Compositions and Methods for Cryopreservation of Mitochondria

Abstract

The present disclosure relates to compositions and methods for cryopreservation of mitochondria, cryopreservation compositions containing mitochondria, and their therapeutic uses.

Description

Technical Field

[0001] The present application relates to compositions and methods for the cryopreservation of compositions containing isolated viable mitochondria. The present application further relates to cryopreservation compositions used in the methods described herein, and cryopreservation compositions containing isolated viable mitochondria obtained by the methods described herein. These cryopreservation compositions containing mitochondria exhibit an advantageous and valuable long shelf life and are immediately available for many purposes, including but not limited to research, diagnostic, and therapeutic applications.

Background Art

[0002] Mitochondria are double-membrane-bound organelles found in the cytoplasm of nucleated eukaryotic cells. They are found in almost all cells of the human body except red blood cells, and their number and location vary depending on the cell type. Mitochondria perform a number of essential tasks in eukaryotic cells, such as pyruvate oxidation, the Krebs cycle (i.e., the citric acid cycle), and the metabolism of amino acids, fatty acids, and steroids. Mitochondria are also involved in processes such as heat production, cell differentiation, cell signaling, calcium ion storage, calcium signaling, and programmed cell death (apoptosis), and have the ability to regulate the cell growth cycle. Mitochondria are the main sites of energy metabolism in cells and produce adenosine triphosphate (ATP) for different cell functions. Since adenosine triphosphate is the energy source for cell activity, mitochondria are also known as the "energy factories of the cell". Mitochondria produce ATP by the electron transport chain and the oxidative phosphorylation system ("respiratory chain"). Typically, more than 90% of the cell's required amount of ATP is supplied by the cell's own mitochondria. (Cell Biology of the Mitochondrion Genetics (Nov 2017), 207(3):843-871, Van der Bliek A.M., Sedensky M.M., Morgan P.G.; Erratum in: Genetics (Apr 2018), 208(4):1673; Mitochondria:in sickness and in health Cell (16 Mar 2012), 148(6):1145-59, Nunnari J, Suomalainen A.)。

[0003] Mitochondria are composed of two concentric membranes with special functions. The inner mitochondrial membrane contains proteins necessary for respiratory chain function and ATP synthesis. The outer mitochondrial membrane, which contains a number of integral membrane proteins, surrounds the entire organelle. The structure of mitochondria has significant similarities to some modern prokaryotes. In fact, mitochondria are thought to be derived from an ancient symbiosis that occurred when a nucleated cell phagocytosed an aerobic prokaryote. In the symbiotic relationship, the host cell became dependent on the prokaryote that was phagocytosed for energy production, and the prokaryotic cell began to depend on the protective environment provided by the host cell (The Origin of Mitochondria. Nature Education (2010), 3(9):58, Martin W. & Mentel M.).

[0004] Mitochondria, ubiquitous semi-autonomous organelles, are separated from the cytoplasm by the outer mitochondrial membrane and the inner mitochondrial membrane. The outer membrane is porous, and ions and small uncharged molecules can pass freely through it via pore-forming membrane proteins (porins) such as the voltage-dependent anion channel (VDAC). Larger molecules, especially proteins, must be introduced by specific translocases. Due to its porosity, there is no membrane potential across the outer membrane. In contrast, the inner membrane is a tight diffusion barrier to all ions and molecules. These can only pass through with the help of specific membrane transport proteins, each of which is selective for a specific ion or molecule. As a result of its ion selectivity, an electrochemical membrane potential accumulates across the inner mitochondrial membrane. The inner membrane is the site where oxidative phosphorylation occurs in a series of membrane protein complexes that either create an electrochemical gradient across the inner membrane or use it for ATP synthesis. The outer membrane separates the mitochondria from the cytoplasm. This surrounds the inner membrane that separates the intermembrane space from the central matrix with a high protein density. The inner membrane is differentiated into the inner boundary membrane and cristae. The two regions are continuous at the crista junctions. The cristae extend more or less deeply into the matrix and are the main sites of mitochondrial energy conversion. Due to the primary function of mitochondria in cell metabolism, damage and dysfunction in mitochondria can cause various human diseases.

[0005] Mitochondria are intracellular organelles essential for the life of cells. Disruption of normal mitochondrial function is harmful to cell viability. Mitochondrial disorders can be caused by mutations (acquired or hereditary) in mitochondrial DNA (mtDNA) or nuclear genes encoding mitochondrial components. Hereditary mitochondrial disorders may include, but are not limited to, mitochondrial encephalopathy, mitochondrial myopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, myoclonic epilepsy with ragged-red fibers (MERRF), neuropathy, ataxia and retinitis pigmentosa (NARP) syndrome, myoneurogastrointestinal encephalopathy (MNGIE), Leber hereditary optic neuropathy (LHON), and mitochondrial-DNA depletion syndrome (Mol Syndromol (2016), 7:122-137, Nyazov D.M.et al.). Damage to mitochondria can also be caused by injury, toxicity, chemotherapy, infections, and age-related changes. A decrease in blood flow in tissues and organs can result in mitochondrial damage. In particular, ischemia / reperfusion injury can cause mitochondrial damage, which has an adverse effect on oxygen consumption and energy synthesis (Annual Review of Pharmacology and Toxicology, Vol. 57, (2017), Lesnefsky, pp 535-565; A Cell Metabolism Review, (2015), Unifying Mechanism for Mitochondrial Superoxide Production during Ischemia-Reperfusion Injury, Chouchan E.T.). Generally, mitochondrial or mitochondrion-related diseases / disorders are chronic (long-term) diseases / disorders. The symptoms of mitochondrial diseases can be diverse. This depends on how many mitochondria are defective and where they are in the body. Sometimes, only one organ, tissue, or cell type is affected. However, in many cases, the problem affects many of them. Muscles and nerve cells are particularly energy-demanding, so muscle and nerve problems are common. The diseases range from mild to severe. Some types can be fatal.Mitochondrial-related diseases, such as acquired mitochondrial-related diseases or dysfunctions, may include, but are not limited to, central nervous system (CNS) diseases such as Alzheimer's disease, Parkinson's disease, Alpers syndrome, Leigh syndrome, and myoclonic epilepsy with ragged red fibers, as well as dysfunctions or diseases after stroke and traumatic brain or spinal cord injury (Mitochondrion (July 2017), 35:70-79, Gollihue and Rabchevsky). Drugs can induce mitochondrial dysfunction through various mechanisms, including inhibition of fatty acid oxidation, impairment of oxidative phosphorylation and respiratory chain activity, and changes in the integrity of the mitochondrial membrane. Some drugs can also impair mitochondrial function through the production of reactive oxygen species and the generation of active metabolites that can covalently bind to important mitochondrial proteins. Drug-induced mitochondrial dysfunction plays an important role in the etiology of adverse effects such as liver injury, myopathy, and cardiotoxicity. Examples of drugs that can induce mitochondrial dysfunction include, for example, acetaminophen, amiodarone, doxorubicin, nucleoside reverse transcriptase inhibitors (e.g., stavudine, didanosine, zidovudine), statins (e.g., atorvastatin, cerivastatin, simvastatin), and valproic acid (Mitochondrial Biology and Experimental Therapeutics, (March 2018), pp 269-295, Massart).

[0006] The isolated viable mitochondria of the present invention are particularly useful for the treatment of mitochondrial or mitochondriarelated diseases and disorders, genetic or acquired mitochondrial dysfunction, cancer, infectious diseases, inflammatory diseases, autoimmune diseases, ischemia, ischemia-related dysfunction, ischemia-reperfusion injury, or thromboembolism as the above other diseases and dysfunctions. Of particular importance are emerging treatments such as therapeutic mitochondrial transplantation (J Transl Med, (17 May 2021), 19(1):214, Hayashida K.; EMBO Rep. 2020 Sep 3; 21(9):e50964, published online 2020 Aug 2, R.N.Lightowlers et al.; J.et al.; Int J Mol Sci. 2021 May; 22(9):4793, published online 2021 Apr 30, A.Park et al.).

[0007] Cryopreservation of intracellular organisms / organelles is a process that enables the preservation of intracellular organisms such as mitochondria after isolation from cells by cooling their samples to very low temperatures. Mitochondria have great potential for use in basic research and many medical applications, but simple cooling or freezing for long-term storage is not possible because ice crystal formation, such as intracellular ice formation and / or dendritic ice crystal formation, osmotic shock, such as solution effect damage described by Peter Mazur (1977), and membrane damage during freezing and thawing cause mitochondrial damage or death. Many attempts have been made to cryopreserve mitochondria using cryoprotectants and temperature control devices (Biochim. Biophys. Acta (1961) 50:233-242, D. Greiff, M. Myers, C. A. Privitera; Nature (1961) 190:1202-1204, Greiff, D. and M. Myers). However, cryopreservation known in the art causes mitochondrial damage during freezing, thawing, and storage at low temperatures. This degradation adversely affects the activity and viability of mitochondria and thus their potential use for research, diagnostic, or therapeutic purposes. The inability to store mitochondria over long periods significantly limits their possible use, especially in clinical settings where there is often not enough time to freshly prepare mitochondria. Furthermore, mitochondrial isolation is a time-consuming process that is difficult to scale up in a single laboratory or hospital, and the preparation costs are high. This is particularly problematic for therapeutic uses where the preparation of products for patient use must comply with pharmaceutical manufacturing regulatory standards. Therefore, there remains a strong need for compositions and methods for cryopreserving mitochondria that can maintain the structural and / or functional integrity of mitochondria, preferably both the structural and functional integrity of mitochondria.

[0008] It would be highly advantageous to be able to freely obtain compositions containing isolated mitochondria that can be stored for long periods and are immediately available as needed for various purposes such as basic research and / or medical applications. By freely obtaining off-the-shelf compositions containing healthy mitochondria, the administration of viable mitochondria to patients (e.g., by mitochondrial transplantation, such as by replacing defective mitochondria within cells or tissues, or by enriching diseased or dysfunctional cells and tissues with healthy mitochondria) would be convenient and effective. Thereby, mitochondrial therapy would become an everyday procedure and could enable the prevention, amelioration, or elimination of symptoms associated with dysfunctional or insufficient numbers of mitochondria. Therapeutic mitochondrial transplantation (TMT) has the potential to continuously affect mitochondrial function and reactivate or amplify cellular energy metabolism. For example, McCully et al. have described that mitochondrial transplantation can be effective in many cell types and diseases. These include myocardial and skeletal muscle, lung and liver tissues and cells, and nervous tissue. McCully has predicted that mitochondrial transplantation will be an important therapy in the armamentarium of all clinicians and surgeons for the treatment of various ischemic disorders, mitochondrial diseases, and related disorders (Clin Trans Med (2016) 5:16, McCully et al.).

[0009] For this type of therapeutic use at the bedside of patients in need of treatment, compositions containing mitochondria that are immediately available would streamline the overall clinical procedure, for example, by being safer, faster, more consistent, and easier to implement.

[0010] Although various cryopreservatives or cryopreservative mixtures for cell preservation are known, since mitochondria have a double membrane structure and are more fragile than cells, these cryopreservatives are not suitable for freezing mitochondria. For this purpose, the industry has been actively attempting to develop mitochondrion-specific cryopreservatives. However, during mitochondrial cryopreservation, mitochondria still very frequently swell, become damaged, or rupture, preventing the mitochondria from functioning properly, for example, after thawing. Therefore, preventing damage to mitochondria during cryopreservation is still an unsolved problem.

[0011] Mitochondria play an important role in the regulation of apoptosis in cells. The permeabilization of the outer mitochondrial membrane is an important step in the apoptotic process. The outer membrane of isolated mitochondria deteriorates over time and tends to rupture during freeze-thaw cycles. By analyzing cytochrome c retention over time at different temperatures, Yamaguchi et al. showed that mitochondria frozen in a buffer containing trehalose preserve the integrity of the outer mitochondrial membrane (Yamaguchi et al., Cell Death and Differentiation (2007), (14):616-624). Yamaguchi et al. also attempted to demonstrate that trehalose-frozen mitochondria are biologically similar to freshly prepared mitochondria. Indeed, Yamaguchi et al. claimed that mitochondria frozen in a buffer containing trehalose retain many biological and bioenergetic functions, such as the ability to synthesize ATP, the ability to respond to an increase in calcium concentration by activating the permeability transition pore (PTP), the ability to maintain the transmembrane inner potential, and the ability to take up and process proteins. However, the data presented by Yamaguchi et al. do not sufficiently and effectively prove that those mitochondrial functions are maintained, and thus the results are not conclusive. For example, significantly decreased rates of both phosphorylating respiration and maximal uncoupled respiration indicate impaired bioenergetic function. Indeed, Yamaguchi et al. admitted that upon more detailed examination, most of the mitochondrial functions appear to be impaired.

[0012] U.S. Patent Application Publication No. 2019 / 0134088 describes partially purified functional mitochondria derived from cells or tissues that have undergone freeze-thaw cycles. The freezing buffer contains a sugar, oligosaccharide, or polysaccharide as a cryoprotectant. U.S. Patent Application Publication No. 2019 / 0134088 discloses that a sufficient sugar concentration that acts to preserve mitochondrial function is in the range of 100 mM to 400 mM. It is also disclosed therein that the partially purified mitochondria that have undergone freeze-thaw cycles can be stored as frozen mitochondria for at least 1, 2, 3 weeks or even up to 1 month before thawing. In particular, the saccharide used in U.S. Patent Application Publication No. 2019 / 013088 is sucrose, and most importantly, the saccharide always had to be other than trehalose or mannitol. However, throughout the entire disclosure of U.S. Patent Application Publication No. 2019 / 0134088, sufficient evidence to support the fact that the partially purified mitochondria remained intact and viable after freeze-thaw cycles or after storage was not provided. Similarly, the finding that sucrose alone could be considered a sufficiently good cryoprotectant for mitochondria seems to be in complete contradiction to the conclusions of Yamaguchi et al.

[0013] International Publication No. WO 2021 / 168764 (Taiwan Mitochondrion Applied Tech Co Ltd) discloses a mitochondrial cryopreservative and a cryopreservation method. A first cryopreservation composition for cryopreserving mitochondria, consisting of 300 mM trehalose, 10 mM 2-[4-(2-hydroxyethyl)-piperazin-1-yl]-ethanesulfonic acid (i.e., 10 mM HEPES), and 0.1 wt% human serum albumin (HSA), is described. This cryopreservation composition does not contain KCl, NaCl, EDTA, EGTA, and ethylene glycol. Comparative cryopreservation composition 1 consists of 300 mM trehalose, 10 mM HEPES, 0.1 wt% HSA, 10 mM KCl, 1 mM EDTA, and 1 mM EGTA. Comparative cryopreservation composition 2 consists of 300 mM trehalose and 0.1 wt% HSA. The experimental data provided in International Publication No. WO 2021 / 168764 attempted to demonstrate that when the first composition is used, the integrity of the mitochondrial outer membrane (MOM) of cryopreserved mitochondria is maintained. However, the data presented in International Publication No. WO 2021 / 168764 never prove that those cryopreserved mitochondria remain viable through freeze-thaw cycles.

[0014] The technical problem is solved by the embodiments / claims provided herein and characterized in the claims. Accordingly, the present invention relates, inter alia, to the following items.

[0015] 1. A composition comprising: (i) an aqueous buffer having a pH of 5.5 to 8.5; (ii) trehalose at a concentration of at least 150 mM; (iii) a first cryoprotectant selected from one or more (a) amino acids at a total concentration of at least 150 mM; and optionally, (iv) a second cryoprotectant selected from one or more (b) sugars, (c) polymers, or combinations thereof; comprising (b) The sugar has a total concentration of at least 150 mM, and (c) The polymer has a total concentration of 2.5% (w / v) to 30% (w / v), Composition.

[0016] 2. A composition comprising: (i) An aqueous buffer having a pH of 5.5 to 8.5, and (ii) Trehalose at a concentration of at least 150 mM, and (iii) A first cryoprotectant selected from one or more (b) sugars having a total concentration of at least 160 mM, and optionally, (iv) A second cryoprotectant selected from (a) amino acids, (c) polymers, or combinations thereof, wherein (a) The amino acids have a total concentration of at least 150 mM, and (c) The polymers have a total concentration of 2.5% (w / v) to 30% (w / v), Composition.

[0017] 3. A composition comprising: (i) An aqueous buffer having a pH of 5.5 to 8.5, and (ii) Trehalose at a concentration of at least 150 mM, and (iii) One or more cryoprotectants selected from one or more (c) polymers, wherein the total concentration of the polymer is 16% (w / v) to 30% (w / v), Composition.

