptides, and other biological organic molecules in plasma.
  • examples of biomolecules also include, for example, immunoglobulins, blood coagulation proteins (both inactive and active forms of the following proteins), and regulator proteins.
  • Biomolecules also include, but are not limited to, albumin, alpha and beta globulins.
  • immunoglobulins include, but are not limited to, IgA, IgD, IgE, IgG, and IgM, or any combination thereof.
  • tissue factor pathway proteins include, but are not limited to, tissue factor pathway (extrinsic) proteins, contact activation pathway (intrinsic) proteins, and final common pathway proteins.
  • tissue factor pathway proteins include, but are not limited to, Tissue Factor (TF), Factor VII, Factor IX, Factor X, thrombin, Factor XI, plasmin, Factor XII, tissue factor pathway inhibitor (TFPI), prothrombinase complex, prothrombin, Factor V, Factor VIII, von Willebrand factor (vWF), and tenase complex.
  • contact activation pathway proteins include, but are not limited to, collagen, high-molecular-weight kininogen (HMWK), prekallikrein, and FXII (Hageman factor).
  • regulator proteins include, but are not limited to, Protein C, activated protein C (APC), thrombomodulin, protein S, antithrombin, serine protease inhibitor (serpin), tissue factor pathway inhibitor (TFPI), plasmin, plasminogen, tissue plasminogen activator (t- PA).
  • the biomolecule is cryoprecipitate, also referred to as "Cryoprecipitated Antihemophilic Factor” or “Cryoprecipitated AHF.”
  • the cryoprecipitate used herein can be obtained from or derived from animals including, but not limited to, reptiles, amphibians, birds, fish, mammals, and the like. Mammals include, but are not limited to, humans, dogs, cats, horses, pigs, cows, rabbits, goats, and the like.
  • cryoprecipitate refers to biologies that precipitate from plasma when the plasma is frozen.
  • cryoprecipitate as it is currently used in the industry, must be maintained as a frozen composition and, therefore, maintained at a freezing temperature when shipped.
  • the present invention circumvents this requirement as the desiccated cryoprecipitate surprisingly has Factor VIII activity similar to fresh cryoprecipitate. Therefore, the present invention provides an advantage that cryoprecipitate can now be shipped at ambient temperature and still maintain activity.
  • cryoprecipitate is the predominant way to treat dogs having hemophilia. Desiccation of the cryoprecipitate, as described herein, enables the cryoprecipitate to be used in more areas with cheaper shipping and storage costs since freezing is no longer required.
  • cryoprecipitate can be used in methods of treating hemophilia, or other blood disorders.
  • the present invention contemplates methods of treating hemophilia in a mammal comprising administering a therapeutically effective amount of cryoprecipitate, which has been desiccated and rehydrated as described herein.
  • a therapeutically effective amount of cryoprecipitate is one skilled in the art, depending upon the extent of the hemophilia in the mammal, will be able to determine a therapeutically effective amount of cryoprecipitate.
  • compositions comprising: one or more biologies; one or more membrane penetrable sugars; and one or more membrane impenetrable sugars; wherein the moisture content of the composition is from about 5% to about 95%.
  • the biologic is a cell.
  • the cell is anucleated.
  • anucleated cells include, but are not limited to, a platelet and a red blood cell.
  • the anucleated cell is present at from about 1 x 10 3 cells/mL to about 1 x 10 10 cells/mL. In some embodiments, the anucleated cell is present at about 1 x 10 9 cells/mL.
  • the cell is nucleated.
  • nucleated cells include, but are not limited to, a white blood cell (i.e., a T cell, a B cell, a macrophage, a neutrophil, a lymphocyte, and the like), a stem cell (i.e, adult and/or neonatal, various tissues or species origin), a stem cell progenitor cell, a gamete (male and/or female), a gamete progenitor cell, and a cell derived from an organ including, but not limited to, various hepatocytes, various kidney cells, various neural cells, various cardiac cells, a muscle cell, an endothelial cell, an epithelial cell, various skin cells, chondrocytes, an erythroblast, a leukoblast, a chondroblast, a pancreatic cell, and the like.
  • the cell is a cell line such as, for example, Chinese hamster ovary (CHO) cells, 3T3 fibroblasts, HEK cells, and the like.
  • the nucleated cell is an islet cell or cord blood cell.
  • the nucleated cell is a human venous, arterial, or capillary endothelial cell, or the like.
  • the cells used herein can be obtained from or derived from animals including, but not limited to, reptiles, amphibians, birds, fish, mammals, and the like. Mammals include, but are not limited to, humans, dogs, cats, horses, pigs, cows, rabbits, goats, and the like.
  • the nucleated cell is present at from about 1 x 10 3 cells/mL to about 1 x 10 10 cells/mL. In some embodiments, the anucleated cell is present at about 1 x 10 7 cells/mL.
  • the tissue is a thin tissue.
  • thin tissues include, but are no limited to, small blood vessel segments (both arteries and veins), segments of mesentery (the connective tissue between loops of intestines), segments of bowel wall, segments of bladder, pieces of meninges (the various coverings of the brain), split-thickness graft segments of human skin, segments of lung, and the like.
  • the biologic is a virus, protein, nucleic acid, carbohydrate, or lipid, or a combination thereof. In some embodiments, the biologic is an antibody or peptide. In some embodiments, the biologic is an antibiotic, a hormone, an enzyme, a clotting factor, or the like. In some embodiments, the biologic is present at from about 0.001 mg/mL to about 50 mg/mL. In some embodiments, the biologic is present at about 5 mg/mL.
  • the membrane penetrable sugar is chosen from trehalose, glucose, sucrose, lactose, maltose, other mycoses, and the like, to protect membrane-bound as well as free cytosolic enzyme systems and other critical cellular metabolic systems and pathways. Additionally, such treatments help ensure that upon water removal, the changes in cell volume and shape , condensation and crowding of the cytoplasm, membrane phase transitions, loss of supercoiling of DNA, oxidative damage, and metabolic arrest can be minimized.
  • the membrane penetrable sugar is trehalose.
  • the membrane penetrable sugar is generally a non-reducing sugar. Such a sugar may act to stabilize the cell for the drying processes described herein.
  • the membrane penetrable sugar can be replaced with other saccharides, proteins, polymers, and agents that function in the same manner.
  • the membrane penetrable sugar is present at from about 0.1% w/v to about 12% w/v. In some embodiments, the membrane penetrable sugar is present at about 2% w/v, about 3% w/v, about 4% w/v, or about 5% w/v.
  • the trehalose is not introduced within a cell by a viral vector.
  • the cells are not thermally shocked to allow trehalose to enter the cells. In some embodiments, the cells are not osmotically shocked to allow trehalose to enter the cells.
  • trehalose is not combined with glycerol or mannitol.
  • the composition comprises a combination of membrane penetrable sugars.
  • the composition can comprise both trehalose and glucose.
  • a composition comprising more than one membrane penetrable sugar can have the membrane penetrable sugars present at concentrations that are independent from one another.
  • a composition can comprises about 3% w/v trehalose and about 2% w/v glucose.
  • the membrane impenetrable sugar is chosen from dextran, starches, amylase, amylopectin, glycogen, polysucrose, and the like. In some embodiments, the membrane impenetrable sugar is dextran.
  • sugars with molecular weight greater than or equal to 50,000 daltons such as polysaccharides having a general formula of C n (H 2 O) n -I where n is from about 200 to about 2500, or (CeHi 0 Os) n where n is from about 40 to about 3000, can be used.
  • mix-type sugars such as, for example, Xanthan gum, guar gum, starch gum, British gum, and the like can be used as membrane impenetrable sugars.
  • the membrane impenetrable sugar is generally neutral.
  • the membrane impenetrable sugar can be replaced with other saccharides, proteins, polymers, and agents that function in the same manner.
  • the membrane impenetrable sugar can be replaced with plasma proteins such as, for example, albumin, soluble starches, glycogen, soluble chitin, and soluble celluloses.
  • the membrane impenetrable sugar can be present in the presence of plasma proteins such as, for example, albumin, soluble starches, glycogen, soluble chitin, and soluble celluloses.
  • the membrane impenetrable sugar is present at from about 0.01% w/v to about 25% w/v. In some embodiments, the membrane impenetrable sugar is present at about 3% w/v.
  • the cells or biomolecules are treated with at least one membrane impenetrable sugar and at least one membrane penetrable sugar in the absence of any polyol (i.e., a polyhydric alcohol, such as glycerol).
  • a polyhydric alcohol such as glycerol
  • the membrane impenetrable sugars be used to ensure that cells can be viable in a depleted state, metabolically adaptive, and maintenance in favorable local hydration conditions.
