Methods and compositions for cold-storage of cells and blood products

Abstract

Disclosed herein are compositions and methods for extending the cold-storage viability and function of cells and blood products.

Description

METHODS AND COMPOSITIONS FOR COLD-STORAGE OF CELLS AND BLOOD PRODUCTS

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Patent Application No. 63 / 756,540, filed February 10, 2025, which is herein incorporated by reference in its entirety.

[0003] ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0004] This invention was made with Government support under W81XWH-21 -P-0014 awarded by the Defense Health Agency. The Government has certain rights in the invention.

[0005] BACKGROUND OF THE INVENTION

[0006] 1. FIELD OF THE INVENTION

[0007] The field generally relates to compositions and methods for cold storage of cells and blood products.

[0008] 2. DESCRIPTION OF THE RELATED ART

[0009] Whole blood transfusions have a long history in military medicine, dating back to World War I, where it was found to decrease mortality in combat casualties with severe blood loss. Chilled whole blood has been used to treat US combat casualties at the point of injury since 2016 and is the preferred resuscitation product for pre-hospital transfusion according to US military clinical practice guidelines. Whole blood may be cold-stored for up to 35 days. Unfortunately, cold storage induces a cascade of detrimental biochemical and biomechanical changes known as the cold storage lesion, which affects all cellular components of whole blood, including red blood cells (RBCs), platelets, and leukocytes. For example, membrane protein degradation of stored red blood cells (RBCs) becomes observable in the first 7 days storage. Cold storage lesion impairs the therapeutic efficacy and safety of cold-stored blood products.

[0010] Furthermore, the current standard for platelet storage is at room temperature for up to 5 days due to the increased risk of bacterial growth. While cold storage can inhibit bacterial growth risk and alleviate the sepsis problem, cold storage causes low- temperature stress which challenges cellular homeostasis by adversely impacting fundamental molecular processes, including a decrease in membrane fluidity, a diminution in protein import and export, and a slowdown in membrane protein folding. Cold-induced platelet activation leads to a loss of its aggregation ability and rapid platelet clearance from circulation upon transfusion, compromising hemostasis. Afterabout 3 days of cold storage, the functional activity of platelets is significantly and irreversibly damaged (i.e., reduced). Consequently, cold-stored platelets more than 3 days “old” (i.e., post-collection) should not be used in the resuscitation of actively bleeding subjects.

[0011] Thus, a need exists for compositions and methods for improving the shelf-life and quality of cold-stored blood products, especially products containing platelets.

[0012] SUMMARY OF THE INVENTION

[0013] In some embodiments, the present invention is directed to a composition comprising a crystalloid solution and a concentration of about 0.05-5,000 pM of one or more UFA-glycerolipids. In some embodiments, the composition is a platelet additive solution comprising a concentration of about 0.05-5,000 pM of one or more UFA- glycerolipids, wherein the platelet additive solution comprises a crystalloid solution and one or more salts selected from sodium chloride, potassium chloride, magnesium chloride, sodium acetate, sodium citrate, and sodium phosphate. In some embodiments, the composition comprises a concentration of sodium chloride of about 65-120 mM, a concentration of potassium chloride of about 2-6 mM, a concentration of magnesium chloride of about 1-4 mM, and / or a concentration of sodium acetate of about 15-35 mM. In some embodiments, the composition comprises 60-120 mM NaCl, 3-7 mM KC1, 0.5- 3.0 mM MgCh, and 20-50 mM sodium acetate. In some embodiments, the composition further comprises 1.5-5.0 mM sodium citrate and / or 20-30 mM sodium phosphate. In some embodiments, the composition further comprises 25-35 mM mannitol. In some embodiments, the one or more UFA-glycerolipids have the following chemical structure:, wherein at least one of Rl, R2, and R3 is a polyunsaturated fatty acid (PUFA) moiety or a monounsaturated fatty acid (MUFA) moiety. In some embodiments, the PUFA moieties are omega-3 PUFAs and / or omega-6 PUFAs. In some embodiments, the PUFA moiety is alpha-linolenic acid (ALA), eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), or linoleic acid (LA); and the MUFA moiety is oleic acid (OA). In some embodiments, R3 is a phosphate moiety thereby resulting in aUFA-phospholipid. In some embodiments, the one or more UFA-glycerolipids are selected from 1,2, 3 -Tri eicosapentaenoyl Glycerol, 1,2,3-Docosahexaenoyl Glycerol, 1,2,3-Tri-alpha-Linolenoyl Glycerol, 1,3-Dieicosapentaenoyl Glycerol, 1,2,3- Trilinoleoyl Glycerol, 1,2,3-Trioleoyl Glycerol, l-Stearoyl-2-Eicosapentaenoyl-sn- glycero-3-PC, l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PE, and l-Palmitoyl-2- Docosahexaenoyl-sn-glycero-3-PC. In some embodiments, the one or more UFA- glycerolipids are provided in the form of a liposome, a lipid drop, an emulsion, a lipid nanoparticle, or an ionizable lipid nanoparticle, which may additionally comprise “free” unsaturated fatty acids, antioxidants, anti-inflammatory compounds, and the like. In some embodiments, the compositions comprising the one or more UFA-glycerolipids further comprise EP A and / or DHA as free fatty acids.

[0014] In some embodiments, the present invention is directed to a composition comprising a crystalloid solution and a concentration of about 0.05-5,000 pM of one or more polyunsaturated fatty acids (PUFAs). In some embodiments, the one or more PUFAs is an omega-3 PUFA. In some embodiments, the one or more PUFAs are selected from eicosapentaenoic acid (EP A) and docosahexaenoic acid (DHA). In some embodiments, the composition is a platelet additive solution comprising a concentration of about 0.05-5,000 pM of one or more PUFAs, wherein the platelet additive solution comprises a crystalloid solution and one or more salts selected from sodium chloride, potassium chloride, magnesium chloride, sodium acetate, sodium citrate, and sodium phosphate. In some embodiments, the composition comprises a concentration of sodium chloride of about 65-120 mM, a concentration of potassium chloride of about 2-6 mM, a concentration of magnesium chloride of about 1-4 mM, and / or a concentration of sodium acetate of about 15-35 mM. In some embodiments, the composition comprises 60-120 mM NaCl, 3-7 mM KC1, 0.5-3.0 mM MgCE, and 20-50 mM sodium acetate. In some embodiments, the composition further comprises 1.5-5.0 mM sodium citrate and / or 20- 30 mM sodium phosphate. In some embodiments, the composition further comprises 25- 35 mM mannitol. In some embodiments, the one or more PUFAs are provided in the form of a liposome, a lipid drop, an emulsion, a lipid nanoparticle, or an ionizable lipid nanoparticle, which may additionally comprise one or more UFA-glycerolipids, antioxidants, anti-inflammatory compounds, and the like. In some embodiments, the compositions comprising EPA and / or DHA further comprise one or more UFA- glycerolipids, which EPA or DHA may be provided as moieties covalently attached to the glycerol backbone.

[0015] In some embodiments, the present invention is directed to methods of preparing cells or a blood product for cold-storage and / or cold storing the cells or the blood product, which comprise adding one or more UFA-glycerolipids to the cells or the blood product. In some embodiments, the present invention is directed to methods of preparing cells or a blood product for cold-storage and / or cold storing the cells or the blood product, which comprise adding EPA and / or DHA to the cells or the blood product. In some embodiments, the present invention is directed to methods of preparing cells or a blood product for cold-storage and / or cold storing the cells or the blood product, which comprise adding one or more UFA-glycerolipids and one or more PUFAs to the cells or the blood product. In some embodiments, the one or more PUFAs is an omega-3 PUFA. In some embodiments, the one or more PUFAs is EPA and / or DHA. Such methods may further comprise storing the cells or the blood product at a temperature of about 1-6°C for a given period of time. In some embodiments, the given period of time is at least 3 days. In some embodiments, the given period of time is at least 3 and up to about 21 days. In some embodiments, the given period of time is at least 3 days and up to more than 56 days. In some embodiments, the blood product comprises platelets, leukocytes, red blood cells (RBCs), hematopoietic stem cells, plasma, and / or whole blood. In some embodiments, the blood product is synthetic, e.g., recombinantly engineered. In some embodiments, the blood product is concentrated platelets. In some embodiments, the blood product is red blood cells (RBCs). In some embodiments, the blood product is whole blood.

[0016] In some embodiments, about 0.05-5,000 pM of one or more UFA-glycerolipids per 1 x 1012 / L of platelets is added to the platelets. In some embodiments, about 0.05- 5,000 pM of one or more UFA-glycerolipids per 6.0 x 1012 / L of red blood cells is added to the red blood cells. In some embodiments, about 0.05-5,000 pM of one or more UFA- glycerolipids per liter of whole blood is added to the whole blood. In some embodiments, an amount of the one or more UFA-glycerolipids is added to the blood product to give a ratio of 0.05-5,000 mg of UFA-glycerolipids to about 3.5-4.0 x 1011platelets. In some embodiments, an amount of the one or more UFA-glycerolipids is added to the blood product to give a ratio of 0.05-5,000 mg of UFA-glycerolipids to about 6.0 x 1012red blood cells (RBCs). In some embodiments, an amount of the one or more UFA-glycerolipids is added to the blood product to give a ratio of 0.05-5,000 mg of UFA-glycerolipids to about 400-500 mL whole blood. In some embodiments, the one or more UFA-glycerolipids have the following chemical structure:, wherein at least one of Rl, R2, and R3 is a polyunsaturated fatty acid (PUFA) moiety or a monounsaturated fatty acid (MUFA) moiety. In some embodiments, the PUFA moieties are omega-3 PUFAs and / or omega-6 PUFAs. In some embodiments, the PUFA moiety is alpha-linolenic acid (ALA), eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), or linoleic acid (LA); and the MUFA moiety is oleic acid (OA). In some embodiments, R3 is a phosphate moiety thereby resulting in a UFA-phospholipid. In some embodiments, the one or more UFA-glycerolipids are selected from 1,2, 3 -Tri eicosapentaenoyl Glycerol, 1,2,3-Docosahexaenoyl Glycerol, 1,2,3-Tri-alpha-Linolenoyl Glycerol, 1,3-Dieicosapentaenoyl Glycerol, 1,2,3-Trilinoleoyl Glycerol, 1,2,3-Trioleoyl Glycerol, l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC, l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PE, and l-Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC. In some embodiments, an amount of one or more UFA-glycerolipids selected from 1,2, 3 -Tridocosahexaenoyl Glycerol; 1,2,3-Trieicosapentaenoyl Glycerol; 1,2,3-Tri-a-Linolenoyl Glycerol; 1,2,3-Trioleoyl Glycerol; 1,3-Dieicosapentaenoyl Glycerol; or 1,2,3-Trilinoleoyl Glycerol is added to bring to the concentration of the one or more UFA-glycerolipids to about 0.5-25 pM, preferably about 1-20 pM, more preferably about 5-15 pM, and most preferably about 10 pM. In some embodiments, an amount of one or more UFA-glycerolipids selected from l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC; and l-Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC is added to bring to the concentration of the one or more UFA-glycerolipids to about 45-85 pM, preferably about 55-75 pM, and more preferably about 65 pM. In some embodiments, an amount of one or more UFA-glycerolipids selected from 1,2,3-Tridocosahexaenoyl Glycerol; 1,2,3-Trieicosapentaenoyl Glycerol; 1,2,3-Tri-a-Linolenoyl Glycerol; 1,2,3-Trioleoyl Glycerol; 1,3-Dieicosapentaenoyl Glycerol; or 1,2,3-Trilinoleoyl Glycerol is added to bring to the concentration of the one or more UFA-glycerolipids to about 0.5-25 pM, preferably about 1-20 pM, more preferably about 5-15 pM, and most preferably about 10 pM, and the concentration of the cells or platelets is about 150,000-450,000 per pL.In some embodiments, an amount of one or more UFA-glycerolipids selected from 1- Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC; and l-Palmitoyl-2-Docosahexaenoyl-sn- glycero-3-PC is added to bring to the concentration of the one or more UFA- glycerolipids to about 45-85 pM, preferably about 55-75 pM, and more preferably about 65 pM, and the concentration of the cells or platelets is about 150,000-450,000 per pL. In some embodiments, the one or more UFA-glycerolipids are provided in the form of a liposome, a lipid drop, an emulsion, a lipid nanoparticle, or an ionizable lipid nanoparticle, which may additionally comprise “free” unsaturated fatty acids, antioxidants, anti-inflammatory compounds, and the like. In some embodiments, the methods further comprise adding one or more PUFAs, such as EPA and / or DHA as free fatty acids, to the blood product.