[0018] 4. The composition according to any one of items 1 to 3, wherein (i) the pH of the aqueous buffer is 6.0 to 8.5, such as 6.5 to 8.2.

[0019] 5. The composition according to item 4, wherein (i) the pH of the aqueous buffer is 6.8 to 8.0, such as 7.2.

[0020] 6. (i) The aqueous buffer contains a buffer, preferably the buffer is selected from 2-[4-(2-hydroxyethyl)-piperazin-1-yl]-ethanesulfonic acid (HEPES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 4-morpholineethanesulfonic acid (MES), bis-(2-hydroxyethyl)amino-tris-(hydroxymethyl)-methane (bis-tris), 2-(N-cyclohexylamino)-ethanesulfonic acid (CHES), N,N-bis-(2-hydroxyethyl)-glycine (bicine), potassium phosphate, sodium cacodylate, tris-(hydroxymethyl)aminomethane hydrochloride (tris), 4-morpholinepropanesulfonic acid (MOPS), 1,3-bis-[tris-(hydroxymethyl)-methylamino]-propane (bis-trispropane), sodium acetate, or a combination thereof, and is the composition according to any one of items 1 to 5.

[0021] 7. The composition according to any one of items 1 to 6, wherein the buffer in the aqueous buffer has a concentration of 0.5 mM to 50 mM.

[0022] 8. The composition according to any one of items 1 to 7, wherein the buffer in the aqueous buffer has a concentration of 2 mM to 35 mM, for example 5 mM to 30 mM.

[0023] 9. The composition according to any one of items 1 to 8, wherein the buffer in the aqueous buffer has a concentration of 10 mM to 25 mM, for example 15 mM.

[0024] 10. (i) The composition according to any one of items 1 to 9, wherein the aqueous buffer has a pH of 7.2 and contains 10 mM of 2-[4-(2-hydroxyethyl)-piperazin-1-yl]-ethanesulfonic acid (HEPES).

[0025] 11. Further comprising a calcium chelating agent, wherein the calcium chelating agent is selected from the group consisting of ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 2,2’,2’’,2’’’-(ethane-1,2-diyl dinitrilo)-tetraacetic acid (EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis-(acetoxymethyl ester) (BAPTA-AM), or a combination thereof, and the composition according to any one of items 1 to 10.

[0026] 12. The composition according to item 11, wherein the calcium chelating agent has a concentration of 0.1 mM to 10 mM, such as 0.2 mM to 5 mM.

[0027] 13. The composition according to item 12, wherein the calcium chelating agent has a concentration of 0.5 to 2 mM, such as 1 mM.

[0028] 14. The composition according to any one of items 11 to 13, wherein the calcium chelating agent is ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) or 2,2’,2’,2’’’-(ethane-1,2-diyl dinitrilo)-tetraacetic acid (EDTA).

[0029] 15. The composition according to any one of items 1 to 14, further comprising an ionic component.

[0030] 16. The composition according to item 15, wherein the ionic component has a concentration of 0.01% (w / v) to 10% (w / v), such as 0.5% (w / v) to 10% (w / v), and more particularly 1% (w / v) to 5% (w / v).

[0031] 17. The composition according to item 15, wherein the ionic component has a concentration of 0.1 mM to 100 mM, such as 1 mM to 30 mM, and more particularly 5 mM to 15 mM.

[0032] 18. The ionic component is Mg 2+ 、Na+ , K + , Cl - , HCO3 - , or a salt, acid, or base selected from combinations thereof, the composition according to any one of items 15 to 17.

[0033] 19. The ionic component is MgCl 2. MgSO4, KCl, KH2PO4, NaHCO3, Na2HPO4, C2H2MgO4 (magnesium formate), C3H3NaO3 (sodium pyruvate), C2H3NaO2 (sodium acetate), or a combination thereof, the composition according to any one of items 15 to 17.

[0034] 20. The ionic component is an organic anion selected from citrate, pyruvate, malate, oxaloacetate, formate, glutamate, α-ketoglutarate, succinate, acetate anion, or a combination thereof, the composition according to any one of items 15 to 17.

[0035] 21. The composition according to any one of items 1 to 20, further comprising albumin at a concentration of 0.01% (w / v) to 10% (w / v), such as 0.1% (w / v) to 5% (w / v).

[0036] 22. The composition according to any one of items 1 to 21, wherein the albumin is bovine serum albumin (BSA), human serum albumin (HSA), or a combination thereof.

[0037] 23. The composition according to item 21 or 22, wherein the albumin is BSA at a concentration of 0.05% (w / v) to 3% (w / v), such as 0.1% (w / v).

[0038] 24. (i) The buffer has a pH of 7.4 and contains 20 mM tris-(hydroxymethyl)aminomethane hydrochloride (Tris), 2 mM 2,2’,2’’,2’’’-(ethane-1,2-diyl dinitrilo)-tetraacetic acid (EDTA), and 10 mM MgCl2, the composition according to any one of items 1 to 3.

[0039] 25. (i) The composition according to any one of items 1 to 3, wherein the buffer has a pH of 7.25 and contains 5 mM of 4-morpholinepropanesulfonic acid (MOPS), 10 mM of 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA), and 5 mM of sodium pyruvate.

[0040] 26. (i) The composition according to any one of items 1 to 3, wherein the buffer has a pH of 7.2 and contains 10 mM of 2-[4-(2-hydroxyethyl)-piperazin-1-yl]-ethanesulfonic acid (HEPES) and 1 mM of ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA).

[0041] 27. The composition according to any one of items 1 to 26, wherein the composition contains less than the amount for cryopreservation of propylene glycol, ethylene glycol, glycerol, and dimethyl sulfoxide (DMSO), or does not contain propylene glycol, ethylene glycol, glycerol, and dimethyl sulfoxide (DMSO).

[0042] 28. The composition according to any one of items 1 to 26, wherein the composition contains less than the amount for cryopreservation of dimethyl sulfoxide (DMSO), or does not contain dimethyl sulfoxide (DMSO).

[0043] 29. (ii) The composition according to any one of items 1 to 28, wherein trehalose has a concentration of 1500 mM or less, for example 1300 mM or less.

[0044] 30. (ii) The composition according to item 29, wherein trehalose has a concentration of 500 mM or less, preferably 450 mM or less.

[0045] 31. (ii) The composition according to any one of items 1 to 29, wherein trehalose has a concentration of at least 250 mM, for example 300 mM.

[0046] 32. (ii) A composition according to any one of items 1 to 29, wherein trehalose has a concentration of 150 mM to 1000 mM, for example 200 mM to 600 mM.

[0047] 33. (a) A composition according to any one of items 1, 2, or 4 to 32, wherein the amino acid is selected from leucine, isoleucine (e.g., L-isoleucine), proline (e.g., L-proline), methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, valine (e.g., L-valine), alanine (e.g., L-alanine), glycine, asparagine (e.g., L-asparagine), aspartic acid (e.g., L-aspartic acid), glutamic acid (e.g., L-glutamic acid), serine (e.g., L-serine), histidine (e.g., L-histidine), cysteine (e.g., L-cysteine), tryptophan (e.g., L-tryptophan), tyrosine (e.g., L-tyrosine), arginine (e.g., L-arginine), glutamine (e.g., L-glutamine), lysine, threonine, selenocysteine, methionine, phenylalanine, creatine (e.g., L-creatine), taurine (e.g., L-taurine), betaine, ectoine, dimethylglycine, ethylmethylglycine, RGD peptide, or a combination thereof.

[0048] 34. (a) A composition according to item 33, wherein the amino acid is selected from leucine, isoleucine (e.g., L-isoleucine), valine (e.g., L-valine), alanine (e.g., L-alanine), glycine, asparagine (e.g., L-asparagine), aspartic acid (e.g., L-aspartic acid), glutamic acid (e.g., L-glutamic acid), serine (e.g., L-serine), histidine (e.g., L-histidine), cysteine (e.g., L-cysteine), tryptophan (e.g., L-tryptophan), tyrosine (e.g., L-tyrosine), arginine (e.g., L-arginine), glutamine (e.g., L-glutamine), or a combination thereof.

[0049] 35. (a) The composition according to item 33, wherein the amino acid is selected from methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof.

[0050] 36. (a) The composition according to item 33, wherein the amino acid is proline, such as L - proline.

[0051] 37. (a) The composition according to any one of items 1, 2, or 4 - 36, wherein the amino acid has a concentration of at least 160 mM, such as at least 180 mM.

[0052] 38. (a) The composition according to any one of items 1, 2, or 4 - 37, wherein the amino acid has a concentration of at least 200 mM, such as at least 250 mM.

[0053] 39. (a) The composition according to any one of items 1, 2, or 4 - 38, wherein the amino acid has a concentration of at least 300 mM, such as at least 500 mM.

[0054] 40. (a) The composition according to any one of items 1, 2, or 4 - 39, wherein the amino acid has a concentration of at least 800 mM, such as at least 1000 mM.

[0055] 41. (a) The composition according to any one of items 1, 2, or 4 - 40, wherein the amino acid has a concentration of at least 1100 mM, such as at least 1200 mM.

[0056] 42. (a) The composition according to any one of items 1, 2, or 4 - 41, wherein the amino acid has a concentration of at least 1300 mM, such as at least 1500 mM.

[0057] 43. (a) The composition according to any one of items 1, 2, or 4 - 42, wherein the amino acid has a concentration of at least 1800 mM, such as 2000 mM.

[0058] 44. (b) The composition according to any one of items 1, 2, or 4 to 43, wherein the sugar is selected from monosaccharides, disaccharides, or trisaccharides, or a combination thereof.

[0059] 45. (b) The composition according to any one of items 1, 2, or 4 to 44, wherein the sugar is selected from maltose, lactose, fructose, sucrose, glucose, dextran, melibiose, raffinose, nigerotriose, maltotriose, maltotriulose, kestose, cellobiose, chitobiose, lactulose, or a combination thereof.

[0060] 46. (b) The composition according to any one of items 1, 2, or 4 to 45, wherein the sugar is selected from sucrose, glucose, or a combination thereof.

[0061] 47. (b) The composition according to item 46, wherein the sugar is a combination of glucose and sucrose.

[0062] 48. (b) The composition according to item 46, wherein the sugar is glucose.

[0063] 49. (b) The composition according to item 46, wherein the sugar is sucrose.

[0064] 50. (b) The composition according to any one of items 1, 2, or 4 to 49, wherein the sugar has a concentration of at least 160 mM, for example at least 180 mM.

[0065] 51. (b) The composition according to item 50, wherein the sugar has a concentration of at least 200 mM, for example at least 250 mM.

[0066] 52. (b) The composition according to any one of items 50 or 51, wherein the sugar has a concentration of at least 300 mM, for example at least 330 mM.

[0067] 53. (b) The composition according to any one of items 1, 2, or 4 to 52, wherein the sugar has a concentration of 2000 mM or less, such as 1800 mM or less, and more particularly 1500 mM or less.

[0068] 54. (b) The composition according to item 53, wherein the sugar has a concentration of 1400 mM or less, such as 1200 mM or less, and more particularly 1000 mM or less.

[0069] 55. (c) The composition according to any one of items 1 to 54, wherein the polymer is a biocompatible, hydrophilic, amphiphilic polymer, or a combination thereof.

[0070] 56. (c) The composition according to any one of items 1 to 55, wherein the polymer is selected from poloxamer, such as poloxamer 142, poloxamer 188, poloxamer 331, or poloxamer 407, alginate, polyethylene glycol (PEG), such as PEG400 or PEG1000, polyglutamic acid, polyvinyl alcohol, polyvinylpyrrolidone, or a combination thereof.

[0071] 57. (c) The composition according to any one of items 1 to 56, wherein the polymer is polyethylene glycol (PEG).

[0072] 58. (c) The composition according to any one of items 1 or 2, wherein the polymer has a concentration of 2.5% (w / v) to 25% (w / v), such as 5% (w / v) to 25% (w / v).

[0073] 59. (c) The composition according to any one of items 1, 2, or 55 to 58, wherein the polymer has a concentration of 10% (w / v) to 25% (w / v), such as 15% (w / v) to 20% (w / v).

[0074] 60. (iii) The first cryoprotectant is (a) an amino acid such as L-proline at a concentration of at least 150 mM, such as at least 200 mM, preferably at least 300 mM. (iv) The second cryoprotectant is selected from one or more of (b) sugars, (c) polymers, or combinations thereof, (b) The sugar is glucose, sucrose, or a combination thereof, and has a total concentration of at least 150 mM, such as at least 200 mM, more specifically at least 300 mM, such as at least 500 mM, (c) The polymer is polyethylene glycol (PEG) at a concentration of 2.5% (w / v) to 30% (w / v), such as 5% (w / v) to 25% (w / v), more specifically 10% (w / v) to 20% (w / v), such as 15% (w / v), The composition according to any one of items 1, 4 to 33 or 36 to 59.

[0075] 61. (iii) The first cryoprotectant is a proline derivative selected from methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or combinations thereof, and is one or more (a) amino acids at a concentration of at least 150 mM, such as at least 200 mM, preferably at least 300 mM, and (iv) The second cryoprotectant is selected from one or more of (b) sugars, (c) polymers, or combinations thereof, (b) The sugar is glucose, sucrose, or a combination thereof, and has a total concentration of at least 150 mM, such as at least 200 mM, more specifically at least 300 mM, such as at least 500 mM, (c) The polymer is polyethylene glycol (PEG) at a concentration of 2.5% (w / v) to 30% (w / v), such as 5% (w / v) to 25% (w / v), more specifically 10% (w / v) to 20% (w / v), such as 15% (w / v), The composition according to any one of items 1, 4 to 33, 35 or 37 to 59.

[0076] 62. (iii) The first cryoprotectant is one or more (b) sugars that are glucose, sucrose, or a combination thereof, at a total concentration of at least 160 mM, such as at least 200 mM, more specifically at least 300 mM, such as at least 500 mM. (iv) The second cryoprotectant is selected from one or more (a) amino acids, (c) polymers, or a combination thereof. (a) The amino acid is selected from the amino acids described in any one of Items 33 to 36, and is at a concentration of at least 150 mM, such as at least 200 mM, preferably at least 300 mM. (b) The polymer is polyethylene glycol (PEG) at a concentration of 2.5% (w / v) to 30% (w / v), such as 5% (w / v) to 25% (w / v), more specifically 10% (w / v) to 20% (w / v), such as 15% (w / v). The composition according to any one of Item 2 or Items 4 to 59.

[0077] 63. The composition according to Item 62, wherein (a) the amino acid is proline, such as L-proline.

[0078] 64. (iii) The cryoprotectant is a (c) polymer that is polyethylene glycol (PEG) at a concentration of 16% (w / v) to 30% (w / v), such as 20% (w / v) to 30% (w / v), such as 25% (w / v). The composition according to any one of Items 3 to 32 or Items 55 to 57.

[0079] 65. (a) The amino acid is proline, such as L-proline, at a concentration of at least 500 mM, such as at least 600 mM, particularly at least 800 mM. The composition according to any one of Item 60, Item 62, or Item 63.

[0080] 66. The amino acid is proline, such as L-proline, at a concentration of at least 1000 mM, such as 1200 mM. The composition according to any one of Item 60, Item 62, or Item 63.

[0081] 67. The amino acid is proline, such as L - proline, at a concentration of at least 1300 mM, for example at least 1500 mM. The composition according to any one of items 60, 62, or 63.

[0082] 68. The amino acid is proline, such as L - proline, at a concentration of at least 1600 mM, for example at least 1800 mM, particularly at least 2000 mM. The composition according to any one of items 60, 62, or 63.

[0083] 69. The amino acid is proline, such as L - proline. The sugar is glucose at a concentration of 200 mM to 1300 mM, for example 300 mM to 1200 mM, particularly 350 mM to 1100 mM. The composition according to any one of items 60, 62, 63, or 65 - 68.

[0084] 70. The amino acid is proline, such as L - proline. The sugar is sucrose at a concentration of 160 mM to 900 mM, for example 200 mM to 800 mM, particularly 250 mM to 650 mM. The composition according to any one of items 60, 62, 63, or 65 - 68.

[0085] 71. The amino acid is proline, such as L - proline. The polymer is polyethylene glycol (PEG) at a concentration of 5% (w / v) to 30% (w / v), 10% (w / v) to 20% (w / v), for example 15% (w / v). The composition according to any one of items 60, 62, 63, or 65 - 68.

[0086] 72. The cryoprotectant consists of proline such as L - proline at the concentration according to any one of items 37 - 43, and is the composition according to any one of items 1, 4 - 33, 36, or 60.