  • Other protective agents include, for example, proteins or the like, and hydrocolloid or the like. Such treatments/processes are intended to stabilize both internal and external membranes.
  • the composition further comprises a "fluidizer” or the like, such as an extremely mild mixture of glycerol or the like with a minimal, but effective, amount of an omega-3 fatty acid, or the like (e.g., EPA, ALA, etc.).
  • a "fluidizer” or the like such as an extremely mild mixture of glycerol or the like with a minimal, but effective, amount of an omega-3 fatty acid, or the like (e.g., EPA, ALA, etc.).
  • an omega-3 fatty acid, or the like e.g., EPA, ALA, etc.
  • Additional fluidizers include, but are not limited to, dimethylsulfoxide (DMSO), glycerin, and various detergents such as Tween-80.
  • the fluidizer is present at from about 1 nM to about 200 mM. In some embodiments, the fluidizer is present at from about 10 ⁇ M to about 50 ⁇ M.
  • the composition further comprises a fixative agent, such as a cross-linker with an aldehyde function such as, for example, paraformaldehyde, glutaraldehyde, or another compound having two terminal aldehyde groups.
  • a fixative agent may provide cells with physical stability such as volume and shape, which may be helpful for the use of cells as control reagents size simulants and provide uniformity across multiple instrument technologies.
  • the fixative agent is present at from about 0.01% to about 10%.
  • the fixative agent is present at about 0.5%.
  • the composition is free of a fixative agent.
  • the moisture content of the composition is from about 5% to about 95%.
  • the moisture content is from about 10% to about 90%. In some embodiments, the moisture content is from about 15% to about 85%. In some embodiments, the moisture content is from about 20% to about 80%. In some embodiments, the moisture content is from about 25% to about 75%. In some embodiments, the moisture content is from about 30% to about 70%, or about 30% to about 50%. In some embodiments, the moisture content is from about 35% to about 65%. In some embodiments, the moisture content is from about 40% to about 60%. In some embodiments, the moisture content is from about 45% to about 55%.
  • the moisture content is from about 5% to about 30%. In some embodiments, the moisture content is from about 5% to about 25%. In some embodiments, the moisture content is from about 5% to about 20%. In some embodiments, the moisture content is from about 5% to about 30%. In some embodiments, the moisture content is from about 20% to about 25% or from about 15% to about 25%. In some embodiments, the moisture content is about 25%. In some embodiments, the moisture content is about 5%, about 10%, or about 15% In some embodiments, the moisture content is less than about 20%, less than about 15%, or less than about 10% (but in no cases is the moisture content zero).
  • platelets are dried to no less than 15% residual moisture.
  • red blood cells are dried to no less than 25% residual moisture.
  • B cells are dried to no less than about 50% to 90% residual moisture.
  • the biologic is a platelet
  • the membrane penetrable sugar is trehalose
  • the membrane impenetrable sugar is dextran
  • the moisture content is about 15%.
  • the biologic is a red blood cell, the membrane penetrable sugar is trehalose, the membrane impenetrable sugar is dextran, and the moisture content is about 25%. In some embodiments, the biologic is a white blood cell, the membrane penetrable sugar is trehalose, the membrane impenetrable sugar is dextran, and the moisture content is about 50%.
  • the biologic is a protein, virus, or plasma
  • the membrane penetrable sugar is trehalose
  • the membrane impenetrable sugar is dextran
  • the moisture content is from about 5% to about 10%.
  • the compositions described herein without the biologic and/or the compositions described herein containing the biologic further contains one or more antimicrobial agents. Any anti-microbial agent will suffice.
  • anti-microbial agents include, but are not limited to, 1) protein synthesis inhibitors such as, for example, amikacin, anisomycin, apramycin, azithromycin, blasticidine S, brefeldin A, butirosin, chloramphenicol, chlortetracycline, clindamycin, clotrimazole, cycloheximide, demeclocycline, dibekacin, dihydrostreptomycin, doxycycline, duramycin, emetine, erythromycin, fusidic acid, G 418, gentamicin, helvolic acid, hygromycin B, josamycin, kanamycin, kirromycin, lincomycin, meclocycline, mepartricin, midecamycin, minocycline, neomycin, netilmicin, nitrofurantoin, nourseothricin, oleandomycin, oxytetracycline, paromomycin, pur
  • the anti-microbial agent can be used in the amount of from about 0.001% to about 0.1%, from about 0.005% to about 0.075%, from about 0.01% to about 0.05%, or from about 0.015% to about 0.025%, or at about 0.02%.
  • the compositions described herein without the biologic and/or the compositions described herein containing the biologic further contains one or more antioxidants. Any anti-oxidant will suffice.
  • anti-oxidants include, but are not limited to, mannitol, and 1) vitamins such as, for example, vitamin A (retinol), vitamin C (L-ascorbate), and vitamin E (tocotrienol, tocopherol, alpha-tocopherol, and vitamin E succinate); 2) vitamin co factors and minerals such as, for example, coenzyme QlO, manganese, superoxide dismutase (SOD), and iodide; 3) hormones such as, for example, melatonin; 4) carotenoid terpenoids such as, for example, carotenoid, alpha-carotene, astaxanthin, beta-carotene, canthaxanthin, lutein, lycopene, and zeaxanthin; 5) flavonoid polyphenolics such as, for example, flavones (apigenin, luteolin, and tangeritin), flavonols (isorhamnetin, kaempfe
  • the antioxidant can be used in the amount of from about 0.1% to about 1.0%, from about 0.25% to about 0.75%, from about 0.4% to about 0.6%, or from about 0.45% to about 0.55%, or at about 0.5%.
  • the anti-oxidant can be used in the amount of from about 0.01 ⁇ g/mL to about 1000 ⁇ g/mL, from about 0.1 ⁇ g/mL to about 100 ⁇ g/mL, from about 1 ⁇ g/mL to about 50 ⁇ g/mL, or from about 5 ⁇ g/mL to about 25 ⁇ g/mL, or at about 10 ⁇ g/mL.
  • L-ascorbate can be used in the following amounts: from about 3.7 mmol to about 37 mmol, or from about 14.8 mmol to about 25.9 mmol, or at 3.7 mmol, 14.8 mmol, 25.9 mmol, or 37 mmol.
  • Alpha-tocopherol can be used in the following amounts: from about 1.6 mmol to about 16 mmol, or from about 6.4 mmol to about 11.2 mmol, or at 1.6 mmol, 6.4 mmol, 11.2 mmol, or 16 mmol.
  • Mannitol can be used in the following amounts: from about 0.11 mmol to about 1.1 mmol, or from about 0.44 mmol to about 0.77 mmol, or at 0.11 mmol, 0.44 mmol, 0.77 mmol, or 1.1 mmol.
  • the present invention also provides methods of preserving a biologic comprising: contacting the biologic with at least one membrane penetrable sugar and at least one membrane impenetrable sugar; optionally, contacting the biologic with a fixative agent; and drying the biologic by vacuum desiccation to a final moisture content of from about 5% to about 90% (see, Figure 1).
  • the biologic being preserved can be any of the cells or biomolecules described herein.
  • the membrane penetrable sugar can be any of the membrane penetrable sugars described herein.
  • the membrane impenetrable sugar can be any of the membrane impenetrable sugars described herein.
  • the fixative agent can be any of the fixative agents described herein.
  • the moisture content can be any of the ranges or values of moisture content described herein.
  • the methods comprise concentrating the cells or biomolecules, and suspending the cells or biomolecules in a dehydrating solution that is comprised of the membrane penetrable sugar and the membrane impenetrable sugar.
  • the cells can be fixed with a fixative agent to provide physical stability prior to the drying process.
  • the cell/biomolecule media compositions are then dried using a desiccator.
  • the growth rate and/or metabolism of a biologic such as a cell, is slowed in the present dehydration (desiccation) solutions described herein. Without being bound to any theory, it is thought that the slowing of the growth rate and/or metabolism of a cell prepares the cell for desiccation and, therefore, helps the cell retain its functions upon being rehydrated.
  • the biologic such as a cell
  • the biologic is washed through the process of centrifugation and resuspension in an appropriate solution.
  • the biologic can be washed in saline.
  • the membrane penetrable and membrane impenetrable sugars are added to the cells.
  • a low concentration of adenosine is added to increase cellular ATP via the purine-based ATP "salvage pathway.”
  • superoxide dismutase (SOD) is added to effectively scavenge cellular oxygen free radicals.
  • the SOD can be in the Mn form or the Cu/Zn form.