[0017] In some embodiments, about 0.05-5,000 pM of a polyunsaturated fatty acid (PUFA), per 1 x 1012 / L of platelets is added to the platelets, preferably said PUFA is an omega-3 PUFA, more preferably EPA and / or DHA. In some embodiments, about 0.05- 5,000 pM of a PUFA per 6.0 x 1012 / L of red blood cells (RBCs) is added to the red blood cells (RBCs), preferably said PUFA is an omega-3 PUFA, more preferably EPA and / or DHA. In some embodiments, about 0.05-5,000 pM of a PUFA per liter of whole blood is added to the whole blood, preferably said PUFA is an omega-3 PUFA, more preferably EPA and / or DHA. In some embodiments, an amount of a PUFA is added to the blood product to give a ratio of 0.05-5,000 mg of the PUFA to about 3.5-4.0 x 1011platelets, preferably said PUFA is an omega-3 PUFA, more preferably EPA and / or DHA. In some embodiments, an amount of a PUFA is added to the blood product to give a ratio of 0.05-5,000 mg of the PUFA, to about 6.0 x 1012red blood cells (RBCs), preferably said PUFA is an omega-3 PUFA, more preferably EPA and / or DHA. In some embodiments, an amount of a PUFA is added to the blood product to give a ratio of 0.05- 5,000 mg of the PUFA to about 400-500 mL whole blood, preferably said PUFA is an omega-3 PUFA, more preferably EPA and / or DHA.

[0018] In some embodiments, the present invention is directed to a kit comprising at least one UFA-glycerolipid unit of one or more UFA-glycerolipids packaged together with a container for cells or a blood product (e.g., a blood or platelet collection or donation bag) and / or a device for adding the UFA-glycerolipid unit to the cells or the blood product, wherein the at least one UFA-glycerolipid unit is about 0.1 mg to about 6 g of the one or more UFA-glycerolipids. In some embodiments, the at least one UFA- glycerolipid unit is provided in the form of an aqueous composition wherein theconcentration of the at least one UFA-glycerolipid unit is 0.05-5,000 pM. In some embodiments, the container or the volume of the cells or the blood product to be treated is about 300-1,000 mL, preferably about 300-500 mL, and the at least one UFA-glycerolipid unit is about 0.1 mg to about 6 g of the one or more UFA-glycerolipids. In some embodiments, the container or the volume of the cells or the blood product to be treated is about 300-500 mL and the at least one UFA-glycerolipid unit of one or more UFA-glycerolipids is selected from 1,2,3-Tridocosahexaenoyl Glycerol; 1,2,3-Trieicosapentaenoyl Glycerol; 1,2,3-Tri-a-Linolenoyl Glycerol; 1,2,3-Trioleoyl Glycerol; 1,3-Dieicosapentaenoyl Glycerol; or 1,2,3-Trilinoleoyl Glycerol is about 0.05-20 mg, preferably about 0.1-15 mg, more preferably about 1-8 mg, and most preferably about 2-5 mg. In some embodiments, the container or the volume of the cells or the blood product to be treated is about 300-500 mL and the at least one UFA-glycerolipid unit of one or more UFA-glycerolipids is selected from l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC; and l-Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC is about 1-50 mg, preferably about 10-35 mg, more preferably about 15-30 mg, and most preferably about 15-25 mg. In some embodiments, the volume to be treated comprises about 150,000-450,000 cells or platelets per pL. In some embodiments, the blood product comprises platelets, leukocytes, red blood cells (RBCs), hematopoietic stem cells, plasma, and / or whole blood. In some embodiments, the blood product is synthetic, e.g., recombinantly engineered. In some embodiments, the blood product is concentrated platelets. In some embodiments, the blood product is whole blood. In some embodiments, the one or more UFA-glycerolipids have the following chemical structure:, wherein at least one of Rl, R2, and R3 is a polyunsaturated fatty acid (PUFA) moiety or a monounsaturated fatty acid (MUFA) moiety. In some embodiments, the PUFA moieties are omega-3 PUFAs and / or omega-6 PUFAs. In some embodiments, the PUFA moiety is alpha-linolenic acid (ALA), eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), or linoleic acid (LA); and the MUFA moiety isoleic acid (OA). In some embodiments, R3 is a phosphate moiety thereby resulting in a UFA-phospholipid. In some embodiments, the one or more UFA-glycerolipids are selected from 1,2, 3 -Tri eicosapentaenoyl Glycerol, 1,2,3-Docosahexaenoyl Glycerol, 1,2,3-Tri-alpha-Linolenoyl Glycerol, 1,3-Dieicosapentaenoyl Glycerol, 1,2,3- Trilinoleoyl Glycerol, 1,2,3-Trioleoyl Glycerol, l-Stearoyl-2-Eicosapentaenoyl-sn- glycero-3-PC, l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PE, and l-Palmitoyl-2- Docosahexaenoyl-sn-glycero-3-PC. In some embodiments, the one or more UFA- glycerolipids are provided in the form of a liposome, a lipid drop, an emulsion, a lipid nanoparticle, or an ionizable lipid nanoparticle, which may additionally comprise “free” unsaturated fatty acids, antioxidants, anti-inflammatory compounds, and the like. In some embodiments, the kits further comprise one or more units of one or more PUFA, preferably one or more omega-3 PUFAs, more preferably EPA and / or DHA..

[0019] In some embodiments, the present invention is directed to a kit comprising at least one unit of a polyunsaturated fatty acid (PUFA) as free fatty acids, packaged together with a container for cells or a blood product (e.g., a blood or platelet collection or donation bag) and / or a device for adding the PUFA to the cells or the blood product, wherein the at least one unit of the PUFA comprises about 0.1 mg to about 6 g of the PUFA, preferably said PUFA is an omega-3 PUFA, more preferably EPA and / or DHA. In some embodiments, the at least one unit is provided in the form of an aqueous composition wherein the concentration of the at least one unit is 0.05-5,000 pM. In some embodiments, the container or the volume of the cells or the blood product to be treated is about 300-1,000 mL, preferably about 300-500 mL, and the at least one unit is about 0.1 mg to about 6 g of the PUFA. In some embodiments, the kit further comprises one or more UFA-glycerolipids as described herein.

[0020] In some embodiments, the present invention is directed to a platelet additive solution comprising EPA and / or DHA, a crystalloid solution, and one or more components selected from sodium chloride, potassium chloride, magnesium chloride, sodium acetate, sodium citrate, sodium phosphate, and mannitol, wherein the EPA and / or the DHA are in the form of: free fatty acids, one or more polyunsaturated fatty acid (PUFA) moieties of a UFA-glycerolipid as described herein, or both. In some embodiments, the concentration of the EPA and / or DHA as free fatty acids is about 0.05- 5,000 pM. In some embodiments, the concentration of the UFA-glycerolipid is about 0.05-5,000 pM. In some embodiments, the concentration of sodium chloride is about 65- 120 mM, the concentration of potassium chloride is about 2-6 mM, the concentration ofmagnesium chloride is about 1-4 mM, and / or the concentration of sodium acetate is about 15-35 mM. In some embodiments, the platelet additive solution comprises 60-120 mM NaCl, 3-7 mM KC1, 0.5-3.0 mM MgCE, and 20-50 mM sodium acetate. In some embodiments, the platelet additive solution comprises 1.5-5.0 mM sodium citrate and / or 20-30 mM sodium phosphate. In some embodiments, the platelet additive solution comprises 25-35 mM mannitol. In some embodiments, the UFA-glycerolipid, EP A, and / or DHA are provided in the form of a liposome, a lipid drop, an emulsion, a lipid nanoparticle, or an ionizable lipid nanoparticle.