[0087] 73. The composition according to item 72, wherein proline, for example L - proline, has a concentration of 600 mM or 1200 mM.

[0088] 74. The cryoprotectant consists of one or more (a) amino acids, and the (a) amino acid is a proline derivative selected from methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof, and the total concentration is at least 200 mM, preferably at least 300 mM, more preferably at least 500 mM. The composition according to any one of items 1, 4 - 32, 35 or 61.

[0089] 75. The cryoprotectant consists of one or more (a) amino acids, The amino acid is a proline derivative selected from methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof, at a concentration of at least 400 mM, preferably at least 600 mM, for example at least 800 mM. The composition according to any one of items 1, 4 - 32, 35, 61, or 74.

[0090] 76. The cryoprotectant consists of one or more (a) amino acids, The amino acid is a proline derivative selected from methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof, at a concentration of at least 1000 mM, for example at least 1200 mM, particularly at least 1500 mM, for example 2000 mM. The composition according to any one of items 1, 4 - 32, 35, 61, 74 or 75.

[0091] 77. A composition according to any one of items 2, 4 to 32, or 62, wherein the cryoprotectant consists of glucose, sucrose, or a combination thereof, at a total concentration of at least 200 mM, such as at least 300 mM, more specifically at least 500 mM, and 2000 mM or less, such as 1700 mM or less.

[0092] 78. A composition according to any one of items 2, 4 to 32, or 62, wherein the cryoprotectant consists of glucose, sucrose, or a combination thereof, at a total concentration of at least 550 mM, such as at least 650 mM, more specifically at least 800 mM, and 2000 mM or less, such as 1650 mM or less.

[0093] 79. A composition according to any one of items 2, 4 to 32, or 62, wherein the cryoprotectant consists of glucose, sucrose, or a combination thereof, at a total concentration of at least 1000 mM, such as at least 1200 mM, particularly at least 1350 mM, and 2000 mM or less, such as 1650 mM or less.

[0094] 80. A composition according to any one of items 2, 4 to 32, or 62, wherein the cryoprotectant consists of glucose at a total concentration of at least 300 mM, such as at least 500 mM, particularly at least 800 mM but 1200 mM or less, such as 1000 mM or less.

[0095] 81. A composition according to any one of items 2, 4 to 32, or 62, wherein the cryoprotectant consists of sucrose at a total concentration of at least 200 mM, such as at least 250 mM, particularly at least 300 mM but 800 mM or less, such as 650 mM or less.

[0096] 82. A composition according to any one of items 1 to 81, further comprising isolated mitochondria, such as isolated viable mitochondria.

[0097] 83. The composition according to item 82, wherein the isolated mitochondria are isolated mammalian mitochondria, such as isolated viable mammalian mitochondria.

[0098] 84. The composition according to item 82, wherein the isolated mitochondria are isolated human mitochondria, such as isolated viable human mitochondria.

[0099] 85. The composition according to any one of items 82 to 84, wherein the mitochondria are isolated from cells, tissues, or organs.

[0100] 86. The composition according to item 85, wherein the tissue is liver tissue, skeletal muscle, heart, brain, kidney, placenta, lung tissue, or adipose tissue.

[0101] 87. The composition according to item 86, wherein the mitochondria are isolated from cultured cells, such as cultured human cells.

[0102] 88. The composition according to any one of items 85 or 87, wherein the mitochondria are isolated from cells selected from the group consisting of placental cells, muscle tissue cells, cardiac fibroblasts, HeLa cells, prostate cancer cells, yeast, or any mixture thereof.

[0103] 89. The composition according to any one of items 82 to 88, wherein the isolated mitochondria have a concentration of at least 0.02 μg / μL, such as at least 0.05 μg / μL, particularly at least 0.1 μg / μL.

[0104] 90. The composition according to item 89, wherein the isolated mitochondria have a concentration of at least 0.2 μg / μL, such as at least 0.5 μg / μL, particularly at least 0.75 μg / μL.

[0105] 91. The composition according to any one of items 89 or 90, wherein the isolated mitochondria have a concentration of at least 1.0 μg / μL, such as at least 1.5 μg / μL, particularly at least 2 μg / μL, such as 2.5 μg / μL.

[0106] 92. A composition according to any one of items 89 to 91, wherein the isolated mitochondria have a concentration of at least 5 μg / μL, such as at least 10 μg / μL, such as 20 μg / μL or 30 μg / μL.

[0107] 93. A composition according to any one of items 82 to 92, wherein the isolated mitochondria have a concentration of 100 μg / μL or less, such as 80 μg / μL or less, particularly 50 μg / μL or less.

[0108] 94. A composition according to any one of items 82 to 93, wherein the isolated mitochondria have a concentration of 25 μg / μL or less, such as 15 μg / μL or less, particularly 10 μg / μL or less, such as 5 μg / μL or less.

[0109] 95. A composition according to any one of items 82 to 94, wherein the isolated mitochondria are linked to a pharmaceutical agent, a diagnostic agent, a contrast agent, a therapeutic agent, or any other biocompatible substance.

[0110] 96. A composition according to any one of items 82 to 94, wherein the mitochondria are linked to an antibody.

[0111] 97. A composition according to any one of items 82 to 94, wherein the mitochondria are linked to an antigen-binding fragment, and the antigen-binding fragment is at least a part of an antibody or TCR or a recombinant variant thereof.

[0112] 98. A composition according to any one of items 95 to 97, wherein the mitochondria are linked to a pharmaceutical agent, an antibody, or an antigen-binding fragment by a covalent bond.

[0113] 99. A composition according to any one of items 95 to 97, wherein the mitochondria are linked to a pharmaceutical agent, an antibody, or an antigen by a non-covalent bond such as an electrostatic bond.

[0114] 100. The composition according to any one of items 95 to 97, wherein the agent, antibody, or antigen is embedded in the mitochondria, embedded in the mitochondrial membrane, substantially encapsulated within the mitochondria, or completely encapsulated by the mitochondria.

[0115] 101. The composition according to any one of items 95 to 99, wherein the agent, antibody, or antigen is linked to the outer membrane of the mitochondria by a non-covalent bond such as a covalent bond or an electrostatic bond.

[0116] 102. The composition according to any one of items 82 to 101, wherein the isolated mitochondria are mitochondria modified by gene editing.

[0117] 103. The composition according to any one of items 82 to 101, wherein the isolated mitochondria contain exogenous mtDNA.

[0118] 104. (a) A step of freezing the composition at a temperature below 0 °C; (b) A step of storing the frozen composition obtained in step (a) at a temperature below 0 °C, for example, -4 °C, preferably -20 °C; A method for cryopreservation of a composition containing isolated mitochondria as described in any one of items 1 to 103, for example, isolated viable mitochondria.

[0119] 105. The method according to item 104, further comprising a further step (c) in which the frozen composition is thawed at a temperature above 0 °C after step (b).

[0120] 106. (a) The freezing is performed at a temperature below -20 °C, for example, below -50 °C, preferably below -70 °C; The method according to any one of items 104 or 105.

[0121] 107. (a) The freezing is carried out at a temperature below -100 °C, for example below -150 °C, particularly below -180 °C. The method according to any one of items 104 or 105.

[0122] 108. (a) The freezing is carried out in liquid nitrogen at a temperature of -196 °C, or in dry ice at a temperature of -78.5 °C, preferably in dry ice at -78.5 °C. The method according to any one of items 104 or 105.

[0123] 109. 5(a) The freezing is rapid freezing. The method according to any one of items 104 or 105.

[0124] 110. (a) The freezing is stepwise freezing at a rate of at least 5 °C / min, for example at least 10 °C / min, for example 20 °C / min or 30 °C / min. The method according to any one of items 104 or 105.

[0125] 111. (b) The storage has a period of at least 24 hours, for example 120 hours, before thawing, preferably (b) The storage has a period of at least one week, for example four weeks. The method according to any one of items 104 to 110.

[0126] 112. (c) The thawing is carried out at a temperature higher than 4 °C and lower than 40 °C, for example at a temperature of 20 °C to 38 °C. The method according to any one of items 104 to 111.

[0127] A composition comprising an isolated mitochondrion as described in any one of items 1 to 112, for example, an isolated viable mitochondrion, or a composition prepared by the method as described in any one of items 104 to 112, in a therapeutically effective amount for treating a disease in a subject in need of treatment by therapeutic mitochondrial transplantation (TMT) or the like.

[0128] The composition according to item 113, for use in treating a mitochondrion or a mitochondrion-related disease in a subject in need of treatment of a mitochondrion or a mitochondrion-related disease.

[0129] The composition according to item 113, for use in treating cancer or a tumor in a subject in need of treatment of cancer or a tumor.

[0130] The composition according to item 113, for use in treating an autoimmune disease.

[0131] The composition according to item 113, for use in treating ischemia-related injuries such as lung, kidney, heart, or brain ischemia-reperfusion injury.

[0132] The composition according to item 114, for use in removing vascular occlusions such as thrombus.

[0133] The composition according to item 114, for use in gene therapy, particularly for gene therapy for treating cancer, infectious diseases, or autoimmune diseases.

[0134] The composition according to item 114, for use in transplanting mitochondria, for example, isolated viable mitochondria, into immune cells for producing mitochondrion-enhanced immune effector cells, such as but not limited to, chimeric antigen receptor (CAR) T cells, CAR-NK cells, CAR-macrophages, neutrophils, tumor-infiltrating lymphocytes (TIL), gamma delta T cells.

[0135] 123. The composition according to item 114 for use in the treatment of a disease in a subject in need thereof, wherein the composition should be administered to the subject in need thereof by topical or parenteral administration.

[0136] 124. The composition according to item 114 for use in the treatment of a disease in a subject in need thereof, wherein the composition should be administered to the subject in need thereof in the form of an aerosol.

[0137] 125. The composition according to item 114 for use in the treatment of a disease in a subject in need thereof, wherein the composition should be administered to the subject in need thereof by direct injection into a blood vessel, tissue, or organ.

[0138] 126. The composition according to any one of items 1 to 125 for use in the treatment of a disease in a subject in need thereof, wherein the composition should be delivered to target cells in vitro in a subject or donor in need thereof.

[0139] 127. The composition according to any one of items 1 to 126, wherein the viable mitochondria comprised in the composition are autologous, allogeneic, or xenogeneic.

[0140] 128. The composition according to any one of items 114 to 127, wherein the composition has undergone a freeze-thaw cycle such as a freeze-thaw cycle according to the method described in any one of items 106 to 114 before being used for the treatment of a disease.

[0141] 129. Use of the composition according to any one of items 1 to 128 for the cryopreservation of viable mitochondria, such as isolated viable mitochondria.

[0142] 130. Use of the composition prepared by the method according to any one of items 105 to 113 for the cryopreservation of viable mitochondria such as isolated viable mitochondria.

[0143] The present invention provides novel and efficient compositions and methods for cryopreserving isolated mitochondria, such as isolated viable mitochondria. The present invention further provides cryopreserved compositions comprising isolated mitochondria, such as isolated viable mitochondria. The cryopreserved mitochondria of the present invention can be utilized in different types of therapies, such as cell therapy and gene therapy after thawing. These therapies require consistent and reproducible results with high mitochondrial viability and functionality for therapeutic efficacy. Mitochondrial damage during cryopreservation and thawing can render the therapy ineffective. Mitochondria are particularly sensitive to the stress of freezing and thawing, and the lack of optimized cryopreservation methods has hindered research and medical applications. Today, there is a strong need for cell and gene therapy as well as therapeutic mitochondrial transplantation (TMT), which may rely on cryopreservation methods and compositions to safely and efficiently preserve mitochondria during the manufacturing process, storage, and transportation. To date, in basic research and medical applications (e.g., in therapy, diagnosis, and imaging), there is still a strong desire to maintain their integrity and viability during the freezing process before use, storage at low temperature, and thawing process even after making isolated mitochondria immediately available for use. Therefore, the technical problem underlying the present invention is to provide novel and innovative methods and compositions suitable for cryopreserving isolated viable mitochondria that enable the maintenance of their structural integrity and / or functional properties. The present invention provides a cryopreserved composition comprising isolated viable mitochondria that are viable, respiration competent, and / or remain intact during storage at low temperature and during freeze-thaw cycles, preferably mitochondria that are viable, respiration competent, and remain intact. The cryopreserved mitochondria of the present invention exhibit structural and functional properties comparable to those of freshly isolated mitochondria and advantageously exhibit a long shelf life. The compositions of the present invention are non-toxic, safe, and physiologically compatible. Therefore, they can be used in vivo.

Prior Art Documents

Patent Documents

[0144] [Patent Document 1] US Patent Application Publication No. 2019 / 0134088 [Patent Document 2] International Publication No. 2021 / 168764 [Non-Patent Document]

[0145] [Non-Patent Document 1] Cell Biology of the Mitochondrion Genetics (Nov 2017), 207(3):843 - 871, Van der Bliek A.M., Sedensky M.M., Morgan P.G.; Erratum in: Genetics (Apr 2018), 208(4):1673 [Non-Patent Document 2] Mitochondria: in sickness and in health Cell (16 Mar 2012), 148(6):1145 - 59, Nunnari J, Suomalainen A. [Non-Patent Document 3] The Origin of Mitochondria. Nature Education (2010), 3(9):58, Martin W. & Mentel M. [Non-Patent Document 4] Mol Syndromol (2016), 7:122 - 137, Nyazov D.M. et al. [Non-Patent Document 5] Annual Review of Pharmacology and Toxicology, Vol. 57, (2017), Lesnefsky, pp 535 - 565; A Cell Metabolism Review, (2015), Unifying Mechanism for Mitochondrial Superoxide Production during Ischemia - Reperfusion Injury, Chouchan E.T.

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Summary of the Invention

[0146] The present invention relates to a composition comprising: (i) an aqueous buffer having a pH of 5.5 to 8.5; (ii) trehalose at a concentration of at least 150 mM; (iii) a first cryoprotectant selected from one or more (a) amino acids at a total concentration of at least 150 mM; and optionally, (iv) a second cryoprotectant selected from one or more (b) sugars, (c) polymers, or combinations thereof, wherein (b) the sugar has a total concentration of at least 150 mM and (c) the polymer has a total concentration of 2.5% (w / v) to 30% (w / v). In the composition of the present invention, (iii) the first cryoprotectant may be an (a) amino acid such as proline, for example L-proline, at a concentration of at least 150 mM, such as at least 200 mM, preferably at least 300 mM; (iv) the second cryoprotectant may be selected from one or more (b) sugars, (c) polymers, or combinations thereof, (b) the sugar may be glucose, sucrose, or a combination thereof, and may have a total concentration of at least 150 mM, such as at least 200 mM, more specifically at least 300 mM, for example at least 500 mM; (c) the polymer may be polyethylene glycol (PEG) at a concentration of 2.5% (w / v) to 30% (w / v), such as 5% (w / v) to 25% (w / v), more specifically 10% (w / v) to 20% (w / v), for example 15% (w / v).Also, (iii) the first cryoprotectant can be one or more (a) amino acids at a concentration of at least 150 mM, such as at least 200 mM, preferably at least 300 mM, selected from methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or combinations thereof; (iv) the second cryoprotectant can be selected from one or more (b) sugars, (c) polymers, or combinations thereof, where (b) the sugar can be glucose, sucrose, or combinations thereof and can have a total concentration of at least 150 mM, such as at least 200 mM, more specifically at least 300 mM, for example at least 500 mM, and (c) the polymer can be polyethylene glycol (PEG) at a concentration of 2.5% (w / v) to 30% (w / v), such as 5% (w / v) to 25% (w / v), more specifically 10% (w / v) to 20% (w / v), for example 15% (w / v).

[0147] The present invention in a further embodiment relates to a composition comprising: (i) an aqueous buffer having a pH of 5.5 to 8.5; (ii) trehalose at a concentration of at least 150 mM; (iii) a first cryoprotectant selected from one or more (b) sugars at a total concentration of at least 160 mM; and optionally (iv) a second cryoprotectant selected from (a) amino acids, (c) polymers, or combinations thereof, wherein the (a) amino acids have a total concentration of at least 150 mM and the (c) polymers have a total concentration of 2.5% (w / v) to 30% (w / v). In the composition (iii), the first cryoprotectant can be one or more sugars that are glucose, sucrose, or a combination thereof at a total concentration of at least 160 mM, such as at least 200 mM, more specifically at least 300 mM, for example at least 500 mM. The (iv) second cryoprotectant can be selected from (a) amino acids, (c) polymers, or combinations thereof. One or more (a) amino acids can be selected from the amino acids described in any one of items 33 to 36 at a concentration of at least 150 mM, such as at least 200 mM, preferably at least 300 mM. The (b) polymer can be polyethylene glycol (PEG) at a concentration of 2.5% (w / v) to 30% (w / v), such as 5% (w / v) to 25% (w / v), more specifically 10% (w / v) to 20% (w / v), for example 15% (w / v). Also, the (a) amino acid can be proline, such as L-proline.