  • a membrane fluidizer such as an extremely mild mixture of glycerol or the like, together with a minimal but effective amount of omega-3 fatty acid or the like (e.g., EPA, ALA, etc.), is added.
  • omega-3 fatty acid or the like e.g., EPA, ALA, etc.
  • adenosine is present at from about 1 nM to about 100 mM.
  • adenosine is present at from about 1 mM to about 5 mM, or from about 1 mM to about 4 mM. In some embodiments, adenosine is present at a concentration from about 0.5 mg/mL to about 5 mg/mL, or from about 1 mg/mL to about 2 mg/ml. In some embodiments, adenosine is present at about 1 mg/mL or at about 3.8 mM. In some embodiments, adenosine is present at about 70 ⁇ M. In some embodiments, SOD is present at from about 1 nM to about 5 mM. In some embodiments, SOD is present at from about 1 ⁇ M to about 3 ⁇ M.
  • albumin is present in the dehydration solution.
  • the percent w/v of albumin in the solution is from about 1% to about 20%, from about 1% to about 10%, from about 5% to about 10%, at about 5%, at about 6%, at about 7%, at about 8%, at about 9%, or at about 10%.
  • the cell is dried by vacuum desiccation at from about 0 0 C to about 40 0 C.
  • the cell or other biologic is dried for about 1 hour to about 4 hours, or for about 1 hour to about 8 hours, or for about 1 hour to about 12 hours, or for about 1 hour to about 16 hours.
  • the cell is dried by vacuum desiccation at from about 32°C to about 34°C for about 3 hours.
  • freezing and thawing of cells should be avoided.
  • Water molecules should be removed at temperatures from about 0 0 C to about 40 0 C, at about atmospheric pressure (i.e., about 760 mmHg) or at pressures reduced from atmospheric pressure (i.e., less than about 760 mmHg, or about 560 mmHg).
  • the rate of water removal should be controlled depending on the cell type. The rate of water removal should not be too fast to cause the overall collapse of the cell structure but not too slow to promote cellular activities that could compromise the cellular integrity and metabolism and defeat the drying process.
  • the final moisture level can be from about 5% to about 95% dependent on cell type and the final use.
  • dehydration solutions for various biologies have been prepared and used to preserve the indicated cell types.
  • the present invention contemplates dehydration solutions with and/or without a biologic. Any of the components listed in the dehydration solutions can, of course, be substituted by any of its suitable options described herein.
  • red blood cells from about 6.0% to about 8.0% or from about 6.5% to about 7.5% (suitably 7%) albumin; from about 0.7% to about 1.1% or from about 0.8% to about 1.0% (suitably 0.9%) NaCl; from about 15.0% to about 25.0% or from about 17.5% to about 22.5% (suitably 20%) dextran-70; from about 1.0% to about 5.0% or from about 2.0% to about 4.0% (suitably 3%) trehalose; from about 1.0% to about 4.0% or from about 1.0% to about 3.0% (suitably 2%) glucose; and from about 0.6 mg/mL to about 1.4 mg/mL or from about 0.8 mg/mL to about 1.2 mg/mL (suitably 1 mg/mL) adenosine. 2) For platelets: from about 6.0% to about 8.0% or from about 6.5% to about 7.5% (suitably 7%) albumin; from about 0.7% to about 1.1% or from about 0.8% to about 1.0% (suitably 0.9%) NaCl; from about 15.0% to about 25.0%
  • albumin (suitably 7%) albumin; from about 0.7% to about 1.1% or from about 0.8% to about 1.0% (suitably 0.9%) NaCl; from about 15.0% to about 25.0% or from about 17.5% to about 22.5% (suitably 20%) dextran-70; from about 0.5% to about 3.0% or from about 0.5% to about 2.0% (suitably 1%) trehalose; and from about 2.0% to about 6.0% or from about 3.0% to about 5.0% (suitably 4%) glucose.
  • CHO, and/or HEK cells from about 4.0% to about 6.0% or from about 4.5% to about 5.5% (suitably 5%) albumin; from about 0.7% to about 1.1% or from about 0.8% to about 1.0% (suitably 0.9%) NaCl; from about 20.0% to about 30.0% or from about 22.5% to about 27.5% (suitably 25%) dextran-70; from about 0.5% to about 3.0% or from about 0.5% to about 2.0% (suitably 1%) trehalose; from about 2.0% to about 6.0% or from about 3.0% to about 5.0% (suitably 4%) glucose; and from about 80 mM to about 120 mM or from about 90 mM to about 110 mM (suitably 100 mM) K 2 HPO 4 (or other equivalent buffer).
  • For cord blood stem cells from about 6.0% to about 8.0% or from about 6.5% to about 7.5% (suitably 7%) albumin; from about 0.7% to about 1.1% or from about 0.8% to about 1.0% (suitably 0.9%) NaCl; from about 15.0% to about 25.0% or from about 17.5% to about 22.5% (suitably 20%) dextran-70; from about 0.5% to about 3.5% or from about 1.0% to about 3.0% (suitably 2%) trehalose; and from about 1.0% to about 5.0% or from about 2.0% to about 4.0% (suitably 3%) glucose.
  • sporozoites from about 0.7% to about 1.1% or from about 0.8% to about 1.0% (suitably 0.9%) NaCl; from about 25.0% to about 35.0% or from about 27.5% to about 32.5% (suitably 30%) dextran-70; from about 0.1% to about 1.0% or from about 0.25% to about 0.75% (suitably 0.5%) trehalose; from about 1.0% to about 4.0% or from about 1.0% to about 3.0% (suitably 2%) glucose; and from about 80 mM to about 120 mM or from about 90 mM to about 110 mM (suitably 100 mM) K 2 HPO 4 (or other equivalent buffer).
  • cryoprecipitate, and/or serum from about 0.7% to about 1.1% or from about 0.8% to about 1.0% (suitably 0.9%) NaCl; from about 25.0% to about 35.0% or from about 27.5% to about 32.5% (suitably 30%) dextran-70; from about 4% to about 8% or from about 5% to about 7% (suitably 6%) trehalose; from about 1.0% to about 4.0% or from about 1.0% to about 3.0% (suitably 2%) glucose; and from about 80 mM to about 120 mM or from about 90 mM to about 110 mM (suitably 100 mM) K 2 HPO 4 (or other equivalent buffer).
  • the cells destined to undergo such treatment can be dried via a process of desiccation such as vacuum drying or convection oven drying.
  • the cells can be transferred to a nitrogen- filled, mildly heated desiccator with less than 5% humidity and gradually dried over a period of time until the composition contains a moisture level consistent with the needs of the specific application.
  • suitable gasses include, but are not limited to, essentially inert gasses such as helium, argon, or xenon.
  • the gasses can be introduced into the chamber at or near the end of the process to drive off any remaining free oxygen.
  • the process begins at ambient humidity, which should be as low as reasonably achievable (e.g., about 50%).
  • the vacuum desiccator keeps the chamber humidity very low (i.e., at about 5%).
  • the methods further comprise storing the cells in a vacuum sealed container in the presence or absence of a desiccant, and the presence or absence of nitrogen or other inert gas.
  • Desiccants are well known to the skilled artisan and are commercially available and include, but are not limited to, silica gel, calcium sulfate, and calcium chloride. Desiccants can be included to mitigate humidity issues and absorb moisture and gases that may be released by the cells during the storage period.
  • the desiccated cells can be stored under vacuum for long-term storage (see, Figure 2). In some embodiments, the cells or biomolecules can be stored for at least 7 days prior to rehydration and subsequent use.
  • the cells or biomolecules can be stored for at least 10 days prior to rehydration and subsequent use. In some embodiments, the cells or biomolecules can be stored for at least 14 days prior to rehydration and subsequent use. In some embodiments, the cells or biomolecules can be stored for at least 21 days prior to rehydration and subsequent use. In some embodiments, the cells or biomolecules can be stored for at least 28 days prior to rehydration and subsequent use. In some embodiments, the cells or biomolecules can be stored for at least 45 days prior to rehydration and subsequent use. In some embodiments, platelets and/or red blood cells can be stored for greater that 45 days.
  • the cells or biomolecules can be stored for at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months prior to rehydration.
  • the biologic (the biologic can be any those described herein) which has been vacuum dried can be subject to room temperature-induced dryness.
  • any biologic having a moisture content of 50% or less is susceptible to room temperature-induced dryness.
  • a barrier overlay material is added to the biologic which has been vacuum dried, thus preventing or reducing room temperature-induced dryness. The barrier forms on top of the biologic within a container.
  • a small amount of oil or lubricant can serve as the barrier overlay material and can be applied to the biologic which has been vacuum dried, such as be creating an overlay, to prevent drying prior to capping the container.