[0021] In some embodiments, the present invention is directed to a method of preparing cells or a blood product for cold-storage, which comprises adding EPA and / or DHA to the cells or the blood product, wherein the EPA and / or the DHA are in the form of: free fatty acids, one or more polyunsaturated fatty acid (PUFA) moieties of a UFA- glycerolipid as described herein, or both. In some embodiments, about 0.05-5,000 pM of the UFA-glycerolipid, EPA, and / or DHA per 1 x 1012 / L of platelets is added to the platelets. In some embodiments, about 0.05-5,000 pM of the UFA-glycerolipid, EPA, and / or DHA per 6.0 x 1012 / L of red blood cells (RBCs) is added to the red blood cells (RBCs). In some embodiments, about 0.05-5,000 pM of the UFA-glycerolipid, EPA, and / or DHA per liter of whole blood is added to the whole blood. In some embodiments, an amount of the UFA-glycerolipid, EPA, and / or DHA is added to the blood product to result in a ratio of 0.05-5,000 mg of the UFA-glycerolipid, EPA, and / or DHA to about 3.5-4.0 x 1011platelets. In some embodiments, an amount of the UFA-glycerolipid, EPA, and / or DHA is added to the blood product to result in a ratio of 0.05-5,000 mg of the UFA-glycerolipid, EPA, and / or DHA to about 6.0 x 1012red blood cells (RBCs). In some embodiments, an amount of the UFA-glycerolipid, EPA, and / or DHA is added to the blood product to result in a ratio of 0.05-5,000 mg of the UFA-glycerolipid, EPA, and / or DHA to about 400-500 mL whole blood. In some embodiments, the UFA- glycerolipid, EPA, and / or DHA are provided in the form of a liposome, a lipid drop, an emulsion, a lipid nanoparticle, or an ionizable lipid nanoparticle.

[0022] In some embodiments, the present invention is directed to a method of storing cells or a blood product, which comprises adding EPA and / or DHA to the cells or the blood product, wherein the EPA and / or the DHA are in the form of: free fatty acids, one or more polyunsaturated fatty acid (PUFA) moieties of a UFA-glycerolipid as described herein, or both, and then storing the cells or the blood product at a temperature of about 1-6°C. In some embodiments, about 0.05-5,000 pM of the UFA-glycerolipid, EPA,and / or DHA per 1 x 1012 / L of platelets is added to the platelets. In some embodiments, about 0.05-5,000 pM of the UFA-glycerolipid, EP A, and / or DHA per 6.0 x 1012 / L of red blood cells (RBCs) is added to the red blood cells (RBCs). In some embodiments, about 0.05-5,000 pM of the UFA-glycerolipid, EP A, and / or DHA per liter of whole blood is added to the whole blood. In some embodiments, an amount of the UFA-glycerolipid, EP A, and / or DHA is added to the blood product to result in a ratio of 0.05-5,000 mg of the UFA-glycerolipid, EP A, and / or DHA to about 3.5-4.0 x 1011platelets. In some embodiments, an amount of the UFA-glycerolipid, EP A, and / or DHA is added to the blood product to result in a ratio of 0.05-5,000 mg of the UFA-glycerolipid, EP A, and / or DHA to about 6.0 x 1012red blood cells (RBCs). In some embodiments, an amount of the UFA-glycerolipid, EP A, and / or DHA is added to the blood product to result in a ratio of 0.05-5,000 mg of the UFA-glycerolipid, EP A, and / or DHA to about 400-500 mL whole blood. In some embodiments, the UFA-glycerolipid, EP A, and / or DHA are provided in the form of a liposome, a lipid drop, an emulsion, a lipid nanoparticle, or an ionizable lipid nanoparticle.

[0023] A kit comprising at least one unit of EPA and / or DHA packaged together with a container for cells or a blood product and / or a device for adding the at least one unit to the cells or the blood product, wherein the at least one unit is at least 0.1 mg to about 6 g, and the EPA and / or the DHA are in the form of: free fatty acids, one or more polyunsaturated fatty acid (PUFA) moieties of a UFA-glycerolipid as described herein, or both. In some embodiments, the UFA-glycerolipid, EPA, and / or DHA are provided in the form of a liposome, a lipid drop, an emulsion, a lipid nanoparticle, or an ionizable lipid nanoparticle. In some embodiments, the kit comprises EPA and / or DHA in the form of a platelet additive solution as described herein packaged together with a container for cells or a blood product and / or a device for adding the platelet additive solution to a blood product.

[0024] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description explain the principles of the invention.

[0025] DESCRIPTION OF THE DRAWINGS

[0026] This invention is further understood by reference to the drawings wherein:

[0027] FIG. 1: Platelet aggregometry induced by agonists collagen, adenosine diphosphate (ADP), and epinephrine were performed for the assessment of human platelet function according to the manufacturer’s guidelines. The maximum amplitude of response to agonists at Day 3 cold storage are shown as 79% for platelet-rich plasma (PRP) treated with 1,2,3-Trieicosapentaenoyl Glycerol (EPA-TG) 10.4 pM in 0.1% ethanol, 99% for PRP treated with 1,2,3-Tridocosahexaenoyl Glycerol (DHA-TG) 10.4 pM in 0.1% ethanol, and 55% for PRP treated with control Ethanol 0.1%. At 2 mins, the lines from top to bottom are Ethanol, DHA-TG, and EPA-TG, and at 5 mins, the lines from top to bottom are Ethanol, EPA-TG, and DHA-TG.

[0028] FIG. 2: The maximum amplitude of response to agonists at Day 6 cold storage are shown as 90% for platelet-rich plasma (PRP) treated with 1,2,3-Trieicosapentaenoyl Glycerol (EPA-TG) 10.4 pM in 0.1% ethanol, 102% for PRP treated with 1,2,3- Tridocosahexaenoyl Glycerol (DHA-TG) 10.4 pM in 0.1% ethanol, and 34% for PRP treated with control (Ethanol, 0.1%). At 2 mins, the lines from top to bottom are Ethanol, DHA-TG, EPA-TG, and at 5 mins, the lines from top to bottom are Ethanol, EPA-TG, and DHA-TG.

[0029] FIG. 3: The maximum amplitude of response to agonists at Day 14 cold storage are shown as 42% for platelet-rich plasma (PRP) treated with 1,2,3-Trieicosapentaenoyl Glycerol (EPA-TG) 10.4 pM in 0.1% ethanol, 53% for PRP treated with 1,2,3- Tridocosahexaenoyl Glycerol (DHA-TG) 10.4 pM in 0.1% ethanol, and 21% for PRP treated with control (Ethanol, 0.1%). At 2 mins, the lines from top to bottom are DHA- TG, Ethanol, EPA-TG, and at 5 mins, the lines from top to bottom are Ethanol, EPA-TG, and DHA-TG.

[0030] FIG. 4: The maximum amplitude of response to agonists at Day 21 cold storage are shown as 22% for platelet-rich plasma (PRP) treated with 1,2,3-Trieicosapentaenoyl Glycerol (EPA-TG) 10.4 pM in 0.1% ethanol, 28% for PRP treated with 1,2,3- Tridocosahexaenoyl Glycerol (DHA-TG) 10.4 pM in 0.1% ethanol, and 18% for PRP treated with control (Ethanol, 0.1%). At 2 mins, the top line is Ethanol, and at 5 mins, the lines from top to bottom are Ethanol, EPA-TG, and DHA-TG.

[0031] FIG. 5: Platelet aggregometry induced by agonists collagen, adenosine diphosphate (ADP), and epinephrine were performed for the assessment of human platelet function according to the manufacturer’s guidelines. The maximum amplitude of response to agonists at Day 3 cold storage are shown as 77% for platelet-rich plasma (PRP) treated with l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PE (EPA-PE) 65 pM,84% for PRP treated with l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC (EPA-PC2) 65 pM, 87% for PRP treated with l-Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC (DHA-PC) 65 pM, and 57% for PRP treated with 4 Celsius degree control (4C PRP CTL). At 2 mins, the top 2 lines are DHA-PC and EPA-PC2 then 4C PRP CTL with the bottom line being EPA-PE, and at 5 mins, the lines from top to bottom are 4C PRP CTL, EPA-PE, EPA-PC2, and DHA-PC.

[0032] FIG. 6: The maximum amplitude of response to agonists at Day 6 cold storage are shown as 70% for platelet-rich plasma (PRP) treated with l-Stearoyl-2- Eicosapentaenoyl-sn-glycero-3-PE (EPA-PE) 65 pM, 75% for PRP treated with 1- Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC (EPA-PC2) 65 pM, 62% for PRP treated with l-Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC (DHA-PC) 65 pM, and 37% for PRP treated with 4 Celsius degree control (4C PRP CTL). At 2 mins, the lines from top to bottom are 4C PRP CTL, the middle 2 lines are DHA-PC and EPA-PC2, and the bottom line is EPA-PE, and at 5 mins, the lines from top to bottom are 4C PRP CTL, DHA-PC, EPA-PE, and EPA-PC2.

[0033] FIG. 7. The maximum amplitude of response to agonists at Day 14 cold storage are shown as 28% for platelet-rich plasma (PRP) treated with l-Stearoyl-2- Eicosapentaenoyl-sn-glycero-3-PE (EPA-PE) 65 pM, 32% for PRP treated with 1- Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC (EPA-PC2) 65 pM, 31% for PRP treated with l-Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC (DHA-PC) 65 pM, and 20% for PRP treated with 4 Celsius degree control (4C PRP CTL). At 3.5 mins, the lines from top to bottom are DHA-PC, EPA-PC2, EPA-PE, and 4C PRP CTL, and at 7 mins, the lines from top to bottom are 4C PRP CTL, EPA-PE, DHA-PC, and EPA-PC2.

[0034] FIG. 8. The maximum amplitude of response to agonists at Day 21 cold storage are shown as 22% for platelet-rich plasma (PRP) treated with l-Stearoyl-2- Eicosapentaenoyl-sn-glycero-3-PE (EPA-PE) 65 pM, 18% for PRP treated with 1- Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC (EPA-PC2) 65 pM, 19% for PRP treated with l-Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC (DHA-PC) 65 pM, and 10% for PRP treated with 4 Celsius degree control (4C PRP CTL). At 4 mins, the lines from top to bottom are 4C PRP CTL, DHA-PC, EPA-PC2, and EPA-PE.

[0035] FIG. 9: Platelet aggregometry induced by agonists collagen, adenosine diphosphate (ADP), and epinephrine were performed for the assessment of human platelet function according to the manufacturer’s guidelines. The maximum amplitude of response to agonists at Day 3 cold storage are shown as 65% for PRP treated with 1-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC (EPA-PC2) 260 pM, 71% for PRP treated with l-Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC (DHA-PC) 260 pM, and 57% for PRP treated with 4 Celsius degree control (4C PRP CTL). At 2 mins, the lines from top to bottom are 4C PRP CTL, DHA-PC, and EPA-PC2, and at 5 mins, the lines from top to bottom are 4C PRP CTL, EPA-PC2, and DHA-PC.

[0036] FIG. 10: The maximum amplitude of response to agonists at Day 6 cold storage are shown as 50% for PRP treated with l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC (EPA-PC2) 260 pM, 47% for PRP treated with l-Palmitoyl-2-Docosahexaenoyl-sn- glycero-3-PC (DHA-PC) 260 pM, and 37% for PRP treated with 4 Celsius degree control (4C PRP CTL). At 5 mins, the lines from top to bottom are 4C PRP CTL, DHA- PC, and EPA-PC2.