[0148] The present invention in a further embodiment relates to a composition comprising: (i) an aqueous buffer having a pH of 5.5 to 8.5; (ii) trehalose at a concentration of at least 150 mM; and (iii) a cryoprotectant selected from one or more (c) polymers, wherein the total concentration of the polymer is 16% (w / v) to 30% (w / v). The (iii) cryoprotectant can be a (c) polymer that is polyethylene glycol (PEG) at a concentration of 16% (w / v) to 30% (w / v), such as 20% (w / v) to 30% (w / v), for example 25% (w / v).

[0149] The present invention relates to a cryopreservation composition comprising: (i) an aqueous buffer having a pH of 5.5 to 8.5; (ii) trehalose at a concentration of at least 150 mM; and (iii) a cryoprotectant selected from the group consisting of (a) an amino acid, (b) a sugar, (c) a polymer, or a combination thereof, wherein the amino acid has a concentration of at least 150 mM, the sugar has a concentration of at least 150 mM, such as at least 160 mM, and the polymer has a concentration of 2.5% (w / v) to 30% (w / v), such as 16% (w / v) to 30% (w / v).

[0150] The present invention provides a cryopreservation composition comprising: (i) an aqueous buffer having a pH of 5.5 to 8.5; (ii) trehalose having a concentration of at least 150 mM; and (iii) a cryoprotectant selected from the group consisting of (a) an amino acid, (b) a sugar, (c) a polymer, or a combination thereof, wherein the amino acid is proline, such as L-proline, having a concentration of at least 150 mM, the sugar is glucose, sucrose, or a combination thereof, having a total concentration of at least 150 mM, such as at least 160 mM, and the polymer is polyethylene glycol (PEG) having a concentration of 2.5% (w / v) to 30% (w / v), such as 16% (w / v) to 30% (w / v).

[0151] The cryopreservation composition of the present invention further comprises a therapeutically effective amount of isolated mitochondria, such as isolated viable mitochondria, for use in treating a subject in need thereof in a condition that would benefit from an increase in mitochondrial function.

[0152] The present invention further provides a method suitable for cryopreserving a composition comprising isolated mitochondria, such as isolated viable mitochondria, the method comprising: (a) freezing the composition at a temperature below 0°C; and (b) storing the frozen composition obtained in step (a) at a temperature below 0°C.

[0153] Within the present invention, the mitochondria can also be subjected to lyophilization and / or spray freezing, such as spray freeze-drying or spray freeze lyophilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0154]

Fig. 1A-D

Fig. 1E-G

Fig. 2A-C

Fig. 2D-G

Fig. 3A

Fig. 3B

Fig. 3C

Fig. 4

Fig. 5

Fig. 6

Mode for Carrying Out the Invention

[0155] The present invention relates to a cryopreservation composition and method suitable for cryopreserving isolated viable mitochondria. In one aspect, the cryopreservation composition of the present invention further comprises isolated viable mitochondria such as human mitochondria. According to the present invention, the cryopreservation composition and the mitochondria contained therein are first frozen and then stored at a low temperature, for example, cryopreserved for several weeks, several months or several years, and after thawing, for example, in the treatment of mitochondrial or mitochondrial-related diseases, ischemia or ischemia-reperfusion-related disorders / injuries, for example, damaged cells or tissues (Methods Mol Biol (2021), 2277:15-37, Weissig V., Edeas M.Editors; J.Thorac.Cardiovasc.Surg.(2017), 154, 286-289, Autologous mitochondrial transplantation for dysfunction after ischemia-reperfusion injury, Emani, S.M., Del Nido, P.J., McCully J.D.et al.; Biochimica et Biophysica Acta (BBA) - Bioenergetics Vol. 1847, Issue 11, (Nov 2015), pp. 1387-1400, R.K.Lane et al.), infectious diseases, inflammatory or autoimmune diseases, by promoting the repair process, in coronary artery intervention (International Publication No. 2017 / 124037, McCully et al.), or are ready to be used for diagnostic or research purposes. The composition of the present invention is also for use in gene therapy. Examples of gene therapy for treating cancer, infectious diseases or autoimmune diseases include, but are not limited to, mitochondrial transplantation into immune cells to produce mitochondrial-enhanced immune effector cells such as chimeric antigen receptor (CAR) T cells, CAR-NK cells, CAR-macrophages, neutrophils, tumor-infiltrating lymphocytes (TIL), gamma-delta T cells, particularly mitochondrial-enhanced chimeric antigen receptor (CAR) T cells or mitochondrial-enhanced tumor-infiltrating lymphocytes (TIL) (International Publication No. 2021203046, Schueller A.et al.).

[0156] The compositions and methods of the present invention provide isolated mitochondria, e.g., isolated viable respiration-competent mitochondria, which can be successfully cryopreserved, stored, transported, and then recovered for later use. The cryopreserved compositions of the present invention have a shelf life of at least 3 months, e.g., at least 6 months, e.g., 12 months or 24 months.

[0157] The cryopreserved isolated mitochondria contained in the compositions of the present invention, e.g., cryopreserved isolated viable mitochondria, are immediately available for use and have advantages with respect to ease of application and effectiveness in research and clinical settings. Indeed, the cryopreserved compositions of the present invention and the isolated mitochondria contained therein, e.g., isolated viable mitochondria, are readily available for use in a variety of biomedical techniques aimed at improving or rescuing mitochondrial function in cells and tissues, particularly damaged cells and tissues.

[0158] Composition Embodiment A1: Cryopreserved Composition In one aspect, the present invention comprises (i) an aqueous buffer having a pH of 5.5 to 8.5, and (ii) trehalose at a concentration of at least 150 mM, and (iii) a cryoprotectant selected from the group consisting of amino acids, sugars, polymers, or combinations thereof, wherein the amino acids have a concentration of at least 150 mM (i.e., the total concentration of amino acids in the composition), the sugars have a concentration of at least 150 mM, e.g., at least 160 mM (i.e., the total concentration of sugars in the composition), and the polymers have a concentration of 2.5% (w / v) to 30% (w / v), e.g., 16% (w / v) to 30% (w / v) (in the composition). The composition is preferably a cryopreserved composition. The composition is preferably for cryopreserving mitochondria as defined herein (suitable). Preferably, the mitochondria are viable mitochondria and / or isolated mitochondria.

[0159] Exemplary cryoprotectants included alone or in combination in the compositions of the above embodiments are listed in Table 1a below. [Table 1a]

[0160] The weight / volume or volume / volume percentage (%(w / v) or %(v / v)), and molar concentration (M) of each cryoprotectant in Table 1a above or each cryoprotectant in Tables 1c and 1b below are all provided for aqueous solutions or suspensions, i.e., solutions or suspensions in which the solvent is water.

[0161] The cryoprotectant is added (i) to the aqueous buffer contained in the cryopreservation composition in an amount such that the desired concentration of the cryoprotectant in the final total volume of the composition is obtained. For example, the cryoprotectant CRYO25, which is a 70% w / v solution of D-(+)-sucrose, contains 700 g of sucrose / 1 liter of water. Since the molecular weight of sucrose is equal to 342.3 g / mol, the 70% w / v solution of D-(+)-sucrose contains sucrose at a concentration measured at approximately 2.0 M. This aqueous solution of 2.0 M sucrose is added to the composition, for example, in an amount such that a concentration of 150 mM is obtained in the final total volume of the composition. For example, the cryoprotectant CRYO33, which is a 70% w / v solution of D-(+)-glucose monohydrate, contains 700 g of glucose monohydrate / 1 liter of water. Since the molecular weight of glucose monohydrate is equal to 198.17 g / mol, the 70% w / v solution of D-(+)-glucose monohydrate contains glucose at a concentration measured at approximately 3.5 M. This aqueous solution of 3.5 M glucose is added to the composition, for example, in an amount such that a concentration of 150 mM is obtained. For example, a composition (e.g., a suspension) containing 150 mM of CRYO6, i.e., 150 mM of L-proline, was prepared by adding 10 μL of a 6 M proline solution (i.e., CRYO6) to a final volume of 400 μL of a composition containing 300 mM of trehalose, 1 mM of EGTA, and 10 mM of HEPES (pH 7.2).

[0162] Exemplary cryoprotectants that can be included in the above composition, either alone or in combination thereof, are shown below: [Table 1c]

[0163] Examples of commercially available cryoprotectants are shown in Table 1c above.

[0164] The cryoprotectants described in Table 1c above are used to prepare the composition by diluting the solution at 5%, 10%, and 20% (v / v) in mitochondrial "trehalose-based" isolation buffer (1) (see CRYO1-CRYO47 in FIGS. 3A and 3B and Table 1b), 5%, 10%, 15%, 20%, 25%, and 30% (v / v) (see FIG. 3C for CRYO6, CRYO12, CRYO15, CRYO22, CRYO25, and CRYO33), and 10%, 20%, and 30% (v / v) (FIGS. 4A-D for CRYO6+CRYO12, CRYO6+CRYO25, CYRO6+CRYO33, and CRYO33+CRYO25).

[0165] Preferred cryoprotectants are CRYO13, CRYO14, CRYO15, CRYO16, CRYO17, CRYO18, CRYO22, CRYO23, CRYO25, CRYO38, CRYO46, and / or CRYO47. More preferably, the composition comprises a cryoprotectant selected from the group consisting of CRYO6, CRYO12, CRYO25, and / or CRYO33.

[0166] In one aspect, the aqueous buffer contained in the composition of the above embodiments has a pH of 5.8 to 8.5, such as 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 or 7.5. In certain embodiments, the aqueous buffer has a pH of 6.5 to 8.5, such as 6.7, 6.9, 7.1, 7.3, 7.7 or 7.9. In certain preferred embodiments, the aqueous buffer has a pH of 6.8 to 8.2, such as 7.0, 7.2, 7.4, 7.6, 7.8 or 8.0, such as pH 7.2. The compositions provided herein are preferably aqueous compositions. Preferably, the (aqueous) composition has the same pH as the aqueous buffer contained therein. The definitions and explanations regarding the pH provided herein for the aqueous buffer are applied to the (aqueous) composition with necessary modifications. Preferably, deionized water and / or water free of RNase / DNase is used to prepare the aqueous composition and / or the aqueous buffer. Further, in one aspect, the (sole) aqueous component of the (aqueous) composition is the aqueous buffer described herein.

[0167] Amino acids, sugars and / or polymers may be (directly) dissolved in the aqueous buffer. Alternatively, amino acids, sugars and / or polymers may first be dissolved in a non-aqueous solution and subsequently added to the aqueous buffer or the aqueous composition.

[0168] In one aspect, the aqueous buffer contained in any one of the compositions of the above embodiments contains a buffer, preferably a pH buffer. The buffer is selected from the group of agents including, but not limited to, 2-[4-(2-hydroxyethyl)-piperazin-1-yl]-ethanesulfonic acid (HEPES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 4-morpholineethanesulfonic acid (MES), bis-(2-hydroxyethyl)amino-tris-(hydroxymethyl)-methane (bis-tris), 2-(N-cyclohexylamino)-ethanesulfonic acid (CHES), N,N-bis-(2-hydroxyethyl)-glycine (bicine), potassium phosphate, sodium cacodylate, tris-(hydroxymethyl)aminomethane hydrochloride (tris), 4-morpholinepropanesulfonic acid (MOPS), 1,3-bis-[tris-(hydroxymethyl)-methylamino]-propane (bis-trispropane), sodium acetate, or combinations thereof. In certain embodiments, the pH buffer has a concentration of 0.5 mM to 50 mM, such as 1 mM to 40 mM, preferably 2 mM to 35 mM, such as 5 mM to 30 mM. In certain embodiments, the pH buffer has a concentration of 10 mM to 35 mM, such as 15 mM, 20 mM, 25 mM, or 30 mM. In certain preferred embodiments, the aqueous buffer contains HEPES, such as 10 mM HEPES (adjusted to pH 7.2 with KOH).

[0169] In another aspect, any one of the compositions of the above embodiments further comprises a calcium chelating agent. In certain embodiments, the calcium chelating agent is ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 2,2’,2’’,2’’’-(ethane-1,2-diyldinitrilo)-tetraacetic acid (EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis-(acetoxymethyl ester) (BAPTA-AM) or a combination thereof. In certain preferred embodiments, the calcium chelating agent is EDTA or EGTA. In certain embodiments, the calcium chelating agent has a concentration of 0.1 mM to 10 mM, such as 0.5 mM to 5 mM, such as 1 mM or 1.5 mM. In a preferred embodiment, the calcium chelating agent is EGTA at a concentration of 0.5 mM to 2 mM, such as 1 mM. In another preferred embodiment, the calcium chelating agent is EDTA at a concentration of 0.5 mM to 2 mM, such as 1 mM.

[0170] In another aspect, any one of the compositions of the above embodiments further comprises an ionic component. In certain embodiments, the ionic component is Mg 2+ , Na + , K + , Cl - , HCO3 -It includes salts, acids, bases, etc. that provide ions such as those, or combinations thereof. In a further embodiment, the ionic component is, for example, an appropriate salt including, but not limited to, MgCl2, MgSO4, KCl, KH2PO4, NaHCO3, Na2HPO4, C2H2MgO4 (magnesium formate), C3H3NaO3 (sodium pyruvate), C2H3NaO2 (sodium acetate), etc., or combinations thereof. It includes, but is not limited to, MgSO4, KCl, KH2PO4, NaHCO3, Na2HPO4, C2H2MgO4 (magnesium formate), C3H3NaO3 (sodium pyruvate), C2H3NaO2 (sodium acetate), etc., or combinations thereof. Alternatively, the ionic component is an organic anion derived from an organic acid. In a specific embodiment, the organic anions include, but are not limited to, citrate anion, pyruvate anion, malate anion, oxaloacetate anion, formate anion, glutamate anion, α-ketoglutarate anion, succinate anion, and acetate anion. In a specific embodiment, the ionic component is selected from the group consisting of lithium acetate dihydrate, lithium chloride, lithium formate monohydrate, lithium nitrate, lithium sulfate monohydrate, sodium malonate pH 7.0, magnesium acetate tetrahydrate, sodium chloride, sodium formate, sodium nitrate, and sodium sulfate decahydrate. In a specific embodiment, the ionic component is in the range of 0.01% (w / v) to 10% (w / v), for example 0.1% (w / v) to 10% (w / v), for example 0.5% (w / v), 1.0% (w / v), 1.5% (w / v), 2.5% (w / v), 3% (w / v), or 5% (w / v). In a specific embodiment, the ionic component is in the range of 0.5% (w / v) to 9% (w / v), 1% (w / v) to 8% (w / v), 2% (w / v) to 6% (w / v), for example 2.5% (w / v), 3% (w / v), or 5% (w / v). In a further embodiment, the ionic component has a concentration (for example, a concentration expressed in molar concentration) in the range of, for example, 0.01 mM to 200 mM, for example 0.1 mM to 180 mM, 0.5 mM to 150 mM, 1 mM to 120 mM, 1 mM to 100 mM, for example 3 mM, 5 mM, 10 mM, 20 mM, 40 mM, 50 mM, 70 mM, or 90 mM.In certain preferred embodiments, the ionic component has a concentration of 1 mM to 30 mM, such as 15 mM. In other preferred embodiments, the ionic component has a concentration of 2 to 20 mM, such as 4 mM, 6 mM, 8 mM, 12 mM, 14 mM, 16 mM, or 18 mM.

[0171] In another aspect, the composition of any one of the above embodiments further comprises albumin. In certain embodiments, the albumin is bovine serum albumin (BSA), human serum albumin (HSA), etc., or a combination thereof. In one embodiment, the albumin is BSA. In another embodiment, the albumin is HSA. In certain embodiments, the albumin has a concentration of 0.01% (w / v) to 10% (w / v), such as 0.05% (w / v) to 5% (w / v), 0.1% (w / v) to 3% (w / v), 0.3% (w / v) to 2% (w / v), 0.5% (w / v) to 1.5% (w / v) or 0.8% (w / v) to 1% (w / v). In a preferred embodiment, the albumin has a concentration of 0.1% (w / v), such as 0.1% (w / v) of BSA.

[0172] Serum albumin mainly regulates the osmotic pressure or tumor pressure. Furthermore, it contributes to the saturation of proteases and the binding of fatty acids.