  • a 5 ⁇ L aliquot of red blood cells which has been vacuum dried in a well of a 96-well plate can be contacted with 1 to 5 ⁇ L of oil.
  • the contacting can be carried out by, for example, spraying the biologic sample with the oil or dropping the oil onto the biologic.
  • the amount of oil applied can vary depending upon the amount of the aliquot of the biologic.
  • Suitable oils include, but are not limited to, immersion oils such as Type NVH, Type 300, Type A, and Type B, olive oil, extra virgin olive oil, or any other form of olive oil.
  • Other barrier overlay materials that may be suitable include, but are not limited to, other organic solvents such as acetic acid, acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 2-butanone, t- butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1 ,2-dichloroethane, diethyl ether, diethylene glycol, diethylene glycol dimethyl ether (diglyme), 1 ,2- dimethoxyethane (glyme, DME), dimethylether, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dioxane, ethanol, ethyl acetate, ethylene glycol, glycerin, hept
  • the methods further comprise rehydrating the biologic.
  • rehydration comprises contacting the biologic with water and, optionally, next with saline.
  • the biologic is also rehydrated in the presence of albumin.
  • rehydrating comprises contacting the biologic with water and/or saline that is free of albumin. Rehydration can also be performed in the presence of an osmotic balancer, such as, albumin.
  • the osmotic balancer is a reagent that affects the osmolarity of the biologic.
  • the osmotic balancer in some embodiments, is present in an amount sufficient to maintain an osmolarity of about 200 mOsm/L to about 4500 mOsm/L, about 200 mOsm/L to about 1000 mOsm/L, about 200 mOsm/L to about 2000 mOsm/L, 200 mOsm/L to about 3000 mOsm/L, or about 200 mOsm/L to about 4000 mOsm/L.
  • the percent w/v of albumin in the solution is from about 1% to about 20%, from about 1% to about 10%, from about 1% to about 5%, from about 1% to about 3%, from about 5% to about 10%, at about 1%, at about 2%, at about 3%, at about 4%, at about 5%, at about 6%, at about 7%, at about 8%, at about 9%, or at about 10%.
  • the rehydration solution comprises sodium chloride in an amount that is from about 0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, from about 0.5% to about 1%, or at about 0.9%.
  • the volume of the fluid added to the cells is equal to the fluid volume of the composition prior to the drying process.
  • Cells and biomolecules can be rehydrated to the concentrations described above.
  • various physiological buffers including, but not limited to, HEPES, phosphate buffered saline (PBS), Tris buffer, and the like, or other such solutions, can be used.
  • the solution comprises potassium phosphate.
  • the solution is free of HEPES, PBS, Tris buffer, and the like.
  • the time and temperature for carrying out the rehydration process can be from about 5 minutes to about 200 minutes at room temperature or temperature up to 37°C.
  • the optimal reconstitution time and temperature will be dependent of cell type and the final use and can be determined by the user.
  • temperatures from about 22°C to about 37°C can be used for rehydration.
  • Rehydration time can vary with the procedural factors, expected cell or protein performance, residual moisture, and volume of dried material.
  • the time for rehydration is from about 1 hour to about 24 hours prior to desired use.
  • the time for rehydration is from about 24 hours to about 48 hours, from about 24 to about 72 hours, from about 48 hours to about 72 hours, from about 1 hour to about 48 hours, from about 1 hour to about 72 hours, at least about 24 hours, at least about 48 hours, or at least about 72 hours.
  • the time for rehydration is from about 5 minutes to about 60 minutes, from about 5 minutes to about 30 minutes, from about 5 minutes to about 20 minutes, from about 10 minutes to about 20 minutes, about 15 minutes, about 30 minutes, or for at least about 6 hours.
  • the volume of rehydration solution to rehydrate the biologic can vary depending upon the preference of one of skill in the art or to an amount such that the rehydrated biologic is present at an effective concentration.
  • the effective concentration is a concentration that is effective for the use of the biologic.
  • the biologic is rehydrated in about 1 mL, about 5 mL, from about 1 mL to about 5 mL, or from about 1 mL to about 10 mL of solution.
  • the biologic can be rehydrated, for example, at room temperature or 37 0 C, or any temperature in between.
  • the viability of the rehydrated cells is about 10% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 60% or greater, about 70% or greater, about 75% or greater, about 80% or greater, about 85% or greater, about 90% or greater, about 95% or greater, or about 99% or greater.
  • the methods comprise preserving a platelet comprising: contacting the platelet with at least one membrane penetrable sugar that is trehalose and at least one membrane impenetrable sugar that is dextran; optionally, contacting the platelet with a fixative agent that is glutaraldehyde or paraldehyde; and drying the platelet by vacuum desiccation to a final moisture content of about 15%.
  • the methods comprise preserving a red blood cell comprising: contacting the red blood cell with at least one membrane penetrable sugar that is trehalose and at least one membrane impenetrable sugar that is dextran; optionally, contacting the red blood cell with a fixative agent that is glutaraldehyde or paraldehyde; and drying the red blood cell by vacuum desiccation to a final moisture content of about 25%.
  • the methods comprise preserving a white blood cell comprising: contacting the white blood cell with at least one membrane penetrable sugar that is trehalose and at least one membrane impenetrable sugar that is dextran; optionally, contacting the white blood cell with a fixative agent that is glutaraldehyde or paraldehyde; and drying the white blood cell by vacuum desiccation to a final moisture content of about 50%.
  • the methods comprise preserving a protein, virus, or plasma comprising: contacting the protein, virus, or plasma with at least one membrane penetrable sugar that is trehalose and at least one membrane impenetrable sugar that is dextran; optionally, contacting the protein, virus, or plasma with a fixative agent that is glutaraldehyde or paraldehyde; and drying the protein, virus, or plasma by vacuum desiccation to a final moisture content of from about 5% to about 10%.
  • the methods comprise preserving cryoprecipitate comprising contacting the cryoprecipitate with at least one membrane penetrable sugar, such as trehalose, and at least one membrane impenetrable sugar, such as dextran; optionally, contacting the cryoprecipitate with a fixative agent, such as glutaraldehyde or paraldehyde; and drying the cryoprecipitate by vacuum desiccation to a final moisture content of from about 5% to about
  • a fixative agent such as glutaraldehyde or paraldehyde
  • the methods comprise contacting the biologic with a 5X solution comprising at least one membrane penetrable sugar, such as trehalose, and at least one membrane impenetrable sugar, such as dextran; optionally, contacting the cryoprecipitate with a fixative agent, such as glutaraldehyde or paraldehyde; and drying the cryoprecipitate by vacuum desiccation to a final moisture content of from about 5% to about 25%, from about 5% to about 15%, or at about 10%.
  • a fixative agent such as glutaraldehyde or paraldehyde
  • the present invention also comprises methods of treating an animal having a need for a biologic comprising administering a biologic described herein.
  • the animal will be a human suffering from a blood disorder whereby the human is in need of a blood product (i.e., whole blood, red blood cells, platelets, plasma, clotting factor(s), etc).
  • the need may arise from the human having a disease, condition, or disorder whereby the particular biologic is not produced or is produced in insufficient amounts.
  • the need may arise from injury, such as a traumatic injury characterized by blood loss. Any of the rehydrated vacuum dried biologies described herein can be administered to such animals.
  • the need can be for any biologic for correlated with appropriate diseases, conditions, or disorders. Exemplary diseases, conditions, or disorders include, but are not limited to, anemia, blood loss, and hemophilia.
  • the present invention also provides any of the compositions described herein for treating an animal in need of a biologic, as described above.
  • the present invention also provides any of the compositions comprising a biologic described herein for use in the manufacture of a medicament, such as a sterile medicament, for the treatment of a disease, condition, or disorder related to the particular biologic.
  • the medicament is a sterile composition comprising whole blood, red blood cells, platelets, plasma, clotting factor(s), etc. for the treatment of someone in need thereof.
  • the vacuum desiccated cells that have been rehydrated show surface marker profiles, such as platelet surface marker, similar to fresh cells.
  • the present invention also provides methods of typing blood.
  • a fresh blood sample is desiccated and stored as described herein for later blood typing using routine blood typing methods.
  • the desiccated blood is rehydrated prior to typing the red blood cells.
  • the desiccated sample can be used to determine the presence or absence of common surface antigens. Examples of common surface antigens include, for example, C, E, c, e, K, M, N, S, s, Fya, Fyb, Jka, Jkb, and the like.
  • the present invention provides a desiccated red blood cell sample that is used as a reference when blood is being typed.
  • the present invention also provides for a kit comprising one or more desiccated biologic samples.