[0037] FIG. 11 : The maximum amplitude of response to agonists at Day 14 cold storage are shown as 32% for PRP treated with l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC (EPA-PC2) 260 pM, 31% for PRP treated with l-Palmitoyl-2-Docosahexaenoyl-sn- glycero-3-PC (DHA-PC) 260 pM, and 20% for PRP treated with 4 Celsius degree control (4C PRP CTL). At 5 mins, the lines from top to bottom are 4C PRP CTL, DHA- PC, EPA-PC2.

[0038] FIG. 12: The maximum amplitude of response to agonists at Day 21 cold storage are shown as 22% for PRP treated with l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC (EPA-PC2) 260 pM, 24% for PRP treated with l-Palmitoyl-2-Docosahexaenoyl-sn- glycero-3-PC (DHA-PC) 260 pM, and 10% for PRP treated with 4 Celsius degree control (4C PRP CTL). At 5 mins, the lines from top to bottom are 4C PRP CTL, EPA- PC2, and DHA-PC.

[0039] FIG. 13: Platelet aggregometry induced by agonists collagen, adenosine diphosphate (ADP), and epinephrine were performed for the assessment of human platelet function according to the manufacturer’s guidelines. The maximum amplitude of response to agonists at Day 3 cold storage are shown as 101% for platelet-rich plasma (PRP) treated with 1,2,3-Tridocosahexaenoyl Glycerol 100 pM (DHA-TG-H) in 0.02% chloroform, 104% for PRP treated with 1,2,3-Tridocosahexaenoyl Glycerol 1 pM (DHA- TG-L) in 0.02% chloroform, and 64% for PRP treated with control chloroform 0.02% (CHL-0.02%). At 2 mins, the lines from top to bottom are 4C PRP CTL, DHA-TG-L, and DHA-TG-H, and at 5 mins, the lines from top to bottom are 4C PRP CTL, DHA- TG-L, and DHA-TG-H.

[0040] FIG. 14: The maximum amplitude of response to agonists at Day 6 cold storage are shown as 76% for platelet-rich plasma (PRP) treated with 1,2,3-Tridocosahexaenoyl Glycerol 100 pM (DHA-TG-H) in 0.02% chloroform, 98% for PRP treated with 1,2,3- Tridocosahexaenoyl Glycerol 1 pM (DHA-TG-L) in 0.02% chloroform, and 52% for PRP treated with control chloroform 0.02% (CHL-0.02%). Just prior to 2 mins, the lines from top to bottom are 4C PRP CTL, DHA-TG-L, and DHA-TG-H, and at 5 mins, the lines from top to bottom are 4C PRP CTL, DHA-TG-H, and DHA-TG-L.

[0041] FIG. 15: The maximum amplitude of response to agonists at Day 13 cold storage are shown as 60% for platelet-rich plasma (PRP) treated with 1,2,3-Tridocosahexaenoyl Glycerol 100 pM (DHA-TG-H) in 0.02% chloroform, 39% for PRP treated with 1,2,3- Tridocosahexaenoyl Glycerol 1 pM (DHA-TG-L) in 0.02% chloroform, and 32% for PRP treated with control chloroform 0.02% (CHL-0.02%). At 5 mins, the lines from top to bottom are 4C PRP CTL, DHA-TG-L, and DHA-TG-H.

[0042] FIG. 16: Platelet aggregometry induced by agonists collagen, adenosine diphosphate (ADP), and epinephrine were performed for the assessment of human platelet function according to the manufacturer’s guidelines. The maximum amplitude of response to agonists at Day 3 cold storage are shown as 109% for platelet-rich plasma (PRP) treated with 1,2,3-Tri-a-Linolenoyl Glycerol (ALA-TG) 1 pM in 0.01% chloroform, 109% for PRP treated with 1,2,3-Trioleoyl Glycerol (OA-TG) 1 pM in 0.01% chloroform, and 86% for PRP treated with control chloroform 0.01% (CHL- 0.01%). At 1 min, the lines from top to bottom are OA-TG, ALA-TG, and CHL, and at 5 mins, the lines from top to bottom are CHL, OA-TG, and ALA-TG.

[0043] FIG. 17: The maximum amplitude of response to agonists at Day 13 cold storage are shown as 34% for platelet-rich plasma (PRP) treated with 1,2,3-Tri-a-Linolenoyl Glycerol (ALA-TG) 1 pM in 0.01% chloroform, 32% for PRP treated with 1,2,3- Trioleoyl Glycerol (OA-TG) 1 pM in 0.01% chloroform, and 22% for PRP treated with control chloroform 0.01% (CHL-0.01%). At 3 mins, the top line is CHL and the bottom 2 lines are ALA-TG and OA-TG.

[0044] FIG. 18: Platelet aggregometry induced by agonists collagen, adenosine diphosphate (ADP), and epinephrine were performed for the assessment of human platelet function according to the manufacturer’s guidelines. The maximum amplitude of response to agonists at Day 3 cold storage are shown as 100% for platelet-rich plasma (PRP) treated with 1,3 -Dieicosapentaenoyl Glycerol (EPA-DG) 1 pM in 0.01% ethanol, 99% for PRP treated with 1,2,3-Trilinoleoyl Glycerol (LA-TG) 1 pM in 0.01% ethanol,and 82% for PRP treated with control Ethanol 0.01% (ETH-0.01%). At 1 min, the lines from top to bottom are ETH, EPA-DG, and LA-TG, and at 5 mins, the lines from top to bottom are ETH, LA-TG, and EPA-DG.

[0045] FIG. 19: Platelet aggregometry induced by agonists collagen, adenosine diphosphate (ADP), and epinephrine were performed for the assessment of human platelet function according to the manufacturer’s guidelines. The maximum amplitude of response to agonists at Day 8 cold storage are shown as 60% for platelet-rich plasma (PRP) treated with Eicosapentaenoic Acid (EP A) 250 pM in 0.1% DMSO, 45% for PRP treated with EPA 25 pM in 0.1% DMSO, and 26% for PRP treated with control DMSO 0.1%. At 4 mins, the lines from top to bottom are EPA 250 pM, DMSO, EPA 25 pM, and at 10 mins, the lines from top to bottom are DMSO, EPA 25 pM, and EPA 250 pM.

[0046] FIG. 20: Cytosolic free calcium within resting platelets were maintained at lower levels treated with omega-3 fatty acid in triglyceride form when compared with control. Platelets were loaded with calcium ion indicator Fluo-3-AM. Fluorescence was measured by flow cytometry. Cytosolic free calcium in platelets treated with 1,2,3- Trieicosapentaenoyl Glycerol (EPA-TG) 10.4 pM in 0.1% ethanol, and 1,2,3- Tridocosahexaenoyl Glycerol (DHA-TG) 10.4 pM in 0.1% ethanol were compared with control (Ethanol, 0.1%).

[0047] FIG. 21 : Cytosolic free calcium within resting platelets were maintained at lower levels treated with omega-3 fatty acid in phospholipid form when compared with control. During cold storage of platelets, lower level of cytosolic free calcium means resting platelets are healthier and more functional. Platelets were loaded with calcium ion indicator Fluo-3-AM. Fluorescence was measured by flow cytometry. Cytosolic free calcium in platelets treated with l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PE (EPAPE) 65 pM, l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC (EPA-PC2) 65 pM, and 1- Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC (DHA-PC) 65 pM were compared with 4 Celsius degree control (4C CTRL).

[0048] FIG. 22: Cytosolic free calcium within resting platelets were maintained at lower levels treated with omega-3 fatty acids EPA and DHA when compared with control. During cold storage of platelets, lower level of cytosolic free calcium means resting platelets are healthier and more functional. Platelets were loaded with calcium ion indicator Fluo-3-AM. Fluorescence was measured by flow cytometry. Cytosolic free calcium in platelets treated with EPA 250 pM, EPA 25 pM, Docosahexaenoic Acid(DHA) 250 pM, EP A 250 pM + DHA 250 pM, and EPA 500 pM + DHA 500 pM were compared with control DMSO 0.1%.

[0049] FIG. 23: Complete Blood Count (CBC) assay showed that omega-3 fatty acids EPA and DHA treatment maintained higher number of platelet cells in cold-stored platelets at Days 3, 6, and 14 compared with control DMSO 0.1%.

[0050] FIG. 24: EPA triglyceride treatment increased platelet viability in cold-stored platelets at Days 6 and 14 compared with control. Adenosine Triphosphate (ATP) Luminescent Cell Viability Assay was performed for quantification of platelet viability by luminescence analysis.

[0051] FIG. 25 : EPA phospholipid treatment increased platelet viability in cold-stored platelets at Days 6 and 14 compared with control. Adenosine Triphosphate (ATP) Luminescent Cell Viability Assay was performed for quantification of platelet viability by luminescence analysis.

[0052] FIG. 26: DHA triglyceride treatment increased platelet viability in cold-stored platelets at Days 3, 6, and 13 compared with control. Adenosine Triphosphate (ATP) Luminescent Cell Viability Assay was performed for quantification of platelet viability by luminescence analysis.

[0053] FIG. 27: EPA and DHA treatment increased platelet viability in cold-stored platelets at Days 7, 14, and 21 compared with control. Adenosine Triphosphate (ATP) Luminescent Cell Viability Assay was performed for quantification of platelet viability by luminescence analysis.

[0054] FIG. 28: Treatment of EPA and DHA in cold-stored platelet-rich plasma (PRP) prevented platelet GPVI loss during 21 days of storage period. GPVI is a crucial collagen receptor on platelets, acting as a primary sensor for damaged vessel walls, triggering platelet activation, aggregation, and thrombus (clot) formation by initiating signaling cascades essential for hemostasis and thrombosis.

[0055] FIG. 29: Treatment of EPA and DHA in cold-stored platelet-rich plasma (PRP) prevented platelet CD42b loss during 21 days of storage period. CD42b is a key transmembrane protein on platelets, forming part of the GPIb-IX-V receptor complex, crucial for binding von Willebrand factor (VWF) and thrombin, mediating platelet adhesion under high shear stress, and initiating coagulation.

[0056] FIG. 30: EPA and DHA inhibit platelet apoptosis in cold-stored platelet-rich plasma (PRP) during 21 days of storage period. "PS+" refers to a marker that specifically detects the presence of phosphatidylserine (PS), a phospholipid typicallyexposed on the surface of a cell undergoing apoptosis, making it a key indicator of cell death when analyzed using flow cytometry.