[0173] In another aspect, the composition of any one of the above embodiments comprises 20 mM Tris (adjusted to pH 7.4 with HCl), 2 mM EDTA and 10 mM MgCl2. In another embodiment, the composition of any one of the above embodiments comprises 5 mM MOPS (adjusted to pH 7.25 with KOH), 10 mM B APTA and 5 mM sodium pyruvate. In still further embodiments, the composition of any one of the above embodiments comprises 10 mM HEPES (adjusted to pH 7.2 with KOH) and 1 mM EGTA.

[0174] In another aspect, any one of the compositions of the above embodiments contains a cryoprotectant in an amount less than the amount for cryopreservation or does not contain a cryoprotectant. The cryoprotectant is selected from the group consisting of, for example, propylene glycol, ethylene glycol, glycerol, and dimethyl sulfoxide (DMSO). In a specific embodiment, any one of the compositions of the above embodiments contains dimethyl sulfoxide (DMSO) in an amount less than the amount for cryopreservation or does not contain dimethyl sulfoxide (DMSO).

[0175] In one aspect, any one of the compositions of the above embodiments contains trehalose at a concentration of at least 160 mM, for example at least 170 mM, at least 180 mM, at least 190 mM, at least 200 mM, at least 210 mM, at least 220 mM, at least 230 mM, or at least 240 mM. In one embodiment, the concentration of trehalose is at least 250 mM, for example 260 mM, at least 270 mM, at least 280 mM, at least 290 mM, or at least 300 mM.

[0176] In another aspect, the composition according to any one of the above embodiments contains trehalose at a concentration of at least 150 mM and 1500 mM or less. In some specific embodiments, the composition according to any one of the above embodiments contains trehalose at a concentration of 1400 mM or less, 1300 mM or less, or 1200 mM or less. In some specific embodiments, the composition according to any one of the above embodiments contains trehalose at a concentration of at least 150 mM and 1100 mM or less, such as 1000 mM or less, 900 mM or less, 800 mM or less, or 700 mM or less. Preferably, the composition according to any one of the above embodiments contains trehalose at a concentration of at least 150 mM and 650 mM or less, such as 640 mM or less, 630 mM or less, 620 mM or less, or 610 mM or less. In another preferred embodiment, the composition according to any one of the above embodiments contains trehalose at a concentration of at least 150 mM and 600 mM or less, such as 590 mM or less, 580 mM or less, 570 mM or less, or 560 mM or less. In a more preferred embodiment, the composition according to any one of the above embodiments contains trehalose at a concentration of at least 150 mM and 550 mM or less, such as 540 mM or less, 530 mM or less, 520 mM or less, or 510 mM or less. In another more preferred embodiment, the composition according to any one of the above embodiments contains trehalose at a concentration of at least 150 mM and 500 mM or less, such as 490 mM or less, 480 mM or less, 470 mM or less, or 460 mM or less. Even more preferably, the composition according to any one of the above embodiments contains trehalose at a concentration of at least 150 mM and 450 mM or less. In some particularly preferred embodiments, the composition according to any one of the above embodiments contains trehalose at a concentration of 150 mM to 450 mM, such as 160 mM to 440 mM, 170 mM to 430 mM, 180 mM to 420 mM, or 190 mM to 410 mM. In some other particularly preferred embodiments, the composition according to any one of the above embodiments contains trehalose at a concentration of 200 mM to 400 mM, such as 220 mM to 380 mM, such as 240 mM, 260 mM, 280 mM, 320 mM, 340 mM, or 360 mM.In some other particularly more preferred embodiments, trehalose has a concentration of 250 mM to 350 mM, for example 300 mM.

[0177] In one aspect, the sugar contained in the composition of any one of the above embodiments, for example, the sugar of the cryoprotectant (iii) contained in the composition, is selected from the group consisting of any suitable monosaccharide, disaccharide, trisaccharide, oligosaccharide, polysaccharide, or a combination thereof. In a preferred aspect, the sugar contained in the composition of any one of the above embodiments, for example, the sugar of the cryoprotectant (iii) contained in the composition, is selected from the group consisting of any suitable monosaccharide, disaccharide, or trisaccharide, or a combination thereof. In one specific embodiment, the sugar is all sugars other than trehalose. In certain embodiments, the sugar is a sugar derivative, such as a sugar alcohol. Exemplary sugar alcohols used herein are sorbitol, erythritol, xylitol (e.g., D-sorbitol, meso-erythritol and / or xylitol, specifically 70% w / v D-sorbitol, 35% w / v meso-erythritol and / or 70% w / v xylitol), inositol, mannitol, arabitol, and / or ribitol. In some other embodiments, the sugar is a modified sugar.

[0178] Preferably, the term "sugar" as used herein refers to a sugar in a narrower sense, namely, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, more preferably monosaccharides and / or disaccharides, as further defined and described herein. In certain embodiments, the sugar is maltose, lactose, fructose, sucrose, glucose, dextran, melezitose, raffinose, nigerotriose, maltotriose, maltotriulose, kestose, cellobiose, chitobiose, lactulose, or a combination thereof. In one embodiment, the sugar is preferably sucrose, glucose, or a combination thereof. In one embodiment, (iii) the sugar of the cryoprotectant is not trehalose. In one aspect, the sugar contained in the composition according to any one of the above embodiments has a concentration of at least 150 mM. In a further aspect, the sugar contained in the composition according to any one of the above embodiments has a concentration of at least 160 mM. In a further aspect, the sugar contained in the composition according to any one of the above embodiments has a concentration of at least 170 mM, such as at least 180 mM, at least 190 mM, at least 200 mM, at least 210 mM, at least 220 mM, at least 230 mM, or at least 240 mM. In a further embodiment, the sugar has a concentration of at least 250 mM, such as at least 260 mM, at least 270 mM, at least 280 mM or at least 290 mM. In a further embodiment, the sugar has a concentration of at least 300 mM, such as at least 310 mM, at least 320 mM, at least 330 mM, at least 340 mM, at least 350 mM, at least 360 mM, at least 370 mM, at least 380 mM, at least 390 mM, at least 400 mM, at least 410 mM, at least 420 mM, at least 430 mM, at least 440 mM or at least 450 mM.In certain preferred embodiments, the sugar has a concentration of 300 mM to 800 mM, such as 320 mM, 350 mM, 400 mM, 410 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 480 mM, 490 mM, 500 mM, 510 mM, 520 mM, 530 mM, 540 mM, 550 mM, 560 mM, 570 mM, 580 mM, 590 mM, 600 mM, 650 mM, 700 mM, or 750 mM. The maximum concentration of the sugar (e.g., the total amount of the sugar) is 2000 mM or less, such as 1900 mM or less, or 1800 mM or less. In one embodiment, the sugar has a maximum concentration of 1700 mM or less, such as 1600 mM or less, 1500 mM or less, 1400 mM or less, 1300 mM or less, or 1200 mM or less. In some preferred embodiments, the maximum concentration of the sugar is 1500 mM or less, such as 1300 mM or less, 1000 mM or less, 950 mM or less, 900 mM or less, 850 mM or less, 800 mM or less, 750 mM or less, or 700 mM or less. The sugar is provided at a concentration within a range having endpoints defined by any of the minimum concentrations listed above and any of the maximum concentrations listed above that are higher than the minimum concentration. When two or more sugars are present in any one of the compositions of the above embodiments, the concentration of the sugar reflects the total concentration of all the sugars in the composition. In other preferred embodiments, the sugar has a concentration of 250 mM to 650 mM, such as 280 mM to 600 mM, such as 300 mM, 400 mM, or 500 mM. Thus, in some embodiments, the sugar is present at a concentration of 150 mM to 1600 mM, such as 200 mM to 1500 mM, 250 mM to 1200 mM, or 300 mM to 1100 mM. In some embodiments, the sugar has a concentration of 250 mM to 650 mM, such as 280 mM to 600 mM, such as 300 mM, 400 mM, or 500 mM. In certain embodiments, the sugar contained in any one of the compositions of the above embodiments is sucrose at a concentration of 150 mM to 700 mM, such as 160 mM to 675 mM, such as 200 mM to 650 mM, such as 250 mM to 620 mM, such as 300 mM to 600 mM, such as 400 mM or 500 mM.In other embodiments, the composition comprises glucose at a concentration of 300 mM to 1200 mM, such as 450 mM to 1100 mM, such as 500 mM to 1000 mM, such as 700 mM or 800 mM. In some specific embodiments, the composition of any one of the above embodiments comprises two or more sugars, such as fructose together with glucose or sucrose, or a combination of fructose, glucose, and sucrose. In some preferred embodiments, the composition of any one of the above embodiments comprises sucrose and glucose at a concentration (e.g., total concentration) of at least 150 mM and not more than 2000 mM. In a preferred embodiment, the sugar is sucrose, glucose, or a combination thereof, and sucrose and glucose have a total concentration of at least 160 mM, such as 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or 1500 mM. In another preferred embodiment, sucrose and glucose have a total concentration of 500 mM to 1800 mM, more preferably 600 mM to 1700 mM, such as 800 mM to 1450 mM, such as 900 mM, 1120 mM or 1320 mM. In one aspect, the sugar contained in the composition that does not contain amino acids preferably has a total concentration of at least 160 mM, such as at least 180 mM or at least 200 mM.

[0179] The sugar is provided at a concentration within a range having endpoints defined by any of the minimum concentrations listed above and any of the maximum concentrations listed above that are higher than the minimum concentration. When two or more sugars are present in the composition of any one of the above embodiments, the concentration of the sugar reflects the total concentration of all the sugars in the composition.

[0180] In one aspect, the amino acids contained in any one of the compositions of the above embodiments, for example, the amino acids of the cryoprotectant (iii) contained in the composition, are any suitable amino acids, amino acid derivatives, oligopeptides, peptides, or combinations thereof. For example, in a specific embodiment, the amino acid component is isoleucine (e.g., L-isoleucine), proline (e.g., L-proline), valine (e.g., L-valine), alanine (e.g., L-alanine), glycine, asparagine (e.g., L-asparagine), aspartic acid (e.g., L-aspartic acid), glutamic acid (e.g., L-glutamic acid), serine (e.g., L-serine), histidine (e.g., L-histidine), cysteine (e.g., L-cysteine), tryptophan (e.g., L-tryptophan), tyrosine (e.g., L-tyrosine), arginine (e.g., L-arginine), glutamine (e.g., L-glutamine), lysine, threonine, selenocysteine, methionine, phenylalanine, creatine (e.g., L-creatine), taurine (e.g., L-taurine), betaine, ectoine, dimethylglycine, ethylmethylglycine, glutathione, RGD peptide (i.e., arginyl-glycyl-aspartic acid peptide), or combinations thereof. In a specific preferred embodiment, the amino acid is proline (e.g., L-proline), glycine or cysteine (e.g., L-cysteine). In other preferred embodiments, the amino acid is proline (e.g., L-proline), a proline derivative, such as methyl proline, benzyl proline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid (e.g., homoproline), oxaproline, thiaproline, or combinations thereof. In an even more preferred embodiment, the amino acid is proline (e.g., L-proline).The amino acids contained in any one of the compositions of the above embodiments have a concentration of at least 160 mM, for example at least 180 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 450 mM, at least 500 mM, at least 550 mM, at least 600 mM, at least 650 mM, at least 700 mM, at least 750 mM, at least 800 mM, at least 850 mM, or at least 900 mM. Preferably, the amino acids have a concentration of at least 1000 mM, for example at least 1100 mM, at least 1200 mM, at least 1300 mM, at least 1400 mM, at least 1500 mM, at least 1600 mM, at least 1700 mM, at least 1800 mM, at least 1900 mM, or at least 2000 mM. The amino acid component can be provided at a maximum concentration of 3000 mM or less, for example 2900 mM or less, 2850 mM or less, 2800 mM or less, 2750 mM or less, 2700 mM or less, 2650 mM or less, 2600 mM or less, 2550 mM or less, 2500 mM or less, 2450 mM or less, 2400 mM or less, 2350 mM or less, 2300 mM or less, 2250 mM or less, 2200 mM or less, 2150 mM or less, 2100 mM or less, or 2050 mM or less. The amino acids are provided at a concentration within a range having endpoints defined by any of the above minimum concentrations and any of the above maximum concentrations higher than the minimum concentration. When two or more amino acids are present in the composition, the concentration of the amino acids reflects the total concentration of all amino acids in the composition. Thus, in some embodiments, the amino acids are present at a concentration of 500 mM to 3000 mM, for example 800 mM to 2800 mM, or 1000 mM to 2500 mM. For example, in a preferred embodiment, the amino acid is proline at a concentration of 600 mM. In other embodiments, the amino acid is proline at a concentration of 650 mM to 2000 mM, for example 900 mM to 1900 mM, for example 1500 mM or 1800 mM. In a preferred embodiment, the amino acid is proline at a concentration of 1000 mM to 1700 mM, for example 1500 mM. In another preferred embodiment, the amino acid is proline at a concentration of 1200 mM.In some other preferred embodiments, the amino acid is a proline derivative such as methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid (e.g., homoproline), oxaproline, thiaproline, etc. at a concentration of 150 mM to 3000 mM, for example 200 mM to 2800 mM, or 300 mM to 2500 mM, but is not limited thereto. In a preferred embodiment, the proline derivative is at a concentration of 500 mM to 2000 mM, for example 600 mM to 1800 mM, for example 800 mM, 1000 mM, 1300 mM, or 1500 mM.

[0181] In one aspect, the polymer contained in any one of the compositions of the above embodiments, for example, the polymer of the cryoprotectant (iii) of the composition, is a polymer having one or more of the following properties: biocompatibility, hydrophilicity or amphiphilicity. For example, the polymer component includes poloxamer (e.g., poloxamer 142, poloxamer 188, poloxamer 331, or poloxamer 407), alginate, polyethylene glycol (PEG), such as PEG400 or PEG1000, polyglutamic acid, polyvinyl alcohol, polyvinylpyrrolidone, or a combination thereof. In certain preferred embodiments, the polymer is alginate or polyethylene glycol (PEG). In a more preferred embodiment, the polymer is polyethylene glycol (PEG). The polymer contained in any one of the compositions of the above embodiments has a concentration of at least 2.5% (w / v), at least 3% (w / v), at least 4% (w / v), at least 5% (w / v), at least 6% (w / v), at least 7% (w / v), at least 8% (w / v), at least 9% (w / v), at least 10% (w / v), at least 11% (w / v), at least 12% (w / v), at least 13% (w / v), at least 14% (w / v), or at least 15% (w / v). The polymer contained in any one of the compositions of the above embodiments has a concentration of at least 16% (w / v), for example, at least 18% (w / v). The polymer is provided at a maximum concentration of 30% (w / v) or less, 25% (w / v) or less, 22% (w / v) or less, 21% (w / v) or less, 20% (w / v) or less, or 18% (w / v) or less. The polymer is provided at a concentration within a range having endpoints defined by any of the minimum concentrations listed above and any of the maximum concentrations listed above that are higher than the minimum concentration. When multiple polymers are present in the composition, the concentration of the polymer reflects the total concentration of all the polymers in the composition. Thus, in some embodiments, the polymer component is present at a concentration of 2.5% (w / v) to 30% (w / v), 2.5% (w / v) to 25% (w / v), 5% (w / v) to 25% (w / v), 10% (w / v) to 25% (w / v), 10% (w / v) to 20% (w / v), or 12% (w / v) to 18% (w / v).For example, in certain embodiments, the polymer has a concentration of 8% (w / v) to 18% (w / v). In further specific embodiments, the polymer has a concentration of 14% (w / v) to 16% (w / v). For example, in a preferred embodiment, the polymer is polyethylene glycol (PEG) at a concentration of 2.5% (w / v) to 30% (w / v), more preferably 5% (w / v) to 25% (w / v), such as 10% (w / v), 15% (w / v) or 20% (w / v). In another preferred embodiment, the polymer is polyethylene glycol (PEG) at a concentration of 16% (w / v) to 30% (w / v), more preferably 18% (w / v) to 25% (w / v), such as 20% (w / v), 21% (w / v), 23% (w / v) or 24% (w / v).

[0182] In one aspect, the polymer contained in the composition is the only cryoprotectant and has a concentration of at least 16% (w / v), such as at least 20% (w / v) or at least 25% (w / v). In a preferred embodiment, the polymer contained in a composition that does not contain amino acids and sugars is polyethylene glycol (PEG) at a concentration of 16% (w / v) to 30% (w / v), such as 18% (w / v) to 28% (w / v), such as 20% (w / v) to 25% (w / v), except for trehalose.