  • the biologic is a cell or a biomolecule as described herein.
  • the kit is used for typing blood.
  • the kit comprises a desiccated red blood cell composition.
  • the desiccated sample comprises from about 0.5 % to about 1.0% sodium chloride, from about 3 to about 4 mM adenosine, from about 1% to about 5 % glucose, about 10% Dextran-70, about 3% Trehalose, and about 7% albumin.
  • the protocol for red blood cell dehydration and re-hydration is as follows: 1) RBCs are washed in 0.9% NaCl via centrifugation at high speed at 1000 x g, for 5 minutes each time until there is no more sign of hemolysis; 2) RBC are washed in reconstitution buffer (RB) (2% Albumin + 0.9% NaCl) twice and centrifuged at 1000 x g, for 5 minutes; 3) the dehydration buffer (DHB) is prepared (in 0.9% NaCl solution, add 20% Dex-70 w/v, add 7% Albumin w/v, add 3% Trehalose w/v, add 2% Glucose w/v, add 1 mg/ml Adenosine) taking time to dissolve all the components as the solution will be very thick.
  • RB reconstitution buffer
  • DHB dehydration buffer
  • the DHB is kept refrigerated (discarded after 6 months); 4)the packed RBCs are re-suspended in 1 :4 volumes of DHB (i.e., 1 ml of packed RBC to 4 mL of DHB) and incubated at room temperature for 1 hour (if needed, incubation can occur at 4°C overnight and the next day, mixed well with gentle pipetting and titling the tube to resuspend the cells); 5) if the cells will be dried in an amber vial, proceed to step 6; if the cells will be dried in a 96 well plate, proceed to step 11; 6) a 0.5mL aliquot of cells is transferred into a "20 mL-amber" vial; 7) for dehydration time, the goal is to achieve 70% final weight (the cells are dried for 1 hour at 32°C, 25 mmHg, then checked for moisture; at this point, the vials will have reduced liquid; after an additional 30 minutes, the cells are checked for moisture; at this point the
  • a sugar such as, for example, polysucrose 400, dextran 70, glycerol, PEG, or combinations of one or more of the sugars such as a mixture of dextran 70 and polysucrose 400, for example
  • the reconstitution buffer saline, or any kind of buffers described herein
  • red cells were dried to 10% residual moisture and were reconstituted with reconstitution buffer (RB) (10 mM HEPES, 0.9% NaCl, and 2% albumin), about 1% cell recovery was achieved.
  • RB reconstitution buffer
  • the osmotic stress on the cells can be reduced upon reconstitution by lowering the water content within the rehydration process by adding one or more high molecular weight carbohydrates, proteins or the like.
  • high molecular weight carbohydrates, proteins or the like that can replace polysucrose 400, for example.
  • the cells when working with a very small volume of cells (such as 20 ⁇ L or less, or 10 ⁇ L or less), water will evaporate and will cause the cells to dry even at room temperature after 30 minutes (to around 10% residual dryness). If the same cell volume is placed in the refrigerator, without closing the lid and leaving the cells exposed to the air in the refrigerator, the cells will come to complete dryness after couple of hours (to around 10% residual dryness as well).
  • the cells When the cells are dried at room temperature and reconstituted with 20% polysucrose 400, for example, the cells reconstituted but remained dark red. When the cells that were dried in the refrigerator were reconstituted with 20% polysucrose 400, however, they turned bright red.
  • the cells or other biologies are cold desiccated in the presence of one or more high molecular weight carbohydrates, proteins or the like, such as polysucrose 400.
  • one way to increase the surface area to volume ratio for the biologic fluid is rotary vacuum evaporation.
  • the inside fluid spreads along the walls of the desiccation chamber (bottle, flask, etc.), moisture is released from the liquid phase into the gas phase and is carried away by the vacuum.
  • the fluid being desiccated accretes on the walls of the chamber in a desiccated form and is covered by more and more desiccated material until the entire volume is desiccated to a desired residual moisture (determined, e.g., by wet weightdry weight ratio).
  • a desired residual moisture determined, e.g., by wet weightdry weight ratio.
  • nitrogen or other inert gas can be vented into the rotary vacuum desiccation chamber, sufficient to drive off all or most of the available oxygen.
  • the residual oxygen like the water vapor, is carried out of the desiccation chamber by the vacuum.
  • the venting of an inert gas through the chamber containing the fluid to be desiccated increases the effectiveness of the desiccation via convective water loss.
  • the vacuum flask/desiccation chamber rotates and rests in a heated cradle; the applied heat facilitating the process (see, Figure 10).
  • a variation of the process described by Figure 10 involves placing the vacuum flasks on a roller system (these are commercially available for either cell culture or "hot dog rollers") and placing that system in a vacuum oven. Connections are required, as they are in the first system, to allow rotation of the flask while supplying nitrogen and applying vacuum; though the vacuum chamber can evacuate vapor through the open vacuum line (see, Figure 11).
  • the temperature can be varied in the vacuum chamber (oven) as can the speed and duration of rotation.
  • the multi-roller bed system housed in the vacuum oven, is considerably less expensive.
  • bags of fluid-mixed biologies will require vacuum desiccation (e.g., blood cells, platelets, plasma, other solutions or cell-solution mixes).
  • Traditional "blood bags” represent a particular problem in they are flat or three- dimensionally rhomboidal and not easily amenable to roller beds.
  • the prolonged rolling may place undue wear on the bag, causing leakage or allowing contamination.
  • a solution to the traditional bag problem would be to place the traditional bag into a properly-sized vacuum bottle, connect the bag-bottle doublet to lines as in Figure 11, and place the roller bed/vacuum flask system into a vacuum oven for desiccation as in Figure 11 (see Figure 12).
  • One advantage of the third system configuration is that it should be easier to address required sterility issues, even if, technically, the system will be "open", i.e., open to the environment.
  • one or more "balls” or spheres may be optionally used. These spheres or balls can be added to the fluid volume to increase additionally the surface area available for water vapor exchange during desiccation. They will remain in the bag during rehydration and administration of the fluid and will be discarded along with the container.
  • the process of isolating and washing red blood cells from whole blood is well known in the art. Thus, numerous methods can be used to generate washed red blood cells and prepare them for the desiccation processes described herein. The following is meant to serve as one example of how the process is typically performed.
  • Blood was obtained in a sterile manner using an anti-coagulating agent such as sodium citrate, heparin, ethylenediaminetetraacetic acid (EDTA), or the like.
  • EDTA ethylenediaminetetraacetic acid
  • a 10 mL aliquot of whole blood was placed into a 15 mL conical tube and then centrifuged at 100 g for 30 minutes to remove the platelet rich plasma.
  • the overall packed RBC volume was determined, and a minimum of three times that volume of saline (0.9% NaCl) was added. For example, if the packed RBC volume is 1 mL, a minimum of 3 mL of saline was added. The cells were suspended by inverting the tube several times. Another centrifugation at 100 g for 30 minutes was performed. The saline supernatant was removed and discarded, and the wash process was repeated again. To get RBCs ready for desiccation, a concentrated dehydration buffer (cDHB) was prepared fresh.
  • cDHB concentrated dehydration buffer
  • cDHB a saline solution (0.9% NaCl) containing 100 mM HEPES was used, to which was added 200 ⁇ M adenosine, 100 mM glucose, 10 mM K 2 HPO 4 , 10% Dextran- 70, and 12% trehalose.
  • the overall packed RBC volume was determined, and multiplied by 4 to obtain the final desired volume.
  • the final volume was obtained by adding in 2/4th the volume with saline and l/4th the volume with cDHB.
  • the final l/4th volume was the cell pellet. For example, if the packed RBC volume was 1 mL, then the final volume should be 4 mL.
  • saline 2 mL of saline and 1 mL of cDHB were added.
  • the cells were resuspended by inverting the tubes several times.
  • the RBC were incubated in the buffer for 1 hour at 32°C-37°C or alternately, in 4°C for 24 hours or up to 48 hours.
  • the weight of the empty container (tare weight) was determined.
  • a vial made from any materials that are non-reactive to cells and proteins, can be used for this purpose.
  • a tall vial with 10 mL capacity can be used. This is to account for the "wicking" of the solution up the walls of the vial in a vacuum environment.
  • Example 1 RBC were processed and prepared as outlined in Example 1. After determining the overall packed RBC volume and multiplying this volume by 4 to obtain the final volume (as described above in Example 1), the final volume was obtained by adding in 2/4th the volume with saline and l/4th the volume with fixative buffer. For example, if the packed RBC volume was 1 mL, then the final volume should be 4 mL. To obtain this volume, 2 mL of saline and 1 mL of fixative buffer was added. The cells were suspended by inverting the tubes several times. The RBCs were incubated in the fixative buffer for as little as one hour at 34°C or as long as 24 hours at 4°C. To prepare the fixative buffer with fixative agent, the fixative agent was added to the cDHB such that the final concentration of the fixative agent was 0.5%. The fixative buffer was kept in the cold at 4°C for at least 30 minutes before use.