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] Provided herein are compositions and methods for the cold storage (e.g., storage at about 1-6°C) of cells and blood products (e.g., platelets, red blood cells (RBCs), leukocytes, hematopoietic stem cells, plasma, whole blood, etc.) which may be naturally occurring (e.g., obtain from an animal or a human) or recombinantly engineered.Specifically, as disclosed herein, polyunsaturated fatty acids (PUFAs, e.g., omega-3 PUFAs such as eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), and alphalinolenic acid (ALA), and omega-6 PUFAs such as linoleic acid) and monounsaturated fatty acids (MUFAs, e.g., oleic acid (OA)) carried on glycerol chemical backbones (which may include a phosphate moiety to thereby give a phospholipid) extend the shelflife of cold-stored platelets by more than 10 days. That is, PUFAs and MUFAs covalently linked to a glycerol chemical backbone (hereafter “UFA-glycerolipids”) inhibit the loss of functional activity of cold-stored platelets over time and thereby extend the cold storage shelf-life of platelets. Additionally, EPA and DHA are shown to inhibit the loss of functional activity of cold-stored platelets and extend their cold storage shelf-life by more than 10 days.

[0059] As provided herein, EPA, DHA, and ALA in the form of UFA-glycerolipids added to platelets increased the viability of cold-stored platelets by more than 20% compared to untreated controls. Specifically, the UFA-glycerolipids increased platelet viability by 23.4% at Day 7 and 38.6% at Day 14 as compared with cold-stored untreated control platelets. As discussed herein, similar results were also obtained when EPA and DHA were added in their “free” forms (i.e., not bound to a glycerolipid). EPA increased platelet viability by 34.3% at Day 3, 52.9% at Day 14, and 114.4% at Day 21 as compared with cold-stored untreated control platelets. DHA increased platelet viability by 37.7% at Day 3, 49.7% at Day 14, and 92.0% at Day 21 as compared with cold-stored untreated control platelets. Additionally, treatment with a UFA-glycerolipid, EPA, and DHA extended platelet cold-storage shelf-life and function by more than 10 days. That is, the aggregation function of Day 14 cold-stored platelets treated with a UFA- glycerolipid alone, EPA, or DHA is equivalent to the aggregation function of Day 3 cold-stored untreated platelet controls. Further, treatment with a UFA-glycerolipid, EPA, and DHA inhibited apoptosis by 71.4%. Particularly, the apoptosis rate of Day 7 cold-stored platelets treated with a UFA-glycerolipid alone, EPA, and DHA wasequivalent to the apoptosis rate of Day 2 cold-stored untreated platelet controls. The addition of 10.4 pM of 1,2, 3 -Tridocosahexaenoyl Glycerol perform exceptionally with 99% at Day 3 and 102% at Day 6 amplitude as a measurement of the maximum clot strength compared with untreated controls 55% at Day 3 and 34% at Day 6. 10.4 pM of 1,2, 3 -Tri eicosapentaenoyl Glycerol, 65 pM l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3- PC, 65 pM l-Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC, and 65 pM l-Stearoyl-2- Eicosapentaenoyl-sn-glycero-3-PE have 70-90% maximum clot strength at Days 3 and 6 compared with untreated controls 55% at Day 3 and 34% at Day 6. The addition of 1 pM of 1,2,3-Tridocosahexaenoyl Glycerol perform exceptionally with 104% at Day 3 and 98% at Day 6 amplitude as a measurement of the maximum clot strength compared with untreated controls 64% at Day 3 and 52% at Day 6. The addition of 1 pM of 1,2,3- Tri-a-Linolenoyl Glycerol or 1,2,3-Trioleoyl Glycerol perform exceptionally with 109% at Day 3 amplitude as a measurement of the maximum clot strength compared with untreated controls 86% at Day 3. The addition of 1 pM of 1,3 -Dieicosapentaenoyl Glycerol or 1,2,3-Trilinoleoyl Glycerol perform exceptionally with 100% or 99% at Day 3 amplitude as a measurement of the maximum clot strength compared with untreated controls 82% at Day 3. Thus, based on the experiments herein, as little as 1 pM of one or more UFA-glycerolipids added to about 1 x 1012 / L of platelets is sufficient to result in an observable improvement in the viability, shelf-life, and function of cold-stored platelets. The addition of 250 pM of EPA perform exceptionally with 60% at Day 8 amplitude as a measurement of the maximum clot strength compared with untreated controls 26% at Day 8. Platelets treated with 250 pM EPA or 250 pM DHA exhibit equivalent or much lower cytosolic free calcium concentrations at Day 14 compared with untreated controls at Day 3 of cold storage, and platelets treated with 250 pM EPA or 250 pM DHA preserve more CD42b from decay at Day 14 than untreated controls at Day 3 of cold storage, thereby indicating that the addition of 250 pM EPA or 250 pM DHA extend the cold-storage shelf-life of platelets by more than 10 days.

[0060] Therefore, the present invention is directed to compositions comprising (a) one or more UFA-glycerolipids, (b) EPA, (c) DHA, or any combination thereof and methods of using the one or more UFA-glycerolipids, EPA, and / or DHA to: increase the viability of cold-stored platelets, increase the shelf-life of cold-stored platelets, increase the functionality (e.g., activation and aggregation functions) of cold-stored platelets, and inhibit platelet apoptosis. The one or more UFA-glycerolipids, EPA, and / or DHA maybe provided in the form of liposomes, lipid drops, emulsion, or lipid nanoparticles such as ionizable lipid nanoparticles.

[0061] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-250 pM, and most preferably about 1-100 pM of one or more UFA- glycerolipids per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0062] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-100 pM, and most preferably about 1-10 pM of a PUFA-glyceride and / or a MUFA-glyceride per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0063] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-250 pM, and most preferably about 10-100 pM of a PUFA- phospholipids and / or a MUFA-phospholipid per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0064] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-100 pM, and most preferably about 1-10 pM of 1,2,3- Docosahexaenoyl Glycerol (DHA-TG) per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0065] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-100 pM, and most preferably about 1-10 pM of 1,2,3- Tri eicosapentaenoyl Glycerol (EPA-TG) per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0066] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-100 pM, and most preferably about 1-10 pM of 1,2,3-Tri-Alpha- Linolenoyl Glycerol (ALA-TG) per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0067] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-100 pM, and most preferably about 1-10 pM of 1,3- Dieicosapentaenoyl Glycerol (EPA-DG) per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0068] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-100 pM, and most preferably about 1-10 pM of 1,2,3-Trilinoleoyl Glycerol (LA-TG) per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0069] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-100 pM, and most preferably about 1-10 pM of 12,3 -Trioleoyl Glycerol (OA-TG) per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0070] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-250 pM, and most preferably about 10-100 pM of l-Stearoyl-2- Eicosapentaenoyl-sn-glycero-3-PC (EPA-PC2) per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0071] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-250 pM, and most preferably about 10-100 pM of l-Stearoyl-2- Eicosapentaenoyl-sn-glycero-3-PE (EPA-PE) per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0072] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1-250 pM, and most preferably about 10-100 pM of l-Palmitoyl-2- Docosahexaenoyl-sn-glycero-3-PC (DHA-PC) per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0073] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-1,000 pM, more preferably about 1-500 pM, and most preferably about 10-250 pM of EPA per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0074] In some embodiments, about 0.05-5,000 pM, preferably about 0.5-1,000 pM, more preferably about 1-500 pM, and most preferably about 10-250 pM of DHA per 1 x 1012 / L of platelets is added to a composition comprising the platelets and then the composition is placed in cold-storage.

[0075] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-2,500 pM, and most preferably about 50-1,000 pM of one or more UFA-glycerolipids per 6.0 x 1012 / L of red blood cells (RBCs) is added to a composition comprising the RBCs and then the composition is placed in cold-storage.

[0076] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-1,000 pM, and most preferably about 50-500 pM of a PUFA- glyceride and / or a MUFA-glyceride per 6.0 x 1012 / L of red blood cells (RBCs) is added to a composition comprising the RBCs and then the composition is placed in cold- storage.

[0077] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-2,500 pM, and most preferably about 500-1,000 pM of PUFA- phospholipids and / or a MUFA-phospholipid per 6.0 x 1012 / L of red blood cells (RBCs) is added to a composition comprising the RBCs and then the composition is placed in cold-storage.

[0078] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-4,000 pM, and most preferably about 100-2,000 pM of EPA per 6.0 x 1012 / L of red blood cells (RBCs) is added to a composition comprising the RBCs and then the composition is placed in cold-storage.

[0079] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-4,000 pM, and most preferably about 100-2,000 pM of DHA per 6.0 x 1012 / L of red blood cells (RBCs) is added to a composition comprising the RBCs and then the composition is placed in cold-storage.

[0080] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-2,500 pM, and most preferably about 50-1,000 pM of one or more UFA-glycerolipids per liter of whole blood is added to whole blood and then the whole blood is placed in cold-storage.

[0081] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-1,000 pM, and most preferably about 50-500 pM of a PUFA- glyceride and / or a MUFA-glyceride per liter of whole blood is added to whole blood and then the whole blood is placed in cold-storage.

[0082] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-2,500 pM, and most preferably about 500-1,000 pM of PUFA- phospholipids and / or a MUFA-phospholipid per liter of whole blood is added to whole blood and then the whole blood is placed in cold-storage.

[0083] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-4,000 pM, and most preferably about 100-2,000 pM of UFA- glycerolipids per liter of whole blood is added to whole blood and then the whole blood is placed in cold-storage.

[0084] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-4,000 pM, and most preferably about 100-2,000 pM of UFA- glycerolipids per liter of whole blood is added to whole blood and then the whole blood is placed in cold-storage.

[0085] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-4,000 pM, and most preferably about 100-2,000 pM of EPA per liter of whole blood is added to whole blood and then the whole blood is placed in cold- storage.

[0086] In some embodiments, about 0.05-5,000 pM, preferably about 5-5,000 pM, more preferably about 50-4,000 pM, and most preferably about 100-2,000 pM of DHA per liter of whole blood is added to whole blood and then the whole blood is placed in cold- storage.