[0183] In one aspect, the composition of any one of the above embodiments is (i) an aqueous buffer having a pH of 5.5 to 8.5, such as a pH of 6.5 to 8.0, such as a pH of 7.2, and (ii) trehalose at a concentration of at least 150 mM, and (iii) a cryoprotectant selected from proline, sucrose, glucose, polyethylene glycol (PEG) or a combination thereof It contains proline which has a concentration of at least 150 mM, for example at least 500 mM, preferably at least 1000 mM, for example 1200 mM, and sucrose and glucose have a total concentration of at least 150 mM, for example at least 200 mM, for example 300 mM or 500 mM, and polyethylene glycol (PEG) has a concentration of 2.5% (w / v) - 30% (w / v), for example 5% (w / v) - 25% (w / v), for example 10% (w / v), 15% (w / v) or 20% (w / v).

[0184] In another aspect, a composition of any one of the above embodiments contains an aqueous buffer as described in any one of the above embodiments.

[0185] In another aspect, a composition of any one of the above embodiments further contains a calcium chelating agent as described in any one of the above embodiments.

[0186] In another aspect, a composition of any one of the above embodiments further contains an ionic component as described in any one of the above embodiments.

[0187] In another embodiment, a composition of any one of the above embodiments further contains albumin as described in any one of the above embodiments.

[0188] In another aspect, a composition of any one of the above embodiments contains 20 mM Tris (adjusted to pH 7.4 with HCl), 2 mM EDTA and 10 mM MgCl2. In another embodiment, a composition of any one of the above embodiments contains 5 mM MOPS (adjusted to pH 7.25 with KOH), 10 mM B APTA and 5 mM sodium pyruvate. In yet another embodiment, a composition of any one of the above embodiments contains 10 mM HEPES (adjusted to pH 7.2 with KOH) and 1 mM EGTA.

[0189] In another aspect, any one of the compositions of the above embodiments contains a cryoprotectant in an amount less than the amount for cryopreservation or does not contain a cryoprotectant. The permeable cryoprotectant is selected from the group consisting of, for example, propylene glycol, ethylene glycol, glycerol, and dimethyl sulfoxide (DMSO). In a specific aspect, any one of the compositions of the above embodiments contains dimethyl sulfoxide (DMSO) in an amount less than the amount for cryopreservation or does not contain dimethyl sulfoxide (DMSO).

[0190] In one aspect, a composition according to any one of the above embodiments contains trehalose at a concentration defined in any one of the above embodiments.

[0191] In one aspect, a composition according to any one of the above embodiments contains sucrose and glucose at a total concentration of at least 150 mM and not more than 2000 mM, for example, 150 mM to 1800 mM, for example, 200 mM to 1750 mM. In a specific embodiment, the composition contains sucrose and glucose at a total concentration of at least 250 mM, for example, 250 mM to 1650 mM. In a specific embodiment, sucrose and glucose have a total concentration of 500 mM to 1500 mM, for example, 600 mM to 1400 mM, for example, 800 mM to 1320 mM, for example, 900 mM, or 1120 mM.

[0192] In one aspect, the proline contained in any one of the compositions of the above embodiments has a concentration of at least 200 mM, for example, 300 mM or 400 mM. In a specific embodiment, proline has a concentration of at least 500 mM, for example, 600 mM, 700 mM, 800 mM, or 900 mM. In a more preferred embodiment, proline has a concentration of 600 mM. In other preferred embodiments, proline contained in any one of the compositions of the present invention as described above has a concentration of at least 1000 mM. In a more preferred embodiment, proline has a concentration of 1200 mM. In other preferred embodiments, proline has a concentration of at least 1300 mM, for example, at least 1500 mM, 1600 mM, 1800 mM, 1900 mM, 2000 mM, or 2200 mM.

[0193] In another aspect, the cryoprotectant of the composition according to any one of the above embodiments is preferably a combination of proline and sucrose. In a particular embodiment, the cryoprotectant is a combination of proline and sucrose, preferably the concentration of proline is at least 500 mM, such as 600 mM, and the concentration of sucrose is 150 mM to 650 mM, such as 200 mM, 300 mM, 400 mM, 500 mM, or 600 mM. In a particular embodiment, the cryoprotectant is a combination of proline and sucrose, preferably the concentration of proline is at least 1000 mM, such as 1200 mM, and the concentration of sucrose is 150 mM to 650 mM, such as 200 mM, 300 mM, 400 mM, 500 mM, or 600 mM.

[0194] In a preferred embodiment, the cryoprotectant of the composition according to any one of the above embodiments consists of proline and sucrose. Proline has a concentration of 10% volume of proline (i.e., 600 mM proline) with respect to the final total volume of the suspension (i.e., %(v CRYO / v tot )) and sucrose has a concentration of 5%, 10%, 15%, 20%, 25%, or 30% volume of sucrose with respect to the final total volume of the suspension (i.e., %(v CRYO / v tot )) i.e., sucrose has a concentration of approximately 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, or 600 mM respectively. In another preferred embodiment, the (iii) cryoprotectant of the composition according to any one of the above embodiments consists of proline and sucrose. Proline has a concentration of 20% volume of proline (i.e., 1200 mM proline) over the final total volume of the suspension (i.e., %(v CRYO / v tot )) and sucrose has a concentration of approximately 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, or 600 mM.

[0195] In another aspect, the cryoprotectant of the composition according to any one of the above embodiments is preferably a combination of proline and glucose. In a particularly preferred embodiment, the cryoprotectant is a combination of proline and glucose, where proline is at a concentration of at least 500 mM, such as 600 mM, and glucose is at a concentration of 180 mM to 1500 mM, preferably 350 to 1200 mM, such as 450 mM, 550 mM, 700 mM, 850 mM, 900 mM, 1000 mM, or 1100 mM. In another particularly preferred embodiment, the cryoprotectant is a combination of proline and glucose, where proline is at a concentration of at least 1000 mM, such as 1200 mM, and glucose is at a concentration of 180 mM to 1500 mM, preferably 350 to 1200 mM, such as 450 mM, 550 mM, 700 mM, 850 mM, 900 mM, 1000 mM, or 1100 mM.

[0196] In a preferred embodiment, the cryoprotectant of the composition according to any one of the above embodiments consists of proline and glucose, and proline is at a concentration of 10% volume of proline (i.e., 600 mM of proline) over the final total volume of the suspension (i.e., %(v CRYO / v tot )) and glucose is at a concentration of 5%, 10%, 15%, 20%, 25%, or 30% volume of glucose over the final total volume of the suspension (i.e., %(v CRYO / v tot )) i.e., glucose is at a concentration of approximately 175 mM, 350 mM, 525 mM, 700 mM, 875 mM, or 1050 mM respectively. In another preferred embodiment, the cryoprotectant of the composition according to any one of the above embodiments (iii) consists of proline and glucose, and proline is at a concentration of 20% volume of proline (i.e., 1200 mM of proline) over the final total volume of the suspension (i.e., %(v CRYO / v tot )) and glucose is at a concentration of approximately 175 mM, 350 mM, 525 mM, 700 mM, 875 mM, or 1050 mM.

[0197] Furthermore, in another aspect, the cryoprotectant of the composition according to any one of the above embodiments is a combination of proline and polyethylene glycol (PEG). In a specific embodiment, the cryoprotectant is proline at a concentration of at least 500 mM, preferably at least 1000 mM, such as 1200 mM, and is combined with polyethylene glycol (PEG) at a concentration of 2.5% (w / v) to 30% (w / v), preferably 5% (w / v) to 25% (w / v), such as 10% (w / v), 15% (w / v) or 20% (w / v).

[0198] In one aspect of the present invention, the cryoprotectant contained in the composition of any one of the above embodiments is sucrose, glucose, or a combination thereof at a concentration of at least 200 mM, such as at least 300 mM, such as 400 mM or 500 mM (i.e., the total concentration of sucrose and glucose). In a specific embodiment, sucrose and glucose have a concentration of at least 600 mM, such as 850 mM to 1550 mM, such as 900 mM to 1400, such as 1000 mM or 1350 mM (i.e., the total concentration of sucrose and glucose). In one embodiment, the cryoprotectant is sucrose alone at a concentration of at least 250 mM and 1200 mM or less, such as at least 300 mM and 650 mM or less, such as 400 mM and 500 mM. In another preferred embodiment, the cryoprotectant is glucose alone at a concentration of at least 450 mM and 1550 mM or less, such as at least 500 mM and 1400 mM or less, such as at least 700 mM to 1200 mM, such as 850 mM or 950 mM. In another preferred embodiment, the cryoprotectant contained in the composition of any one of the above embodiments is a combination of sucrose and glucose at a total concentration of 550 mM to 1850 mM, such as 800 mM to 1700 mM, such as 900 mM, 1150 mM, 1400 mM, or 1500 mM. For example, the combination of sucrose and glucose includes sucrose at a concentration of 200 mM to 650 mM, such as 400 mM or 500 mM, and glucose at a concentration of 350 mM to 1350 mM, such as 530 mM, 880 mM or 1100 mM.

[0199] In one aspect of the present invention, the cryoprotectant contained in the composition according to any one of the above embodiments is polyethylene glycol (PEG) at a concentration of 2.5% (w / v) to 25% (w / v), for example 5% (w / v) to 25% (w / v), for example at a concentration of 10% (w / v), 15% (w / v), or 20% (w / v), such as PEG400 or PEG1000.

[0200] In another aspect of the present invention, the only cryoprotectant contained in the composition according to any one of the above embodiments is preferably polyethylene glycol (PEG) at a concentration of 16% (w / v) to 30% (w / v), for example 18% (w / v) to 28% (w / v), for example at a concentration of 20% (w / v), 22% (w / v), 24% (w / v), 25% (w / v), or 26% (w / v), such as a polymer like PEG400 or PEG1000.

[0201] In one aspect of the present invention, the cryoprotectant contained in the cryopreservation composition according to any one of the above embodiments is proline at a concentration of at least 150 mM. In certain embodiments, proline has a concentration of at least 300 mM, for example at least 500 mM, for example 600 mM. In a preferred embodiment, proline has a concentration of at least 600 mM, for example 700 mM, 800 mM, or 900 mM. In another preferred embodiment, proline has a concentration of at least 1000 mM, for example 1200 mM. In other certain embodiments, proline has a concentration of at least 1300 mM, for example 1500 mM or 1800 mM. In a specific preferred embodiment, proline has a concentration of at least 600 mM and 3000 mM or less, for example at least 1000 mM and 2500 mM or less, for example 1300 mM to 2200 mM, for example 1500 mM, 1600 mM, 1700 mM, 1800 mM, 1900 mM, or 2000 mM.

[0202] In one aspect of the present invention, the cryopreservation composition is (i) an aqueous buffer having a pH of 6.0 to 8.0, for example pH 7.2, and (ii) trehalose at a concentration of 300 mM, and (iii) A cryoprotectant containing proline in combination with sucrose or glucose, comprising - Proline has a concentration of 1200 mM, - Sucrose has a concentration of 200 mM to 1700 mM, such as 300 mM to 1500 mM, such as 400 mM to 1300 mM, such as 500 mM or 600 mM, and - Glucose has a concentration of 300 mM to 1700 mM, such as 450 mM to 1500 mM, such as 600 mM to 1300 mM, such as 700 mM or 800 mM.

[0203] In one aspect of the present invention, the cryopreservation composition (i) An aqueous buffer having a pH of 6.0 to 8.0, such as 7.2, and (ii) Trehalose at a concentration of 300 mM, and (iii) A cryoprotectant containing proline in combination with polyethylene glycol (PEG), comprising - Proline has a concentration of 1200 mM, - Polyethylene glycol (PEG) has a concentration of 5% (w / v) to 20% (w / v), such as 10% (w / v) or 15% (w / v).

[0204] In one aspect of the present invention, the cryopreservation composition (i) An aqueous buffer having a pH of 6.0 to 8.0, such as pH 7.2, and (ii) Trehalose at a concentration of 300 mM, and (iii) A cryoprotectant containing proline in combination with sucrose or glucose, comprising - Proline has a concentration of 600 mM, - Sucrose has a concentration of 200 mM to 1700 mM, such as 300 mM to 1500 mM, such as 400 mM to 1300 mM, such as 500 mM or 600 mM, and - Glucose has a concentration of 300 mM to 1700 mM, such as 450 mM to 1500 mM, such as 600 mM to 1300 mM, such as 700 mM or 800 mM.

[0205] In one aspect of the present invention, the cryopreservation composition (i) an aqueous buffer having a pH of 6.0 to 8.0, such as 7.2, (ii) trehalose at a concentration of 300 mM, (iii) a cryoprotectant containing proline in combination with polyethylene glycol (PEG), and includes - Proline has a concentration of 600 mM, - Polyethylene glycol (PEG) has a concentration of 5% (w / v) to 20% (w / v), such as 10% (w / v) or 15% (w / v).

[0206] In another aspect of the present invention, the composition of any one of the above embodiments includes HEPES, such as 10 mM HEPES (adjusted to pH 6.5 to 7.5, such as pH 7.2 with KOH).

[0207] In another embodiment, the composition of any one of the above embodiments includes a calcium chelating agent, such as EGTA or MEDTA, such as 1 mM EGTA or 1 mM EDTA.

[0208] In another embodiment, the composition of any one of the above embodiments further includes ionic components such as, for example, Mg 2+ , Na + , K + , Cl - , HCO3 - , HPO4 2- , C3H3O3 - (pyruvate anion), C2H2O4 2- (formate anion), C2H3O2 - (acetate anion), or a combination thereof.

[0209] In another embodiment, any one of the compositions of the above embodiments further comprises albumin, such as BSA or HAS.

[0210] Embodiment A2: A cryopreservation composition containing isolated viable mitochondria In another aspect of the present invention, any one of the compositions of the above embodiments further comprises isolated mitochondria, such as isolated viable mitochondria. The composition can be obtained, for example, by adding isolated mitochondria, such as isolated viable mitochondria, to any one of the compositions of the above embodiments. In a preferred embodiment, the isolated mitochondria are mammalian mitochondria, such as isolated viable mammalian mitochondria. In an even more preferred embodiment, the isolated mitochondria are human mitochondria, such as isolated viable human mitochondria.

[0211] In another aspect, the composition of the above embodiments contains mitochondria, such as human mitochondria, such as isolated viable human mitochondria, and its source is an organ, tissue, blood, such as cells circulating in the blood, or cells, such as cultured cells. Exemplary cells include, but are not limited to, placental cells, muscle tissue cells, skin fibroblasts, cardiac fibroblasts, cardiomyocytes, cultured cells, HeLa cells, prostate cancer cells, cancer cell lines, such as, among others, HEPG2, A549, MCF7, or RKO, yeast, and any mixture thereof. Exemplary tissues include, but are not limited to, skeletal muscle, liver, heart, brain, kidney, placenta, lung, prostate, and adipose tissue. The source of the cells and tissues is yeast or an animal, preferably a mammal, more preferably a human.

[0212] In another aspect, any one of the compositions of the above embodiments contains isolated mitochondria, such as isolated viable mitochondria, at a concentration of at least 0.02 μg / μL, for example at least 0.05 μg / μL, at least 0.1 μg / μL, at least 0.2 μg / μL, at least 0.5 μg / μL, at least 0.75 μg / μL, at least 1 μg / μL, at least 1.5 μg / μL, or at least 2 μg / μL. In one embodiment, the isolated mitochondria, such as isolated viable mitochondria, are at a concentration of at least 5 μg / μL, for example at least 10 μg / μL. In a further embodiment, the isolated mitochondria, such as isolated viable mitochondria, contained in any one of the compositions of the above embodiments are at a concentration of at least 20 μg / μL, for example 25 μg / μL, 30 μg / μL, 35 μg / μL, 40 μg / μL, 45 μg / μL, or 50 μg / μL. The isolated mitochondria, such as isolated viable mitochondria, are provided at a maximum concentration of 100 μg / μL or less, for example 80 μg / μL or less, 70 μg / μL or less, or 60 μg / μL or less. In one embodiment, the isolated mitochondria, such as isolated viable mitochondria, are contained in the composition at a concentration of 0.02 μg / μL to 10 μg / μL, for example 0.05 μg / μL to 8 μg / μL, for example 0.1 μg / μL to 5 μg / μL. In a further embodiment, the isolated mitochondria, such as isolated viable mitochondria, are contained in the composition at a concentration of 0.1 μg / μL to 1 μg / μL, for example 0.2 μg / μL to 0.75 μg / μL.