  • the cells were centrifuged at 100 g for 30 minutes to remove the fixative buffer.
  • the overall packed RBC volume was determined and multiplied by 4 to obtain the final volume.
  • the final volume was obtained by adding in 2/4th the volume with saline and l/4th the volume with cDHB. For example, if the packed RBC volume was 1 mL, then the final volume should be 4 mL. To obtain this, 2 mL of saline and 1 mL of cDHB were added.
  • the cells were suspended by inverting the tubes several times.
  • the RBCs were desiccated as described above in Example 1. Vials were sealed under vacuum and/or under nitrogen gas. Samples were packed under vacuum with appropriate gas as well as having a desiccant to control and absorb moisture or gas that may be released by cells under storage.
  • the dried RBC vials were kept at 4°C or at room temperature.
  • the process of isolation of PRP from whole blood is well known in the art. Thus, numerous methods can be used to generate PRP and prepare them for the desiccation process. The following is meant to serve as one example of how the process is typically performed.
  • Blood was obtained in sterile manner using an anti-coagulating agent such as sodium citrate, heparin, EDTA, or the like.
  • a lO mL aliquot of whole blood was placed into a 15 mL conical tube.
  • the whole blood was centrifuged at 100 g for 30 minutes to separate PRP from white blood cells and red blood cells.
  • the PRP was decanted from the centrifuge tube containing blood cells to a new tube with no red or white blood cells.
  • cDHB was prepared as described in Example 1.
  • the overall PRP volume was determined, and l/4th of the cell volume, as cDHB, was added. For example, if the PRP volume is 4 mL, 1 mL of cDHB was added and then mixed by inverting the tubes several times. The PRP solution was incubated at 34°C for 1 hour with mixing every 10 minutes.
  • the weight of the empty container was determined.
  • a vial made from any materials that are non-reactive to cells and proteins was used for this purpose.
  • To desiccate 1 mL of PRP solution a vial with a 10 mL capacity was used.
  • a 1 mL aliquot of PRP solution was placed into the vial, which was then weighed (pre- dehydration weight).
  • the temperature of the dehydration chamber was adjusted to 32°C-37°C and dehydrated under vacuum at -560 mmHg open system for 90 minutes or more.
  • the final moisture content was about 15%.
  • the formula in Example 1 was used to calculate the final % moisture.
  • Figure 4 depicts typical size distribution of fresh PRP (labeled as fresh platelets) and the same cells which were reconstituted after being desiccated (Des Platelets) or freeze-dried (FD Platelets).
  • Des Platelets desiccated cells
  • FD Platelets freeze-dried platelets
  • Example 4 Desiccation of Platelet with Fixative Agent (actual example) PRP was processed and prepared as outlined in Example 3.
  • cDHB was prepared as described in Example 1. The overall PRP volume was determined as described in Example 3, and l/5th that volume of cDHB was added. For example, if the final PRP volume was calculated to be 4 mL, 1 mL of cDHB was added and mixed by inverting the tube several times. The PRP solution was incubated at 34°C for 1 hour with mixing every 10 minutes. To fix PRP, glutaraldehyde was added to a final concentration of 0.01% and the PRP was incubated for 1 hour at 34°C with mixing every 10 minutes. The PRP was then desiccated as described in Example 3.
  • Vials were sealed under vacuum and/or under nitrogen gas. Samples were packed under vacuum with the appropriate gas as well as having desiccant to control and absorb moisture or gas that may be released by cells under storage. The dried PRP vials were kept at 4°C or at room temperature.
  • Cells that are naturally non-adherent include B-cells or cells that have been treated with an agent such as EDTA or trypsin that detach them from binding surfaces.
  • Representative cell types include, but are not limited to: stem cells (adult and neonatal, various tissue or species origin), stem cell progenitor cells, gametes (male and female), gamete progenitor cells, endothelial cells, erythroblasts, leukoblasts, chondroblasts, hepatocytes, etc.
  • stem cells adult and neonatal, various tissue or species origin
  • stem cell progenitor cells gametes (male and female), gamete progenitor cells, endothelial cells, erythroblasts, leukoblasts, chondroblasts, hepatocytes, etc.
  • B-cells and stem cells were washed through the process of centrifugation and suspended in fresh media.
  • the membrane penetrable sugar such as the non-reducing sugar trehalose (5 to 250 mM)
  • a membrane "fluidizer” such as a mild mixture of glycerol (0.1 ⁇ M to 20 mM) with a minimal, but effective amount of omega- 3 fatty acid (0.1 to 10 ⁇ M) is also added to the cell media. Cells were incubated at 37°C overnight.
  • the buffer in this example was 0.1 M HEPES with salt components such as 20-60 mM NaCl, 1-5 mM K 2 HPO 4 , adenosine at 70 ⁇ M and glucose at 2-5 mM added to the buffer. Also, 5- 250 mM trehalose was added to the buffer and also, a membrane impenetrable sugar, such as a neutral dextran 70 (mol. wt. 70 kilodaltons) at 0.1-5% weight by volume was added to the buffer. Alternatively, a fixative agent such as glutaraldehyde at 0.1-0.5% may also be added to the process to stabilize the volume, size and shape of the cells. Cells were incubated for 1 hour at 37°C prior to desiccation.
  • salt components such as 20-60 mM NaCl, 1-5 mM K 2 HPO 4 , adenosine at 70 ⁇ M and glucose at 2-5 mM added to the buffer.
  • 5- 250 mM trehalose was
  • Cells were washed through the process of centrifugation with media containing 5 to 250 rnM trehalose and neutral dextran 70 at 0.1-5% weight by volume.
  • Cells were suspended in buffer at a concentration of 1,000 cells per mL to 100,000,000 cells per mL.
  • the cells were suspended in a volume of 50 ⁇ L to 1000 ⁇ L of cDHB, or at any volume and concentration suitable for drying.
  • the cells were transferred to a desiccator with a relative humidity level of 5% or less and heated to 35-45°C.
  • the desiccator was flushed with nitrogen gas and was maintained under nitrogen gas for the duration of the drying process.
  • the dehydration rate was controlled so that the water evaporation was about 0.1-100.0 ⁇ L per minute.
  • the dehydration rate can be faster or slower depending on the cell type.
  • the process of drying was considered complete when the relative levels of moisture in the dried cells was suitable for cells to function upon reconstitution.
  • the residual moisture in cells can be 5% to 95%. Dried cells are those at moisture level of 5% to 20%, whereas semi-dried cells are those at moisture level of >20%.
  • Cells were sealed under vacuum and possibly under nitrogen gas. Samples were packaged under vacuum with appropriate gas as well as having desiccant to control and absorb moisture and/or gas that may be released by cells under storage. The dried cells were kept at 4°C or at room temperature.
  • Example 6 Desiccation of Adherent Nucleated Cells
  • Representative cell types include: stem cells (adult and neonatal, various tissue or species origin), stem cell progenitor cells, gamete progenitor cells, endothelial cells, erythroblasts, leukoblasts, chondroblasts, hepatocytes, etc.
  • endothelial cells were grown in appropriate containers that allowed cells to attach and proliferate to an appropriate density. Then, 5-250 niM trehalose was added to the cell media and cells were incubated at 37°C overnight.
  • a desiccation buffer such as, for example, 0.1 M HEPES with salt components such as 20-60 mM NaCl, 1-5 mM K 2 HPO 4 , adenosine at 70 ⁇ M and glucose at 2-5 mM
  • a fixative agent such as glutaraldehyde at 0.1-0.5% can be added to the process to stabilize the volume, size and shape of the cells. Cells were incubated for 1 hour at 37°C prior to desiccation.
  • the buffer was aspirated and cell media was added containing 5-250 mM trehalose and neutral dextran 70 at 0.1-5% by weight.
  • the cells were transferred to a desiccator with a relative humidity level of 5% or less and heated to 35-45°C.
  • the desiccator was flushed with nitrogen gas and was maintained under nitrogen gas for the duration of the drying process.
  • the dehydration rate was controlled so that the water evaporation was about 0.1-100.0 ⁇ L per minute.
  • the dehydration rate can be faster or slower depending on the cell type.
  • the process of drying was considered complete when the relative level of moisture in the dried cells was suitable for the cells to function upon reconstitution.