[0087] In some embodiments, one or more of the UFA-glycerolipids, EPA, and / or DHA are provided in the form of liposomes, lipid drops, emulsion, and lipid nanoparticles such as ionizable lipid nanoparticles, which may be PEGylated. In some embodiments, the UFA-glycerolipids are provided in the form of a composition, which may comprise a mixture of two or more UFA-glycerolipids. In some embodiments, the one or more UFA-glycerolipids are provided in the form of a composition, which comprises the one or more UFA-glycerolipids in combination with EPA and / or DHA. In some embodiments, EPA is provided in the form of a composition, which comprises EPA in combination with a UFA-glycerolipid and / or DHA. In some embodiments, DHA is provided in the form of a composition, which comprises DHA in combination with a UFA-glycerolipid and / or EPA. In some embodiments, the UFA-glycerolipids are provided in the form of a composition, which comprise one or more UFA-glycerolipids in combination with one or more of the following: “free” unsaturated fatty acids, antioxidants, anti-inflammatory compounds, and the like. As used herein, “free” unsaturated fatty acids refer to unsaturated fatty acids (including PUFAs, e.g., EPA and DHA) that are not covalently attached to another chemical moiety or backbone, e.g., a glycerol backbone.

[0088] Compositions

[0089] Platelet additive solutions comprising one or more UFA-glycerolipids, EPA, and / or DHA are contemplated herein. Such platelet additive solutions include those known in the art, e.g., PAS-I, PAS-II, and PAS-III, PAS-C, SSP+, Composol PS, T- PAS+, and the like, with the one or more UFA-glycerolipids, EPA, and / or DHA added thereto, wherein the concentration of the one or more UFA-glycerolipids, EPA, and / or DHA is about 0.05-5,000 pM, preferably about 0.5-500 pM, more preferably about 1- 250 pM, and most preferably about 1-100 pM. In some embodiments, the concentration of the one or more UFA-glycerolipids, EPA, and / or DHA is about 0.05-5,000 pM,1preferably about 0.5-500 pM, more preferably about 1-500 pM, and most preferably about 1-250 pM. The one or more UFA-glycerolipids, EP A, and / or DHA may be provided in the form of liposomes, lipid drops, emulsion, or ionizable lipid nanoparticles.

[0090] Kits

[0091] In some embodiments, the present invention provides kits for preparing cells and blood products for cold-storage. In some embodiments, the kits comprise a platelet additive solution comprising one or more UFA-glycerolipids, EP A, and / or DHA packaged together with a platelet collection (or donation) bag and / or a device, e.g., a syringe, for adding the platelet additive solution to, e.g., a bag of platelets. In some embodiments, the kits comprise at least one unit of one or more UFA-glycerolipids, EP A, and / or DHA individually packaged together with a platelet or blood collection (or donation) bag and / or a device, e.g., a syringe, for adding the one or more UFA- glycerolipids, EP A, and / or DHA to a given composition (e.g., cells or a blood product). In some embodiments, the unit of the one or more UFA-glycerolipids, EP A, and / or DHA is provided prepackaged in a single-use syringe. As used herein, a “unit” of one or more UFA-glycerolipids, EP A, and / or DHA (e.g., a “UFA-glycerolipid unit”, a unit of EP A, a unit of DHA, etc.) refers a single “dose” of the given agent(s) to be added to 3.5-4.0 x 1011platelets, 6.0 x 1012red blood cells (RBCs), or 400-500 mL whole blood. Thus, in some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about 1-250 mg, and most preferably about 1-100 mg, of one or more UFA-glycerolipids. In some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about 1- 100 mg, and most preferably about 1-10 mg, of a PUFA-glyceride and / or a MUFA- glyceride. In some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about 1-250 mg, and most preferably about 10-100 mg, of a PUFA-phospholipid and / or a MUFA-phospholipid. In some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about 1-100 mg, and most preferably about 1-10 mg of EPA-TG. In some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about 1-100 mg, and most preferably about 1-10 mg of DHA-TG. In some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about 1-100 mg, and most preferably about 1-10 mg of ALA-TG. In some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about1-100 mg, and most preferably about 1-10 mg of EPA-DG. In some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about 1-100 mg, and most preferably about 1-10 mg of LA-TG. In some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about 1-100 mg, and most preferably about 1-10 mg of OA-TG. In some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about 1-250 mg, and most preferably about 10-100 mg of EPA-PC2. In some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about 1- 250 mg, and most preferably about 10-100 mg of EPA-PE. In some embodiments, a UFA-glycerolipid unit comprises about 0.05-5,000 mg, preferably about 0.5-500 mg, more preferably about 1-250 mg, and most preferably about 10-100 mg of DHA-PC. In some embodiments, a unit of EPA comprises about 0.05-5,000 mg, preferably about 0.5- 1,000 mg, more preferably about 1-500 mg, and most preferably about 10-250 mg, of the EPA. In some embodiments, a unit of DHA comprises about 0.05-5,000 mg, preferably about 0.5-1,000 mg, more preferably about 1-500 mg, and most preferably about 10-250 mg of DHA. In some embodiments, a unit of EPA and DHA comprises about 0.05-5,000 mg, preferably about 0.5-1,000 mg, more preferably about 1-500 mg, and most preferably about 10-250 mg, of EPA and DHA.

[0092] In some embodiments, the kits include a carrier, package, or container that may be compartmentalized to receive one or more containers, such as vials, tubes, and the like. In some embodiments, the kits optionally include an identifying description or label or instructions relating to its use. In some embodiments, the kits include information prescribed by a governmental agency that regulates the manufacture, use, or sale of compounds and compositions as contemplated herein.

[0093] The following examples are intended to illustrate but not to limit the invention.

[0094] EXAMPLES

[0095] Exemplary UFA-glycerolipids

[0096] The PUFA-glycerides exemplified herein are:

[0097] 1,2,3-Trieicosapentaenoyl Glycerol (EPA-TG):

[0098] 1,2, 3 -Docosahexaenoyl Glycerol (DHA-TG):

[0099] 1,2,3-Tri-Alpha-Linolenoyl Glycerol (ALA-TG):

[0100] 1,2,3-Trilinoleoyl Glycerol (LA-TG)

[0101] The PUFA-glyceride exemplified herein is:

[0102] 1,3-Dieicosapentaenoyl Glycerol (EPA-DG)

[0103] The PUFA-phospholipids exemplified herein are:

[0104] l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC (EPA-PC2):

[0105] l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PE (EPA-PE):

[0106] l-Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC (DHA-PC):

[0107] The MUFA-glyceride exemplified herein is:

[0108] 1,2,3-Trioleoyl Glycerol (OA-TG):

[0109] Optical Aggregometry Assay

[0110] Optical Aggregometry was performed to assess the efficacy of the omega-3 fatty acids in platelet function recovery from cold storage lesion. Chrono-Log® Model 700 Lumi-Aggregometer (Chrono-Log Corp.) was used to evaluate platelet function by measuring platelet aggregation of platelets suspended in plasma via optical density. Thus, this assay quantifies platelet function via their ability to form clots.

[0111] Platelet aggregation was measured using optical aggregometry on a Chrono-Log 700 aggregometer. A combination of ADP, collagen, and epinephrine (5 pM ADP + 1 pg / mL collagen + 2 pM EPI) was used as a combined agonist for each sample.Aggregometer and all agonists were obtained from Chrono-Log Corp. (Havertown, PA). Agonist response was recorded for 6 minutes according to manufacturer’s recommendations, and the maximum amplitude of response to agonists was reported.

[0112] FIGS. 1 to 19 indicate that UFA-glycerolipids and EPA extend platelet cold- storage shelf-life and function by more than 10 days. Particularly, the platelet aggregation assays indicate that the aggregation function of Day 14 cold-stored platelets treated with UFA-glycerolipids is equivalent to the aggregation function of Day 3 cold- stored control platelets (platelets not treated with UFA-glycerolipids).

[0113] Cytosolic Free Calcium Assays

[0114] Cytosolic free calcium (Ca2+) was assayed as an indicator of platelet health and activation. Cytosolic free calcium was measured using Fluo-3-AM. During cold storage, healthy, resting platelets should be inactive and thereby the amount of cytosolic free calcium should be low (e.g., the cytosolic calcium concentration in a resting platelet is typically between 40-100 nanomolar (nM). This is much lower than the concentration of calcium outside the cell (plasma), which is around 2 millimolar (mM). Activated platelets, e.g., by agonists such as convulxin and thrombin, will have increased amounts of cytosolic free calcium. Thus, upon activation, higher amounts of Fluo-3-AMfluorescence, i.e., higher amounts of cytosolic free calcium, indicates healthier, more functional platelets.

[0115] Fresh platelet rich plasma (PRP) samples were collected on the day of each experiment to serve as the low and high controls. Measurements from these controls will be used to further calculate calcium concentrations for other samples.

[0116] PRP samples were prepared by Trima Accel 7 apheresis system. Platelet counts in PRP were adjusted by dilution to 300 x 103platelets / pL.

[0117] Test PRP samples were then cold-stored at about 3-5°C and assayed on Days 0- 21. Fc receptors on platelets were blocked with Human TruStain FcX™ (Catalogue No.422302, Biolegend, San Diego, CA) and then stained with CD41a and Fluo-3-AM for either 1 hour at room temperature or 30 minutes at 37°C before evaluated via flow cytometry using a FACS Symphony™ A5 SE flow cytometer (BD Biosciences, San Diego, CA). A baseline fluorescence intensity signal for Fluo-3-AM was collected for one minute. Then the samples were removed from the flow cytometer and spiked with a mixture of 50 ng / mL convulxin and 0.05 U thrombin, vortexed, and then immediately assayed via flow cytometry for 15 minutes of data acquisition.

[0118] For Low Control PRP samples, fresh PRP samples were incubated with 2 mM EGTA for 20 minutes and then EGTA-AM for 20 minutes in order to chelate outer and intra-cellular calcium. After incubation, the PRP samples were stained with CD41a and Fluo-3-AM and evaluated via flow cytometry in the same manner as the test PRP samples.

[0119] For High Control PRP samples, fresh PRP samples were incubated with 2 mM CaCh for 5 minutes and then stained with CD41a and Fluo-3-AM and evaluated via flow cytometry in the same manner as the test PRP samples except, the High Control PRP samples were spiked with 2 pM Calcium Ionophore A23187 (Calcimycin) instead of convulxin and thrombin.

[0120] Cytosolic Free Calcium was calculated as , wherein, Kd = Dissociation constant for Fluo-3-AM, F = Fluorescence intensity of Fluo-3-AM of the given sample, Fmin = Fluorescence intensity for Low Control post agonist spike, Fmax = Fluorescence intensity for High Control post agonist spike, whereby calcium concentration is reported in Kd units (nM).