[0213] In one aspect, a composition of any one of the above embodiments includes isolated mitochondria, such as isolated viable mitochondria, linked to a pharmaceutical agent, a diagnostic agent, an imaging agent, a therapeutic agent, or any other biocompatible agent. In some further embodiments, the mitochondria are linked to an antibody or antigen, such as an antibody or antigen-binding fragment. In another embodiment, the mitochondria are linked to a nucleic acid such as DNA or RNA. Other exemplary nucleic acids include, but are not limited to, double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, or triple-stranded nucleic acid molecules. In certain cases, the nucleic acid polymers are DNA, interfering RNA (siRNA), and microRNA. In one particular embodiment, the agent and the mitochondria are in physical contact with each other, e.g., the agent is linked to the mitochondria, such as by a covalent bond, embedded in the mitochondria, added to the mitochondria, embedded in the mitochondrial membrane, substantially encapsulated within the mitochondria, or completely encapsulated by the mitochondria. In another particular embodiment, the agent and the mitochondria are electrostatically linked. In another particular embodiment, the agent is covalently, e.g., chemically, linked to the outer membrane of the mitochondria or non-covalently, e.g., electrostatically, linked. The mitochondria linked to the agents described herein are referred to as "complex mitochondrial agents."

[0214] In one aspect, the compositions of the above embodiments include, but are not limited to, inorganic or organic compounds, small molecules (less than 500 Daltons) or macromolecules; proteinaceous molecules such as peptides, polypeptides, proteins, post-translationally modified proteins, or antibodies; or nucleic acid molecules such as double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, or triple-stranded nucleic acid molecules, and are linked to a therapeutic agent selected from the group consisting of mitochondria, such as isolated viable mitochondria. In some embodiments, the therapeutic agent is a natural product derived from a library of any known organism (e.g., from animals, plants, bacteria, fungi, protists, or viruses) or synthetic molecules. In some embodiments, the therapeutic agent is a monomeric compound or a polymeric compound. Some exemplary therapeutic agents include cytotoxic agents, DNA vectors, small interfering RNAs (siRNAs), microRNAs (miRNAs), reactive peptides, nanoparticles, microspheres, and fluorescent molecules. In another aspect, the composition of any one of the above embodiments includes mitochondria, and the mitochondria are linked to an imaging agent that is a radioactive substance, a fluorescent substance, or any substance detectable by any imaging technique such as, but not limited to, X-ray, magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), micro-computed tomography (μCT), PET / CT, PET / MRI, fluorescence molecular tomography (FMT), FMT / CT, scintigraphy, or ultrasound. Exemplary contrast agents include, but are not limited to, Mito Tracker fluorophores (Thermo Fisher Scientific Inc.), CellLight® RFP, BacMam 2.0 (Thermo Fisher Scientific Inc.), pH-sensitive pHrodo fluorescent dyes (Thermo Fisher Scientific Inc.). In another aspect, the composition of any one of the above embodiments includes mitochondria linked to a diagnostic agent designed to determine, for example, intracellular conditions such as intracellular pH and oxidative stress, such as a fluorescent dye or a molecule containing a radioisotope of an element.

[0215] In one aspect, any one of the compositions of the above embodiments comprises isolated mitochondria modified by gene editing, such as isolated viable mitochondria. Non-limiting examples of gene editing techniques for modifying mitochondrial DNA (mtDNA) are, for example, gene editing techniques by restriction endonuclease (RE) technology, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, CRISPR system, and pAgo-based system. Alternatively, any one of the compositions of the above embodiments comprises mitochondria containing exogenous mtDNA, such as mitochondria modified by transplanting exogenous mtDNA into autologous mitochondria (Trends Genet. (Feb 2018), 34(2):101-110, Payam A, Gammage et al.).

[0216] In another aspect, the viable mitochondria contained in the composition according to any one of the above embodiments are isolated according to any isolation method known in the art. In a particular embodiment, the viable mitochondria are isolated by density gradient centrifugation or ultracentrifugation, differential centrifugation, cell-specific mitochondrial affinity purification (CS-MAP) (STAR Protocols, Vol. 2, Issue 4, (17 Dec 2021), 100952, A. Ahier, T. Onraet, S. Zuryn), magnetic bead affinity purification, such as irreversible binding to antibody-coated magnetic beads (Hornig-Do, H. T. et al., (2009), Isolation of functional pure mitochondria by superparamagnetic microbeads. Anal. Biochem. 389:1-5), or differential filtration (e.g., as described in Preble et al., JOVE (2014): “Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration” and International Publication No. 2015 / 192020, McCully et al.). Preferably, the mitochondria, such as viable mitochondria, are separated by differential filtration operation in a filtration device including, for example, two or more filters, such as operation in a cylindrical filtration device including three filters. The filter at the upper end of the device has pores with a pore diameter of 30 μm to 50 μm, the filter at the lower end of the device has pores with a pore diameter of 5 μm to 20 μm, and the filter disposed between the upper filter and the bottom filter has pores with a pore diameter of 15 μm to 50 μm. In a particular embodiment, the mitochondria contained in the composition according to any one of the above embodiments are newly isolated from cells or tissues before incorporation (i.e., suspension) into the composition of the present invention.

[0217] Method Embodiment B: Method for Cryopreserving Mitochondria The isolated mitochondria of the present invention, such as isolated viable mitochondria, are subjected to freeze-thaw cycles. The freeze-thaw cycle includes freezing the isolated mitochondria in any one of the above-described cryopreservation compositions (i.e., contained therein). Without wishing to be bound by any theory or mechanism, the possibility of freezing mitochondria contained in any one of the compositions disclosed herein and the possibility of thawing the mitochondria without impairing their functionality and structural integrity during the freeze-thaw cycle enable easy storage and use of the mitochondria with reproducible results even after long-term storage.

[0218] In one aspect, the present invention provides a method for cryopreserving a composition comprising isolated mitochondria, such as isolated viable mitochondria, according to any one of the above embodiments, the method comprising: (a) freezing the composition at a temperature below 0 °C; (b) storing the frozen composition obtained in step (a) at a temperature below 0 °C; and comprising.

[0219] In another aspect, the method according to any one of the above embodiments further comprises a further step (c) after step (b), which consists of thawing the frozen composition at a temperature above 0 °C.

[0220] In another aspect, the present invention provides a method for cryopreserving a composition, (a.1) adding isolated mitochondria, such as isolated viable mitochondria, to the cryopreservation composition according to any one of the above embodiments; (a.2) freezing the composition at a temperature below 0 °C; (b) storing the frozen composition obtained in step (a.2) at a temperature below 0 °C; (c) optionally, thawing the frozen composition at a temperature above 0 °C; and comprising.

[0221] In another aspect, the freezing step (a) or (a.2) of the method according to any one of the above embodiments is completed in less than 10 hours, for example, 8, 6, 4, 5, or 3 hours. In one embodiment, the freezing step (a) or (a.2) is completed in less than 2.5 hours, for example, 2 hours or 1.5 hours. In another embodiment, the freezing step (a) or (a.2) of the method according to any one of the above embodiments is completed in less than 60 minutes, for example, less than 50, 45, or 35 minutes. In a preferred embodiment, the freezing step (a) or (a.2) is completed in less than 30 minutes, for example, 25, 20, or 15 minutes. In another preferred embodiment, the freezing step (a) or (a.2) of the method according to any one of the above embodiments is completed in less than 10 minutes, more preferably less than 5 minutes, for example, 3 or 2 minutes.

[0222] In another aspect, the freezing step (a) or (a.2) of the method according to any one of the above embodiments is carried out at a temperature of at least -200°C (i.e., at a temperature of -200°C or less). In some specific embodiments, the freezing step (a) or (a.2) of the method according to any one of the above embodiments is carried out at a temperature of at least -170°C (i.e., lower than -170°C), for example, in liquid nitrogen (for example, in liquid nitrogen at a temperature of about -196°C). In some embodiments, the freezing step (a) or (a.2) is carried out at a temperature of at least -90°C or -100°C, etc., at least -80°C (i.e., lower than -80°C). In some specific embodiments, the freezing step (a) or (a.2) of the method according to any one of the above embodiments is carried out at a temperature of at least -70°C (i.e., lower than -70°C), for example, at the temperature of dry ice (for example, freezing is carried out in dry ice at a temperature of about -78.5°C). In some embodiments, the freezing step (a) or (a.2) is carried out at a temperature of at least -4°C (i.e., lower than -4°C). In a specific embodiment, the freezing step (a) of the method according to any one of the above embodiments is carried out at a temperature of at least 0°C (for example, 0°C or lower). Preferably, the freezing step (a) or (a.2) of the method according to any one of the above embodiments is carried out in dry ice or in liquid nitrogen, more preferably in dry ice.

[0223] In another aspect, the freezing of an isolated mitochondria, such as an isolated viable mitochondria, contained in a composition implemented in step (a) or (a.2) by any one of the methods of the above embodiments is rapid freezing. An example of rapid freezing may be spray freezing as described herein. In another embodiment, the freezing in step (a) or (a.2) of any one of the methods of the above embodiments is stepwise. In another embodiment, the freezing in step (a) or (a.2) is gentle at a rate of at least 5 °C / min, such as 8 °C / min or 10 °C / min. In a preferred embodiment, the freezing rate of the freezing step (a) or (a.2) is at least 15 °C / min, such as 20 °C / min, 25 °C / min, 30 °C / min, 35 °C / min or 40 °C / min.

[0224] In a specific embodiment, the storage in step (b) of the method for cryopreserving isolated mitochondria, such as isolated viable mitochondria, is at a temperature below 0 °C, such as -4 °C, -8 °C, -10 °C, -15 °C, -20 °C or -25 °C. In one embodiment, the storage temperature in step (b) of any one of the methods of the above embodiments is less than at least -10 °C, such as -20 °C or -30 °C. In one embodiment, the storage temperature in the storage step (b) of any one of the methods of the above embodiments is the temperature of dry ice (-78.5 °C). In yet another embodiment, the storage temperature in the storage step (b) is the temperature of liquid nitrogen (e.g., -196 °C).

[0225] In another aspect, the storage of step (b) by any one of the methods of the above embodiments (e.g., storage at a low temperature of a frozen composition containing isolated mitochondria, e.g., isolated viable mitochondria) has a period of at least 2 hours before thawing, such as 3, 4, 5, 6, 7, 8, 10, 12, 15, or 18 hours (i.e., the storage time of the frozen composition). In some embodiments, the storage of step (b) has a period of at least 24 hours, such as 48, 72, 96, or 120 hours before thawing. In some embodiments, the storage period of step (b) is at least 5 days (i.e., 120 hours) before thawing, such as 6 days (i.e., 144 hours) before thawing. In some other embodiments, the storage period of step (b) is at least 1 week (i.e., 7 days) before thawing, such as 2, 3, or 4 weeks. In some other embodiments, the storage of the composition by step (b) of any one of the methods of the above embodiments has a period of at least 1 month, such as 3 months, 6 months, 12 months, 18 months, 24 months or more before thawing. Each possibility represents a separate embodiment of the present invention.

[0226] In another aspect, the method according to any one of the embodiments of step (c) comprises thawing the frozen composition at a temperature higher than 4°C, for example higher than 10°C. In another embodiment, the thawing in step (c) is carried out at a temperature higher than 18°C, for example 20°C or 22°C. In a specific embodiment, step (c) comprises thawing the frozen composition at a temperature of 40°C or lower, for example 39°C or 38.5°C. In a specific embodiment, the thawing in step (c) is preferably carried out at a temperature of 20°C to 38°C, for example 37°C (for example, in a water bath at 37°C). In another specific embodiment, the thawing is carried out at body temperature. In another specific embodiment, the thawing in step (c) is carried out at a temperature that enables the administration of mitochondria to a patient in need of treatment. In another specific embodiment, the thawing in step (c) is carried out gradually. For example, in some embodiments, the thawing is carried out at a temperature that rises at a rate of at least 1°C / min (i.e., 1°C / min), for example 2°C / min, 3°C / min or 4°C / min. In one preferred embodiment, the thawing is carried out at a temperature that rises at a rate of at least 5°C / min, for example 6°C / min, 7°C / min, 8°C / min or 9°C / min, and more preferably at a temperature that rises at a rate of at least 10°C / min, for example 15°C / min, 20°C / min or 30°C / min.

[0227] In another aspect, the thawing step (c) of the method according to any one of the above embodiments is carried out for a period of up to 6 hours, for example 5, 4 or 3 hours. In one embodiment, the thawing step (c) has a period of up to 2.5 hours, for example 2 hours or 1.5 hours. In a specific embodiment, the thawing is carried out for up to 60 minutes, for example 50, 45, 40, or 35 minutes. In a preferred embodiment, the thawing step (c) has a period of up to 30 minutes, for example 25 minutes or 20 minutes. In another preferred embodiment, the thawing step (c) has a period of up to 15 minutes, for example 10 minutes. In a more preferred embodiment, the thawing step (c) has a period of up to 5 minutes, for example 3 minutes.

[0228] A further method for cryopreserving mitochondria included in a composition according to any one of the above embodiments includes a freezing step, particularly a spray freezing (SF) step, optionally followed by a drying step (i.e., freeze-drying method (SFD)). Spray freezing (SF) and spray freeze-drying (SFD) directly address the problem of preserving thermally unstable mitochondria containing mitochondrial subparticles. Spray freezing first involves spraying a composition containing mitochondria into a liquid cryogenic medium (e.g., liquid nitrogen contained in a chamber or container), and then maintaining the composition at a temperature below 0°C, e.g., the temperature of the cryogenic medium itself. The composition may be sprayed by any suitable spraying device, such as a spray gun, which may be appropriately pre-cooled before use. In the SFD process, the composition is first sprayed into and frozen in a cryogenic medium, e.g., liquid nitrogen. The shortest time between the isolation of fresh mitochondria and the spray freezing of a composition containing these isolated mitochondria, i.e., the cryopreservation composition of the present invention containing isolated mitochondria, is preferably 120 minutes or less, more preferably 60 minutes or less, still more preferably 30 minutes or less after the isolation of fresh mitochondria. According to this method, the freezing step requires a very short time, is rapid, and is almost immediate (e.g., rapid freezing). The droplets of the sprayed composition containing mitochondria according to any one of the above embodiments, when frozen, can be held / stored in a cryogenic medium such as liquid nitrogen or any other suitable cryogenic agent, e.g., dry ice, for at least 2 minutes, 5 minutes, 10 minutes, 30 minutes, or 60 minutes. The droplets have a size of 1 μm to 100 μm, more generally 3 μm to 75 μm or 5 μm to 55 μm. The spray-frozen composition can be stored at a temperature below 0°C for at least 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 1 week, 2 weeks, 3 weeks, or 4 weeks. The spray-frozen composition can also be stored for a period of 1 year or 2 years.

[0229] After spray-freezing, the composition can be separated from the cryopreservation medium, for example liquid nitrogen, by evaporating the cryopreservation medium at a temperature above 0°C, for example at room temperature (for example, between 20 and 25°C). Evaporation of the cryopreservation medium may be carried out at atmospheric pressure (for example, 1 atm or 1.01325 bar) or at a pressure lower than atmospheric pressure.

[0230] After separation from the cryopreservation medium, for example, the composition obtained after the cryopreservation medium has been removed at a pressure lower than atmospheric pressure may be a lyophilized composition.

[0231] The spray-frozen composition may be thawed before use (for example, lyophilized).

[0232] In another aspect, the present invention provides a method for cryopreserving a composition comprising isolated mitochondria according to any one of the above embodiments, for example isolated viable mitochondria, the method comprising (i) spray-freezing the composition in a cryopreservation medium, for example liquid nitrogen, at a temperature of about -196°C; (ii) storing the frozen composition obtained in step (i) at a temperature below 0°C, for example the temperature of liquid nitrogen; and comprising.

[0233] In another aspect, the present invention provides a method for cryopreserving a composition comprising isolated mitochondria according to any one of the above embodiments, for example isolated viable mitochondria, (i) spray-freezing the composition in a cryopreservation medium, for example liquid nitrogen, at a temperature of about -196°C; (ii) drying / evaporating the cryopreservation medium such as liquid nitrogen; (iii) storing the lyophilized composition obtained in step (ii) at a temperature below 0°C; and comprising.

[0234] In one aspect, the present invention provides a method for cryopreserving a composition according to any one of the above embodiments, wherein the composition contains isolated viable mitochondria in an amount of at least 0.1 μg / μL, such as 0.2 μg / μL, 0.5 μg / μL, 0.7 μg / μL, 1 μg / μL, 1.5 μg / μL or 2.0 μg / μL, and steps (a) and (b) of the method are (a) freezing the composition in liquid nitrogen (e.g., at a temperature of about -196 °C) or dry ice (e.g., at a temperature of -78.5 °C), preferably in dry ice, for at least 1 minute; (b) storing the frozen composition obtained in step (a) at a temperature below 0 °C; and the method includes.