  • the residual moisture in the cells can be about 5% to about 95%.
  • Dried cells are those at moisture levels of 5-20%, whereas semi-dried cells are those at moisture levels of >20%-95%.
  • Cells were sealed under vacuum and/or under nitrogen gas. Samples were packaged under vacuum with the appropriate gas as well as having desiccant to control and absorb moisture or gas that may be released by cells under storage. The dried cells were kept at 4°C or at room temperature.
  • Example 7 Desiccation of Proteins, Nucleic Acids and Viruses (macromolecules) (actual example)
  • Desiccation of macromolecules was conducted by adding trehalose (5-250 mM) and neutral dextran-70 (l%-6% w/v) into the buffer defined for the macromolecules by the end user.
  • the buffer or solution used is determined by the end user and can be any desired solution or buffer such as saline or PBS.
  • the weight of the empty container was determined.
  • a vial made from any material that is non- reactive to cells and proteins was used for this purpose.
  • To desiccate 1 mL of macromolecule solution a vial with 10 mL capacity was used.
  • a 1 mL aliquot of macromolecule solution was placed into the vial and the vial was weighed again (pre-dehydration weight).
  • the temperature of the dehydration chamber was adjusted to 32°C-37°C and dehydrated under vacuum at -560 mmHg open system for 90 minutes or more. The final moisture content was about 5%- 15%.
  • Vials were capped and sealed under vacuum and nitrogen atmosphere. Vials were stored at 4°C or ambient temperature.
  • the recommended volume of distilled water was gently pipetted onto the wall of the vial and allowed to contact the dried sample by gravity. In general, 0.85-0.05 mL of water was used for reconstitution. The reconstituted vial was left at 34°C for 30 minutes with frequent mixing.
  • Example 8 Desiccation of Whole Blood With and Without Fixative Agent (actual example)
  • cDHB The volume of whole blood was determined, and l/5th the calculated final volume was added as cDHB.
  • a saline solution (0.9% NaCl) containing 100 mM HEPES was used. To this solution was added 100 mM Glucose, 10 mM K 2 HPO 4 , 10% w/v Dextran-70, and 12% w/v Trehalose.
  • the whole blood solution was incubated at 34°C for 1 hour with mixing every 10 minutes.
  • glutaraldehyde can be added to a final concentration of 0.1% and the whole blood incubated for 1 hour at 34°C with mixing every 10 minutes.
  • the weight of the empty container was determined.
  • a vial made from any material that is non-reactive to cells and proteins was used for this purpose.
  • a vial with 10 mL capacity was used.
  • a 1 mL aliquot of whole blood solution was placed into the vial and the weight of the vial was determined again (pre-dehydration weight).
  • the temperature of the dehydration chamber was adjusted to 32°C-37°C and dehydrated under vacuum at -560 mmHg open system for 90 minutes.
  • the final moisture content was about 25%.
  • Example 9 Desiccation and Activity of Rehydrated Cryoprecipitate (actual example) Canine cryoprecipitate was prepared by routine methodology. The cryoprecipitate was added to 5X DHB-2 solution, which comprised 15% Dextran-70 and 15% Trehalose in saline. The final concentration of the DHB-2 solution was IX upon addition of the cryoprecipitate. An aliquot of the cryoprecipitate-DHB-2 mixture (e.g., 5 mL) was placed into a vial and desiccated until dryness with a residual moisture of 10%. The vial was vacuum sealed. To reconstitute the desiccated cryoprecipitate, 5 ml of sterile water was added to the vial. The vial was allowed to rest for 15 minutes with frequent swirling.
  • 5X DHB-2 solution which comprised 15% Dextran-70 and 15% Trehalose in saline.
  • the final concentration of the DHB-2 solution was IX upon addition of the cryoprecipitate.
  • the sample was analyzed for Factor VIII activity.
  • the undiluted sample contained 1.77 U/mL of Factor VIII activity, and the 5X dilution sample contained 0.21 U/mL of Factor VIII activity.
  • Activity of a fresh sample that had not been desiccated and rehydrated contained about 1.8 U/mL of Factor VIII activity.
  • Example 10 Desiccated Plasma Retains Clotting Properties (actual example)
  • Bovine plasma was desiccated according to a procedure described herein.
  • the desiccated plasma was rehydrated in water.
  • the desiccated plasma properties were compared to fresh plasma in Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT), tests designed to measure clotting and to identify defects or deficits in Factors VII, X, V, II, fibrinogen, other circulating inhibitors, and platelet disorders of primary hemostasis.
  • PT Prothrombin Time
  • aPTT Activated Partial Thromboplastin Time
  • the PT test for fresh bovine plasma was 38 seconds and for desiccated and rehydrated plasma was 34 seconds, which are not statistically different.
  • the aPTT test for fresh and desiccated plasma were also not statistically different. Therefore, the results of these tests demonstrate that plasma, desiccated as described herein, is functional and works similarly to fresh plasma.
  • Red blood cells were either frozen and then thawed or desiccated as described herein and rehydrated. The number of viable cells was determined and percent recovered was calculated. The data is shown in Table 1. Table 1
  • FIG. 6 shows the gross macroscopic examination of rehydrated (A) and thawed (B) red blood cells under 400X magnification. Representative samples of both rehydrated and thawed cell preparations were mounted on microscope slides and immediately inspected under the microscope.
  • the desiccated red bloods cells were also analyzed for the ability to retain surface antigens. Rehydrated cells were analyzed and it was determined that the common surface antigens (C, E, c, e, K, M, N, S, s, Fya, Fyb, Jka, and Jkb) were conserved in the process. Therefore, desiccated/rehydrated red blood cells can be used as a control in the typing of red blood cells.
  • Example 12 Dehydration Solutions for Various Biologies and Recovery, Viability, and Activity Thereof (actual example) The following dehydration solutions were prepared.
  • red blood cells 7% albumin,
  • cord blood stem cells 7% albumin, 0.9% NaCl, 20% dextran-70, 2% trehalose, and 3% glucose.
  • sporozoites 0.9% NaCl, 30% dextran-70, 0.5% trehalose, 2% glucose, and 100 mM K 2 HPO 4 .
  • plasma, cryoprecipitate, and/or serum 0.9% NaCl, 30% dextran-70, 6% trehalose, 2% glucose, and 100 mM K 2 HPO 4 .
  • enzymes 5% albumin, 0.9% NaCl, 30% dextran-70, 6% trehalose, 2% glucose, and 100 mM K 2 HPO 4 .
  • Table 2 presents representative data for recovery, viability, and activity for each of the indicated biologicals using the above-mentioned dehydration solutions.
  • Red Blood Cells In-dated human blood was obtained from the Red Cross as packed red cells. Cells were checked for count per unit volume, morphology, lysis (inspection of the supernatant for hemoglobin), crenated cells, etc. Evaluations were performed according to standard manual methods for counting (hemocytometer), microscopy and digital photomicroscopy.
  • Preparation for Desiccation Red blood cells were mixed in a proprietary fashion with HeMemics' desiccation buffer (described herein). The buffer moiety in these experiments was K 2 HPO 4 and there was adenosine in the buffer to replenish lost ATP through an adenine nucleotide salvage pathway. Preparation for desiccation included an incubation/stabilization period in the desiccation buffer.
  • Stabilization of the red blood cells in the desiccation buffer allows an adaptation of the cells to the new intra- and extracellular milieu created by the buffer components and provides sufficient time for the transmembrane movement of certain of those components.
  • An optimized incubation period allows for the development of a reasonable steady state, if not equilibration per se, for the intracellular and extracellular environments.
  • water enters, swelling the cell transiently.
  • Various homeostatic mechanisms serve to adapt to or regulate this process, including production of ATP, ion pump function, osmosis, etc. It has been observed, even in reasonably fresh, unprocessed blood, the presence of numerous crenated red cells.
  • Desiccation Cells were desiccated by vacuum drying according to methods described herein, involving "strength" of vacuum, timing, temperature, and allowances for residual moisture. Over-drying renders the cells useless, or at least useless for certain functions. Therefore, the process was constantly monitored manually.
  • the desiccation has a high surface area to volume ratio, to adequately dry, but not damage the cells. Currently, and in these experiments, 0.5 ml volumes were dried in 20 ml screwtop glass vials.
  • Rehydration The desiccated cells were rehydrated by a proprietary process and in a proprietary rehydration buffer (different from the dehydration buffer). Rehydration buffer was added to the cells in the vial and allowed to incubate and rehydrate the cells. Gentle swirling or vial rotation helped insure complete rehydration. Complete rehydration takes approximately 1 to 60 minutes. Attempts to speed up the process, e.g., by vortex mixing, shaking or vigorous vial inversion, all cause damage and frothing of the cell-buffer mix, rendering it useless. To ensure sufficient volume for testing, once rehydration was completed, the contents of several vials were combined.