[0121] The data in FIGS. 20 to 22 indicate that UFA-glycerolipids, EP A, and DHA improve the cold-storage shelf-life of platelets. While all the UFA-glycerolipids loweredthe cytosolic free calcium concentrations of cold-stored platelets, as shown in FIG. 20, platelets treated with triglyceride linked EPA and DHA exhibit lower cytosolic free calcium concentrations at Day 14 than untreated controls at Day 3 of cold storage, thereby indicating that triglyceride linked PUFAs extend the cold-storage shelf-life of platelets by at least 11 days. As shown in FIG. 22, platelets treated with EPA or DHA exhibit equivalent or much lower cytosolic free calcium concentrations at Day 14 compared with untreated controls at Day 3 of cold storage, thereby indicating that EPA and DHA extend the cold-storage shelf-life of platelets by at least 10 days.

[0122] Complete Blood Count (CBC) Assay

[0123] Complete Blood Count (CBC) assay was performed on Siemens AD VIA 2120i Hematology System. Platelet counting was measured for each platelet-rich plasma (PRP) samples from the time-course cold storage.

[0124] As shown in FIG. 23, higher number of platelet cells are preserved by omega-3 fatty acids EPA and DHA treatment during platelets cold-storage period as compared to untreated control.

[0125] Adenosine Triphosphate Assay

[0126] Adenosine triphosphate (ATP) was measured via luminescence analysis as an indicator of platelet viability. Specifically, Lower ATP levels in stored platelets can lead to impaired platelet aggregation and function, affecting their ability to clot effectively. Luciferin reacts with ATP, O2, and Mg2+to generate oxyluciferin which emits luminescence. Samples of platelets suspended in media are treated with reagent containing luciferin (e.g., Promega® CellTiter-Glo). This assay is performed in a microplate, which is then analyzed for luminescence in a plate reader.

[0127] Normalized cell numbers of platelets are treated with a luciferase-luciferin reagent containing a lysate. Platelets are lysed to release their intracellular ATP and the luciferase-luciferin reagent causes a luminescent reaction proportional to the amount of ATP present. An aliquot of 100 pL diluted PRP from each sample tube are transferred to one well of a 96-well multiwell plate for each replicate of the sample. A diluent treatment of 70 pL Isoplate (PAS-F) is transferred to each well. The reaction is initiated with 30 pL CellTiter-Glo 2.0 Reagent transferred to each Isoplate-and-sample-loaded well. The plate is mixed on an orbital shaker at 600 RPM for 2 minutes and then incubated at RT without exposure to light for 10 minutes. The luminescence is then measured using a luminometer GloMax Discover multimode multiplate reader, allowingfor quantification of the ATP concentration. Higher luminescence readings indicate a higher concentration of ATP, signifying a greater number of viable platelets.

[0128] As shown in FIGS. 24 to 27, cold-stored platelets treated with UFA- glycerolipids, EP A, and DHA exhibit higher concentrations of ATP as compared to untreated controls.

[0129] Glycoprotein VI (GPVI) and CD42b Assay

[0130] GPVI on the surface of platelets was assayed as an indicator of platelet cold storage lesion. Acting as the primary receptor for collagen, GPVI mediates platelet adhesion and aggregation upon vascular injury. CD42b (also known as GPIba) mediates platelet adhesion to damaged blood vessels via von Willebrand factor (VWF) and helps trigger platelet activation and clot formation. GPVI and CD42b were measured via fluorescently labeled antibodies thereagainst. The antibodies used were GP6 Monoclonal Antibody (HY101), eFluor™ 660, eBioscience™ (Catalogue No. 50-9813- 42) from ThermoFisher Scientific (Waltham, MA) and BD OptiBuild™ BUV805 Mouse Anti -Human CD42b (Catalog No. 742018) from BD Biosciences. Platelet samples were assessed for various activation, apoptosis, and constitutive markers using flow cytometry. Briefly, a 300 pL diluted sample of platelets was incubated with 10 pL of Human TruStain FcXTM receptor block (BioLegend, San Diego, CA) for 5 min at 22°C. After this time, 100 pL of the blocked sample was added to a tube containing the following: GPVI, CD42b, CD62P (P-Selectin, BD Biosciences, Franklin Lakes, NJ), CD41a (GPIIb, BioLegend), lactadherin (phosphatidyl serine exposure, Hematological Technologies, Inc., Essence Junction, VT), and Hank's Balanced Salt Solution (HBSS, ThermoFisher). Samples were incubated with antibodies for 15 minutes at room temperature in the dark, washed twice using a lyse wash assistant (BD Biosciences), and analyzed on a BD FACSCanto I flow cytometer (BD Biosciences). Forward scatter and side scatter were used for identification of the platelet population, and 100,000 events were recorded.

[0131] The data in FIG. 28 and FIG. 29 indicate that treatment with EPA and DHA inhibited platelet GPVI shedding and prevented CD42b loss during 21 days of cold- storage. As shown in FIG. 29, platelets treated with EPA and DHA preserve more CD42b from decay at Day 14 than untreated controls at Day 3 of cold storage, thereby indicating that EPA and DHA extend the cold-storage shelf-life of platelets by at least 11 days.

[0132] Phosphatidylserine Assay

[0133] Phosphatidylserine (PS) is a marker on the surface of cells that is a key indicator of platelets undergoing apoptosis. This assay was performed using flow cytometry as described above for the Glycoprotein VI (GPVI) and CD42b Assay. The results are reported as the percentage of platelets with external PS. That is, the greater the percentage of platelets with surface PS, the greater the percentage of platelets undergoing apoptosis.

[0134] As shown in FIG. 30, treatment with EP A, DHA, and EP A and DHA inhibited platelet apoptosis during 21 days of cold-storage.

[0135] REFERENCES

[0136] The following references are herein incorporated by reference in their entirety with the exception that, should the scope and meaning of a term conflict with a definition explicitly set forth herein, the definition explicitly set forth herein controls:- Vanderspurt, et al. The use of whole blood in US military operations in Iraq, Syria, and Afghanistan since the introduction of low-titer Type O whole blood: feasibility, acceptability, challenges. Transfusion. 2019 Mar;59(3):965-970. doi:10.1111 / trf.l 5086.- Edwards, et al. Lessons Learned From the Battlefield and Applicability to Veterinary Medicine - Part 2: Transfusion Advances. Front Vet Sci. 2021 May 7;8:571370. doi: 10.3389 / fvets.202L 571370.- Hanna, et al. The Use of Whole Blood Transfusion in Trauma. Curr Anesthesiol Rep.2022;12(2):234-239. doi: 10.1007 / s40140-021-00514-w.- D' Amici, et al. Proteomic analysis of RBC membrane protein degradation during blood storage. J Proteome Res. 2007 Aug;6(8):3242-55. doi: 10.1021 / pr070179d. - Rajashekaraiah & Rajanand. Platelet storage: Progress so far. J Thromb Thrombolysis. 2023 Jan;55(l):9-17. doi: 10.1007 / sl 1239-022-02716-3.- Miles, et al. Storage temperature determines platelet GPVI levels and function in mice and humans. Blood Adv. 2021 Oct 12;5(19):3839-3849. doi:10.1182 / bloodadvances.2021004692. Erratum in: Blood Adv. 2022 May 24;6(10):3102-3105. doi: 10.1182 / bloodadvances.2021006681.Maillot, etal. Cold adaptation in the environmental bacterium Shewanella oneidensis is controlled by a J-domain co-chaperone protein network. Commun Biol. 2019 Aug 29;2:323. doi: 10.1038 / s42003-019-0567-3.- D' Alessandro, et al. Protect, repair, destroy or sacrifice: a role of oxidative stress biology in inter-donor variability of blood storage? Blood Transfus. 2019 Jul;17(4):281-288. doi: 10.2450 / 2019.0072-19.- Jabbur, et al. Bacteria can anticipate the seasons: Photoperiodism in cyanobacteria.Science. 2024 Sep 6;385(6713): 1105-1111. doi: 10.1126 / science.ado8588.- Kawamoto, et al. Eicosapentaenoic acid plays a beneficial role in membrane organization and cell division of a cold-adapted bacterium, Shewanella livingstonensis AclO. JBacteriol. 2009 Jan;191(2):632-40. doi: 10.1128 / JB.00881- 08.- Dai, et al. Eicosapentaenoic acid facilitates the folding of an outer membrane protein of the psychrotrophic bacterium, Shewanella livingstonensis AclO. Biochem Biophys Res Commun. 2012 Aug 24;425(2):363-7. doi: 10.1016 / j.bbrc.2012.07.097.- Jiang, et al. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021 Apr;22(4):266-282. doi: 10.1038 / s41580-020-00324-8.- Liu, et al. The platelet storage lesion, what are we working for? J Clin Lab Anal.2024 Jan;38(l-2):e24994. doi: 10.1002 / jcla.24994.- Reddoch-Cardenas, et al. Cold-stored platelets: A product with function optimized for hemorrhage control. Transfus Apher Sci. 2019 Feb;58(l): 16-22. doi:10.1016 / j. transci.2018.12.012.- Sanchez, et al. Weathering the Cold: Modifying Membrane and Storage Fatty Acid Composition of Seeds to Improve Cold Germination Ability in Upland Cotton (Gossypium hirsutum L.). Agronomy. 2019; 9(11):684. doi:10.3390 / agronomy9110684.- Hou, et al. Lipid signalling in plant responses to abiotic stress. Plant Cell Environ.2016 May;39(5): 1029-48. doi: 10.1111 / pce.12666.- Welti, et al. Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis. J Biol Chem. 2002 Aug 30;277(35):31994-2002. doi: 10.1074 / jbc.M205375200.- Trend, et al. Insect cold-tolerance and lipidome: Membrane lipid composition of two chironomid species differently adapted to cold. Cryobiology. 2022 Jun; 106:84-90. doi: 10.1016 / j.cryobiol.2022.03.004.- Barria, et al. Bacterial adaptation to cold. Microbiology (Reading). 2013 Dec;159(Pt 12):2437-2443. doi: 10.1099 / mic.0.052209-0.- Phadtare S. Recent developments in bacterial cold-shock response. Curr Issues Mol Biol. 2004 Jul;6(2): 125-36.- Eastridge, et al. Death on the battlefield (2001-2011): implications for the future of combat casualty care. J Trauma Acute Care Surg. 2012 Dec;73(6 Suppl 5):S431-7. doi: 10.1097 / TA.0b013e3182755dcc. Erratum in: J Trauma Acute Care Surg. 2013 Feb;74(2):706. Kotwal, Russell S [corrected to Kotwal, Russ S],

[0137] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.