  • Post-Rehydration Evaluation for Hb-Oxygen Dissociation Curve Analysis, etc. Post rehydration evaluation was conducted at USAISR. Complete blood counts and other standard laboratory evaluations were conducted by the clinical lab at the USAISR using automated equipment and standard lab procedures. Hb-oxygen dissociation curves were determined using a Hemox oxygen analyzer, standard procedures, and conducting the determination as a true dissociation of oxygen from fully oxygenated hemoglobin. Equilibration gases were 100% N 2 (for deoxygenation) and medical grade air (20% O 2 for oxygenation). Such determinations require 50 ⁇ l of blood, and mixture in the Hemox proprietary buffers and de-foaming agents. All dissociation curves were run in duplicate to assure valid p50 determinations.
  • CBC complete blood count
  • microhematocrit % packed red blood cells in relation to extracellular fluid volume
  • pH pH
  • blood gas analysis p ⁇ 2
  • oxygen saturation concentration of methemoglobin
  • concentration in the supernatant indicating hemolysis during or following the rehydration processing
  • red blood cell elasticity indicating hemolysis during or following the rehydration processing
  • red blood cell elasticity indicating hemolysis during or following the rehydration processing
  • Red blood cell morphology is one of the easiest characteristics for monitoring of process success. Typical, healthy red blood cells are round, smooth bi-concave disks with a pink or red color. Other forms of red cells are seen, even in unprocessed blood; most typically, crenated (or partially shrunken) red cells and spherocytes (essentially "balls" instead of disks). In our experience, these are the two most common appearances seen. Crenated cells are thought to occur in response to high osmotic conditions or drying, and this is certainly borne out with our preliminary experiments. While the natural tendency when seeing crenated cells is to consider the cells "bad” or worthless, often crenation is a temporary, reversible condition.
  • Microhematocrit (HCT) was 14.0%, the pH was 7.325, the p ⁇ 2 was 81.2 mmHg (Torr), oxygen saturation was 99.4%, the methemoglobin was 1.5%, and the residual hemoglobin concentration (supernatant) was 0.15 g/dl (with a total hemoglobin concentration of 5.73 g/dl) or 2.62% hemolysis.
  • the original hematocrit was calculated to be 50% (for a unit of packed red cells), corresponding to a calculated hemoglobin concentration of 20 g/dl.
  • the overall hemolysis for the procedure was likely 0.8%.
  • the RBC elasticity curves for normal human blood and rehydrated human blood were very similar and are presented in Figure 7.
  • the determination of the ability to properly bind, carry and release oxygen are conducted by exposure of the red cells to oxygen, to assure complete loading, and then exposure to complete nitrogen, to determine the continuous unloading of the oxygen from the oxyhemoglobin — the "oxyhemoglobin dissociation curve".
  • whole blood oxygen dissociation curves were determined in duplicate for the rehydrated red blood cells and singly for fresh whole human blood.
  • Typical oxyhemoglobin dissociation curve determinations, from the earliest samples, are presented in Figures 8 and 9. While the curves can be, and often run in the reverse direction (“association curves"), the "normality" of the unloading ability was desired, and therefore only the dissociation curves were observed.
  • Figure 8 presents a process "failure", the curves are instructive, as the experimentally processed red cells, which all lysed, demonstrated a typical oxygen dissociation curve for the predominantly free human hemoglobin. This appears to be a normal Hb-oxygen dissociation curve for free, unmodified human hemoglobin. Thus, the dehydration process can keep the hemoglobin protein subunits, (and by extension, the normal tetromeric configuration) intact and undisturbed.
  • a subsequent set of dissociation curves, for a later desiccation/rehydration preparation of red cells shows typical oxygen binding and release, with a mid-point (p50) closer to the normal physiologic range.
  • the blood cells almost completely hemolysed (ruptured, releasing hemoglobin into the media).
  • the curves on the left represent primarily free hemoglobin oxygenation for the Hb protein itself, not intact cells.
  • the curves on the left are completely consistent with normal extracellular hemoglobin, demonstrating that the desiccation/rehydration process protects and maintains normal protein function.
  • Hb is hemoglobin concentration
  • HCT is microhematocrit (% blood volume that is red cells)
  • MCV is mean corpuscular volume
  • MCH is mean corpuscular hemoglobin
  • MCHC is mean corpuscular hemoglobin concentration
  • p50 is the oxygen tension at which 50% of the hemoglobin is saturated with oxygen. Also shown is the methemoglobin concentration, a factor that can affect the p50.
  • anucleated cells such as red blood cells: 3% albumin, 0.9% NaCl, 20% Dextran-70, 3% trehalose, 1% glucose, 1 mg/mL adenosine, 0.9 mg/mL K 2 HPO 4 , 0.5% mannitol, 10 ⁇ g/mL vitamin E, 0.02% sulfanilamide, and 10 mM EDTA.
  • nucleated cells high strength: 3% albumin, 0.9% NaCl, 10% Dextran-70, 3% trehalose, 1% glucose, 0.9 mg/mL K 2 HPO 4 , 0.5% mannitol, 10 ⁇ g/mL vitamin E, 0.02% sulfanilamide, and 10 mM EDTA.
  • nucleated cells For nucleated cells (middle strength): 3% albumin, 0.9% NaCl, 6% Dextran-70, 3% trehalose, 1% glucose, 0.9 mg/mL K 2 HPO 4 , 0.5% mannitol, 10 ⁇ g/mL vitamin E, 0.02% sulfanilamide, and 10 mM EDTA.
  • nucleated cells low strength: 3% albumin, 0.9% NaCl, 3% Dextran-70, 3% trehalose, 1% glucose, 0.9 mg/mL K 2 HPO 4 , 0.5% mannitol, 10 ⁇ g/mL vitamin E, 0.02% sulfanilamide, and 10 mM EDTA.

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Abstract

La présente invention concerne des compositions comportant des substances biologiques desséchées comprenant une cellule, une protéine, un virus, un acide nucléique, un glucide ou un lipide, ou l'une quelconque de leurs combinaisons, associées à au moins un sucre capable de pénétrer la membrane, et au moins un sucre ne pénétrant pas la membrane, la teneur en humidité étant comprise entre 5 % et 95 %. L'invention concerne également des procédés pour les préparer, et des procédés de traitement d'animaux les employant.
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US11613727B2 (en) 2010-10-08 2023-03-28 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
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US11613727B2 (en) 2010-10-08 2023-03-28 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
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US11667876B2 (en) 2013-11-16 2023-06-06 Terumo Bct, Inc. Expanding cells in a bioreactor
US11708554B2 (en) 2013-11-16 2023-07-25 Terumo Bct, Inc. Expanding cells in a bioreactor
US11795432B2 (en) 2014-03-25 2023-10-24 Terumo Bct, Inc. Passive replacement of media
US12065637B2 (en) 2014-09-26 2024-08-20 Terumo Bct, Inc. Scheduled feed
US11667881B2 (en) 2014-09-26 2023-06-06 Terumo Bct, Inc. Scheduled feed
US11608486B2 (en) 2015-07-02 2023-03-21 Terumo Bct, Inc. Cell growth with mechanical stimuli
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11634677B2 (en) 2016-06-07 2023-04-25 Terumo Bct, Inc. Coating a bioreactor in a cell expansion system
US12077739B2 (en) 2016-06-07 2024-09-03 Terumo Bct, Inc. Coating a bioreactor in a cell expansion system
US11999929B2 (en) 2016-06-07 2024-06-04 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion
US11702634B2 (en) 2017-03-31 2023-07-18 Terumo Bct, Inc. Expanding cells in a bioreactor
US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
US12234441B2 (en) 2017-03-31 2025-02-25 Terumo Bct, Inc. Cell expansion
US12359170B2 (en) 2017-03-31 2025-07-15 Terumo Bct, Inc. Expanding cells in a bioreactor
US12043823B2 (en) 2021-03-23 2024-07-23 Terumo Bct, Inc. Cell capture and expansion
US12152699B2 (en) 2022-02-28 2024-11-26 Terumo Bct, Inc. Multiple-tube pinch valve assembly
US12209689B2 (en) 2022-02-28 2025-01-28 Terumo Kabushiki Kaisha Multiple-tube pinch valve assembly
USD1099116S1 (en) 2022-09-01 2025-10-21 Terumo Bct, Inc. Display screen or portion thereof with a graphical user interface for displaying cell culture process steps and measurements of an associated bioreactor device

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