[0138] As used herein, “UFA-glycerolipids” refer to glycerolipids containing unsaturated fatty acid (UFA) moieties. Particularly, UFA-glycerolipids are compounds having a glycerol backbone and 1, 2, or 3 fatty acid moieties selected from polyunsaturated fatty acids (PUFAs, e.g., omega-3 and omega-6 PUFAs) and monounsaturated fatty acids (MUFAs) covalently attached thereto. That is, UFA- glycerolipids have the following chemical structure:, wherein at least one of the R groups (i.e., Rl, R2, or R3) is a polyunsaturated fatty acid (PUFA) moiety and / or a monounsaturated fatty acids (MUFA) moiety. The PUFA moiety is preferably selected from omega-3 PUFAs and omega-6 PUFAs. UFA-glycerolipids include “UFA-phospholipids” which are UFA-glycerolipids, wherein one of the R groups, e.g., R3, is a phosphate moiety, and “UFA-glycerides” which are UFA-glycerolipids, wherein none of the R groups is a phosphate moiety. In some embodiments, all three R groups are each independently a PUFA moiety. In someembodiments, R1 is a saturated or monounsaturated fatty acid moiety, R2 is a PUFA moiety, and R3 is a phosphate moiety. In some embodiments, the hydrocarbon chains of the saturated fatty acid, MUFA moieties, and PUFA moieties are independently 14-22 carbon atoms in length. In some embodiments, the PUFA moieties are selected from alpha-linolenic acid (ALA), eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), and linoleic acid (LA). In some embodiments, the MUFA moieties are oleic acid (OA). In some embodiments, at least one of the R groups is EPA. In some embodiments, at least one of the R groups is DHA. In some embodiments, at least one of the R groups is ALA. In some embodiments, at least one of the R groups is LA. In some embodiments, at least one of the R groups is OA. In some embodiments, the phosphate moiety is, wherein R4 is H, -N+(CH3)3, -CH2-CH2-N+H3, -CH2-CH2-N+(CH3)3, -CH2-CH(N+H3)-COO , -CH2-CH(OH)-CH2(OH), -CH2(COO )H-N+H3, an inositol moiety, or -CH2-CH(OH)-CH2-phosphatidic acid. In some embodiments, the UFA- glycerolipids are selected from 1,2,3-Trieicosapentaenoyl Glycerol, 1,2,3- Docosahexaenoyl Glycerol, 1,2,3-Tri-alpha-Linolenoyl Glycerol, 1,3- Dieicosapentaenoyl Glycerol, 1,2,3-Trilinoleoyl Glycerol, 1,2,3-Trioleoyl Glycerol, 1- Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC, l-Stearoyl-2-Eicosapentaenoyl-sn- glycero-3 -PE, and 1 -Palmitoyl-2-Docosahexaenoyl-sn-glycero-3 -PC .

[0139] As used herein, a “PUFA-glyceride” is a UFA-glyceride, wherein none of the R groups is a MUFA moiety. As used herein, a “MUFA-glyceride” is a UFA-glyceride, wherein none of the R groups is a PUFA moiety. As used herein, a “PUFA- phospholipid” refers to a UFA-phospholipid, wherein none of the R groups is a MUFA moiety. As used herein, a “MUFA-phospholipid” refers to a UFA-phospholipid, wherein none of the R groups is a PUFA moiety.

[0140] As used herein, “free” unsaturated fatty acids refer to unsaturated fatty acids (including MUFAs and PUFAs) that are not covalently attached to another chemical moiety or backbone, e.g., a glycerol backbone. Usage of “EPA” and “DHA” refer to their "free" forms unless the context indicates that the referenced EPA or DHA is attached to another chemical moiety, e.g., a glycerol backbone as in UFA-glycerides.

[0141] As used herein, “and / or” means “and” or “or”. For example, “A and / or B” means “A, B, or both A and B” and “A, B, C, and / or D” means “A, B, C, D, or a combination thereof’ and said “A, B, C, D, or a combination thereof’ means any subset of A, B, C,and D, for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D).

[0142] As used herein, the phrase “one or more of’, e.g., “one or more of A, B, and / or C” means “one or more of A”, “one or more of B”, “one or more of C”, “one or more of A and one or more of B”, “one or more of B and one or more of C”, “one or more of A and one or more of C” and “one or more of A, one or more of B, and one or more of C”.

[0143] The phrase “comprises or consists of A” is used as a tool to avoid excess page and translation fees and means that in some embodiments the given thing at issue: comprises A or consists of A. For example, the sentence “In some embodiments, the composition comprises or consists of A” is to be interpreted as if written as the following two separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition consists of A.”

[0144] Similarly, a sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself. For example, the sentence “In some embodiments, the composition comprises A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C ” As another example, the sentence “In some embodiments, the composition comprises at least A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises at least A. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C ”

[0145] To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

[0146] Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.

Claims

What is claimed is:

1. A platelet additive solution comprising a concentration of about 0.05-5,000 pM of one or more UFA-glycerolipids, wherein the platelet additive solution comprises a crystalloid solution and one or more salts selected from sodium chloride, potassium chloride, magnesium chloride, sodium acetate, sodium citrate, and sodium phosphate.

2. The platelet additive solution according to claim 1, wherein the concentration of sodium chloride is about 65-120 mM, the concentration of potassium chloride is about 2-6 mM, the concentration of magnesium chloride is about 1-4 mM, and / or the concentration of sodium acetate is about 15-35 mM.

3. The platelet additive solution according to claim 1 or claim 2, which comprises 60-120 mM NaCl, 3-7 mM KC1, 0.5-3.0 mM MgCh, and 20-50 mM sodium acetate.

4. The platelet additive solution according to any one of claims 1 ~ 3, which further comprises 1.5 -5.0 mM sodium citrate and / or 20-30 mM sodium phosphate.

5. The platelet additive solution according to any one of claims 1 - 4, which further comprises 25-35 mM mannitol.

6. A method of preparing cells or a blood product for cold-storage, which comprises adding one or more UFA-glycerolipids to the blood product.

7. A method of storing cells or a blood product, which comprises preparing the cells or the blood product according to the method of claim 6 and then storing the cells or the blood product at a temperature of about 1-6°C.

8. The method according to claim 6 or claim 7, wherein the blood product comprises platelets, leukocytes, red blood cells, hematopoietic stem cells, plasma, and / or whole blood.

9. The method according to claim 8, wherein- about 0.05-5,000 pM of the one or more UFA-glycerolipids per 1 x 1012 / L of platelets is added to the platelets;- about 0.05-5,000 pM of the one or more UFA-glycerolipids per 6.0 x 1012 / L of red blood cells is added to the red blood cells; or- about 0.05-5,000 pM of the one or more UFA-glycerolipids per liter of whole blood is added to the whole blood.

10. The method according to any one of claims 6 - 9, wherein- an amount of the one or more UFA-glycerolipids is added to the blood product to result in a ratio of 0.05-5,000 mg of UFA-glycerolipids to about 3.5-4.0 x 1011platelets;- wherein an amount of the one or more UFA-glycerolipids is added to the blood product to result in a ratio of 0.05-5,000 mg of UFA-glycerolipids to about 6.0 x 1012red blood cells (RBCs);- wherein an amount of the one or more UFA-glycerolipids is added to the blood product to result in a ratio of 0.05-5,000 mg of UFA-glycerolipids to about 400-500 mL whole blood.

11. A kit comprising at least one UFA-glycerolipid unit of one or more UFA-glycerolipids packaged together with a container for cells or a blood product and / or a device for adding the UFA-glycerolipid unit to the cells or the blood product, wherein the at least one UFA-glycerolipid unit is about 0.1 mg to about 6 g of the one or more UFA-glycerolipids.

12. The platelet additive solution according to any one of claims 1 - 5, the method according to any one of claims 6 - 10, or the kit according to claim 11, wherein the one or more UFA-glycerolipids have the following chemical structure:, wherein at least one of Rl, R2, and R3 is a polyunsaturated fatty acid (PUFA) moiety (e.g., an omega-3 and / or an omega-6 polyunsaturated fatty acid), or a monounsaturated fatty acid (MUFA) moiety.

13. The platelet additive solution, the method, or the kit according to claim 12, wherein the PUFA moiety is alpha-linolenic acid (ALA), eicosapentaenoic acid (EP A), docosahexaenoic acid (DHA), or linoleic acid (LA), and the MUFA moiety is oleic acid (OA).

14. The platelet additive solution, the method, or the kit according to claim 12 or claim 13, wherein R3 is a phosphate moiety.

15. The platelet additive solution, the method, or the kit according to any one of the preceding claims, wherein the one or more UFA-glycerolipids are selected from 1,2,3-Trieicosapentaenoyl Glycerol, 1,2, 3 -Docosahexaenoyl Glycerol, 1,2,3-Tri-alpha-Linolenoyl Glycerol, 1,3-Dieicosapentaenoyl Glycerol, 1,2,3-Trilinoleoyl Glycerol, 1,2,3-Trioleoyl Glycerol, 1-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PC, l-Stearoyl-2-Eicosapentaenoyl-sn-glycero-3-PE, and 1-Palmitoyl-2-Docosahexaenoyl-sn-glycero-3-PC.

16. The platelet additive solution, the method, or the kit according to any one of the preceding claims, wherein the one or more UFA-glycerolipids are provided in the form of a liposome, a lipid drop, an emulsion, or a lipid nanoparticle.

17. A platelet additive solution comprising eicosapentaenoic acid (EP A) and / or docosahexaenoic acid (DHA), a crystalloid solution, and one or more components selected from sodium chloride, potassium chloride, magnesium chloride, sodium acetate, sodium citrate, sodium phosphate, and mannitol, wherein the EPA and / or the DHA are in the form of: free fatty acids, one or more polyunsaturated fatty acid (PUFA) moieties of a UFA-glycerolipid, or both.

18. A method of preparing cells or a blood product for cold-storage, which comprises adding eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA) to the cells or the blood product, wherein the EPA and / or the DHA are in the form of: free fatty acids, one or more polyunsaturated fatty acid (PUFA) moieties of a UFA-glycerolipid, or both.

19. A method of storing cells or a blood product, which comprises preparing the cells or the blood product according to the method of claim 18, and then storing the cells or the blood product at a temperature of about 1-6°C.

20. A kit comprising at least one unit of eicosapentaenoic acid (EP A) and / or docosahexaenoic acid (DHA) packaged together with a container for cells or a blood product and / or a device for adding the at least one unit to the cells or the blood product, wherein the at least one unit is at least 0.1 mg to about 6 g, and the EPA and / or the DHA are in the form of: free fatty acids, one or more polyunsaturated fatty acid (PUFA) moieties of a UFA-glycerolipid, or both.

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