[0235] In a specific embodiment, the freezing step (a) or (a.2) of the method according to any one of the above embodiments is carried out in dry ice for a maximum of 5 minutes, and the storage step (b) is carried out at a temperature below -5 °C, such as -4 °C, -8 °C, -15 °C, -20 °C, -25 °C, -50 °C or -78.5 °C, for at least 1 week, such as 4 weeks or 8 weeks, preferably at least 12 weeks or 24 weeks, more preferably at least 32 weeks, such as 36 weeks or 72 weeks.

[0236] In one aspect, the method of the present invention is a method for cryopreserving a composition according to any one of the above embodiments, wherein the composition contains isolated mitochondria, such as isolated viable mitochondria, in an amount of at least 5 μg / μL, such as at least 10 μg / μL, at least 15 μg / μL, at least 20 μg / μL, at least 25 μg / μL, or at least 30 μg / μL. Further, in a further embodiment, the present invention provides a method for cryopreserving a composition according to any one of the above embodiments, wherein the composition contains isolated mitochondria, such as isolated viable mitochondria, in an amount of at least 35 μg / μL, such as 40 μg / μL, 50 μg / μL, 60 μg / μL, 70 μg / μL or 80 μg / μL.

[0237] Therapeutic treatment Embodiment C: Cryopreserved composition containing mitochondria for use in the treatment of diseases The present invention provides herein a cryopreserved composition containing mitochondria, such as human mitochondria, for use as a pharmaceutical, or prepared by any of the above methods. In particular, the present invention provides a composition according to any one of the above embodiments in a therapeutically effective amount for use in the treatment of conditions that would benefit from an increase or restoration of mitochondrial function in a subject in need thereof, as well as conditions that would benefit from the combined use of mitochondria with another pharmaceutical agent, such as a molecule, antibody, oligonucleotide, peptide, or extracellular vesicle. The combined use may include simultaneous application or administration of the pharmaceutical agent and mitochondria, as well as solutions prepared as a single therapeutic entity, such as where the pharmaceutical agent is introduced into the mitochondria or linked to the mitochondria before or after cryopreservation, such that the pharmaceutical agent and mitochondria are a single therapeutic agent.

[0238] The present invention provides, in the present specification, a cryopreserved composition comprising isolated mitochondria, such as isolated viable mitochondria, in a therapeutically effective amount according to any one of the above embodiments for treating a disease or condition in a subject in need thereof. In certain embodiments, the mitochondria included in the composition for treating a disease are human isolated mitochondria, such as human isolated viable mitochondria, such as wild-type human mitochondria (e.g., naturally occurring human mitochondria), or modified human mitochondria, such as mitochondria modified by gene editing. In another particular embodiment, the mitochondria included in the composition for use in treating a disease are a complex mitochondrial agent, such as viable mitochondria that have been previously isolated and then conjugated to a pharmaceutical, therapeutic, diagnostic, or imaging agent. A complex mitochondrial agent includes isolated mitochondria artificially combined with a therapeutic agent, a pharmaceutical agent, a diagnostic agent, an imaging agent, or any other agent. The agent is combined with the mitochondria in any manner, for example, conjugated to the mitochondria (e.g., chemically or electrostatically conjugated), added to the mitochondria, for example, added to the outer membrane of the mitochondria, embedded in the mitochondrial membrane, substantially encapsulated within the mitochondria, or completely encapsulated by the mitochondria, as long as the mitochondria and the agent are in physical contact with each other.

[0239] Accordingly, the present specification provides a composition according to any one of the above embodiments for use in treating a disease of a patient in need thereof by delivering the composition and the mitochondria included therein to the cells and / or tissues of the patient or to cells derived from an allogeneic donor. A composition comprising isolated mitochondria, such as isolated viable mitochondria, according to any one of the above embodiments for use in treating a disease or disorder that benefits from an increase in mitochondrial function is directly frozen, stored (i.e., cryopreserved), and thawed prior to use.

[0240] The mitochondria contained in the composition according to any one of the above embodiments for use in the treatment of various diseases or disorders including, but not limited to, cancer, tumors, autoimmune diseases, age-related diseases, metabolic diseases, ischemia-related or ischemia-reperfusion injury and / or diseases, or various forms of wounds, are frozen, stored, and then thawed according to the cryopreservation method of any one of the above embodiments prior to administration to a subject in need thereof.

[0241] In one aspect, the present invention provides a composition according to any one of the above embodiments in a therapeutically effective amount for use in the treatment of a disease, such as treatment by therapeutic mitochondrial transplantation (TMT), in a subject in need of treatment of the disease.

[0242] In some embodiments, the mitochondria contained in the composition according to any one of the above embodiments, such as isolated viable mitochondria, are autologous (i.e., autogenic or autologous). In some embodiments, the mitochondria are autologous mitochondria or autologous-derived mitochondria having genetic modification. In some other embodiments, the mitochondria are autologous-derived and are linked to imaging, diagnosis, or a pharmaceutical agent. In some other embodiments, the agent is embedded or incorporated into the autologous mitochondria. In some other embodiments, the mitochondria are allogeneic. In some embodiments, the mitochondria are allogeneic mitochondria having genetic modification. In some other embodiments, the mitochondria are allogeneic mitochondria linked to imaging, diagnosis, or a pharmaceutical agent. In some other embodiments, the agent is embedded or incorporated into the allogeneic mitochondria. In some other embodiments, the mitochondria are xenogeneic. In some embodiments, the mitochondria are xenogeneic mitochondria having genetic modification. In some other embodiments, the mitochondria are xenogeneic mitochondria linked to imaging, diagnosis, or a pharmaceutical agent. In some other embodiments, the agent is embedded or incorporated into the xenogeneic mitochondria.

[0243] In one aspect, any one of the compositions of the above embodiments has undergone a freeze-thaw cycle before being used for the treatment of a disease.

[0244] In a further aspect, any of the compositions of the above embodiments has undergone a freeze-thaw cycle by any one of the methods of the above embodiments before being used for the treatment of a disease.

[0245] In another aspect, the present invention provides a composition comprising isolated mitochondria, such as isolated viable mitochondria, according to any one of the above embodiments for use in the treatment of a disease in a subject (e.g., a patient) in need thereof, the composition being administered in a therapeutically effective amount to the subject in need thereof by various routes, such as by topical or parenteral administration, such as by dermal or subcutaneous administration, by aerosol administration, by direct injection, by vascular infusion, such as by intravenous injection (i.e., iv injection), or by injecting the composition into a blood vessel of the subject (e.g., by intra-arterial injection or infusion into a blood vessel), the blood vessel being part of the vascular system of the subject and carrying blood to a target site. The blood flowing through the blood vessel carries the isolated viable mitochondria, such as isolated viable mitochondria modified by gene editing, or a combined mitochondrial agent, to the target site, such as an organ, tissue, or damaged site. The target site is any part of the subject, such as the heart, kidney, pancreas, liver, lung, bladder, gonad, placenta, optic nerve, acoustic nerve, brain, or skeletal muscle. In a particular embodiment, the blood vessel is the coronary artery of the subject. In some embodiments, the mitochondria or combined mitochondrial agent contained in any one of the compositions of the above embodiments is delivered to target cells both in vitro, ex vivo, and in vivo. In a particular embodiment, a composition comprising isolated viable mitochondria (e.g., mitochondria modified by gene editing, or mitochondria linked to a therapeutic, diagnostic, or imaging agent) according to any one of the above embodiments is for use in the treatment of ischemia-reperfusion injury, such as lung, kidney, heart, or brain ischemia-reperfusion injury, the composition being administered in a therapeutically effective amount to the subject in need thereof, such as by direct injection or by intravenous or intra-arterial injection.

[0246] In one aspect, the composition according to any one of the above embodiments is for use in treating mitochondrial or mitochondrial-related diseases in a subject in need thereof. In one embodiment, the composition according to any one of the above embodiments is for use in treating mitochondrial diseases caused by mutations in mitochondrial DNA (mtDNA) or nuclear genes encoding mitochondrial components, such as acquired or hereditary mutations. In another embodiment, the composition according to any one of the above embodiments is for use in treating hereditary mitochondrial disorders, including but not limited to mitochondrial encephalopathy, mitochondrial myopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, myoclonic epilepsy with ragged red fibers (MERRF), neuropathy, ataxia and retinitis pigmentosa (NARP) syndrome, myoneurogastrointestinal encephalopathy (MNGIE), Leber's hereditary optic neuropathy, myoclonic epilepsy with ragged red fibers (MERRF) disorder, maternally inherited diabetes and deafness (MIDD), Leigh syndrome, and Huntington's disease (HD). In another embodiment, the composition according to any one of the above embodiments is for use in treating acquired mitochondrial-related diseases caused by toxicity, chemotherapy, or iatrogenic diseases. In another embodiment, the composition according to any one of the above embodiments is for use in treating acquired mitochondrial-related diseases or dysfunctions, including but not limited to central nervous system (CNS) diseases such as Alzheimer's disease and Parkinson's disease. In some embodiments, the mitochondrial and mitochondrial-related diseases / disorders are chronic.

[0247] In another aspect, a composition according to any one of the above embodiments is for use in the treatment of ischemia or ischemia-reperfusion related injury, for example, for use in promoting the repair process of damaged cells or tissues in a subject in need thereof. In one embodiment, a composition according to any one of the above embodiments is for use in promoting the repair process of damaged cells or tissues, where the damage is caused by stroke, traumatic brain or spinal cord injury. In one embodiment, a composition according to any one of the above embodiments is for use in the treatment of non-ischemic injury.

[0248] In another aspect, a composition according to any one of the above embodiments is for use, for example, for angiography, in increasing blood flow in the vasculature of a patient in need thereof. In some embodiments, the composition is for use in dilating blood vessels such as arteries or veins of a patient in need thereof. In some other embodiments, the composition is for use in reducing vascular resistance in an organ (e.g., heart, kidney, liver, or lung). In certain embodiments, the composition is for use in, for example, localizing, identifying, and / or removing blockages in blood vessels such as blood clots.

[0249] In another aspect, the composition according to any one of the above embodiments is for use in the treatment of wounds, such as wounds showing impaired wound healing, such as wounds in diabetic patients. In another aspect, the composition according to any one of the above embodiments is for use in promoting the repair response in acute wounds. In another aspect, the composition according to any one of the above embodiments is for use in promoting the repair response in chronic wounds. (Arch Dermatol Res. (May 2016), 308(4): 239-48, Janda J.et al.; Cell Metabolism (December 7, 2021) 33, 2398-2414, Willenborg et al.; Biochem Soc Trans.(30 Oct 2020); 48(5):1995-2002, Horn A.et al.; Cell Regeneration volume 10, Article number:5 (2021), Ma Y. et al.)

[0250] In another aspect, the composition according to any one of the above embodiments is for use in the treatment of age-related changes, disorders, or diseases. Non-limiting examples of age-related dysfunctions / diseases are neurodegenerative diseases (e.g., Morbus Parkinson's disease (PD), Alzheimer's disease (AD), dementia), skin aging, retinal dysfunction, hearing impairment, age-related metabolic diseases, such as diabetes or cachexia, and muscle weakness, reduced muscle tone, dystonia, dystrophy and / or atrophy (Biology (2019), 8, 48, R. H. Haas). Another example of an age-related disease is Hutchinson-Gilford progeria syndrome, a progressive genetic disorder that rapidly ages children.

[0251] In another aspect, the composition according to any one of the above embodiments is for use in the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) or multiple sclerosis (MS).

[0252] In another aspect, the composition according to any one of the above embodiments can be used, for example, for the treatment of sarcopenia such as "primary" (or age-related) sarcopenia that can be measured according to the diagnostic criteria of the European Working Group on Sarcopenia in Older People (EWGSOP), or for the treatment of "secondary" sarcopenia. Non-limiting examples of secondary sarcopenia include: (a) sarcopenia due to sedentary, sitting lifestyle and immobility; (b) disease-related sarcopenia associated with advanced organ failure (heart, lung, liver, kidney, brain), inflammatory diseases, malignancies or endocrine diseases; and (c) nutrition-related sarcopenia (caused by insufficient dietary intake of energy and / or protein, for example, malabsorption, gastrointestinal disorders, or use of drugs that cause loss of appetite).

[0253] In another aspect, a composition according to any one of the above embodiments is for use in the treatment of cancer in a human subject in need thereof. Exemplary suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumor, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, bronchial tumor, cancer of unknown primary, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, fetal tumor, endometrial cancer, epithelioma, esophageal cancer, neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma (HL), hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma with occult primary, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, polypoid mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal and paranasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

[0254] In another aspect, a composition according to any one of the above embodiments is for use in the treatment of an infectious disease in a human subject in need thereof.

[0255] In another aspect, the composition according to any one of the above embodiments is for use in the treatment or amelioration of immunological dysfunction or disease. In one aspect, the composition according to any one of the above embodiments is for use in mediating and regulating an immunological response. In a particular embodiment, the composition according to any one of the above embodiments is for use in the treatment or amelioration of immunological dysfunction or disease, and the mitochondria contained in the composition according to any one of the above embodiments are transplanted into naturally occurring immune cells to produce mitochondria-enhanced immune cells. Without intending to be limited by any theory, in some embodiments, immune system disorders (e.g., immunological disorders or dysfunctions) may be due to a decrease in the immunological diversity of naive T cells and an increase in the number of senescent effector cells associated with aging. In a particular aspect, the composition according to any one of the above embodiments is for use in the treatment of autoimmune diseases. Non-limiting examples of autoimmune diseases are systemic lupus erythematosus (SLE), rheumatoid arthritis, multiple sclerosis, autoimmune vasculitis, myasthenia gravis, pernicious anemia, Hashimoto's thyroiditis, type 1 diabetes, inflammatory bowel disease (IBD), autoimmune Addison's disease, Graves' disease, Sjogren's syndrome, psoriasis, and celiac disease. Another example is autoimmune lymphoproliferative syndrome (ALPS), which is a genetic disorder in which the body cannot properly regulate the number of immune system cells (lymphocytes). Without wishing to be bound by any theory or mechanism, mitochondria play an important role not only in the proliferation but also in the regulation of the function of immune cells. In addition to providing energy to support the synthesis of macromolecules essential for immune cell proliferation, mitochondria also act as signaling organelles and drive the activation of immune cells via metabolic intermediates, mitochondrial DNA (mtDNA), and reactive oxygen species (ROS). Furthermore, mitochondrial dynamics (fusion and fission), biogenesis (synthesis of new mitochondria), and mitophagy (degradation of damaged mitochondria) also play important roles in the regulation of immune cell function. The relationship between immune cell function and mitochondrial function is known in the art as "immunometabolism".The relationship between the immune pathway and the metabolic pathway is important, for example, in many dysfunctions of the immune response in severe diseases, particularly sepsis and the like.

[0256] In another aspect, the composition according to any one of the above embodiments is for use in gene therapy. In certain embodiments, the gene therapy is for the treatment of cancer.

[0257] In yet another specific embodiment, gene therapy is for the treatment of infectious diseases. In yet another embodiment, gene therapy is for the treatment of diseases of the heart, lung, kidney, bladder, prostate, uterus, pancreas or kidney. In yet another embodiment, gene therapy is for the treatment of hereditary genetic deficiencies or acquired genetic disorders. In a specific embodiment, gene therapy is for the treatment of autoimmune diseases. In yet another embodiment, it is gene therapy for the treatment of mitochondrial-related diseases. Mitochondrial diseases, such as acquired mitochondrial-related diseases or dysfunctions, include, but are not limited to, central nervous system (CNS) diseases such as Alzheimer's disease, Parkinson's disease, Alpers syndrome, Leigh syndrome, and myoclonic epilepsy with ragged red fibers, as well as those following stroke and traumatic brain or spinal cord injury (Mitochondrion (July 2017), 35: 70-79, Gollihue and Rabchevsky). Drugs can induce mitochondrial dysfunction through various mechanisms including inhibition of fatty acid oxidation, impairment of oxidative phosphorylation and respiratory chain activity, and changes in mitochondrial membrane integrity. Some drugs can also impair mitochondrial function through the production of reactive oxygen species and the generation of active metabolites that can covalently bind to important mitochondrial proteins. Drug-induced mitochondrial dysfunction plays an important role in the etiology of adverse effects such as liver injury, myopathy and cardiotoxicity. Examples of drugs that can induce mitochondrial dysfunction are, for example, acetaminophen, amiodarone, doxorubicin, nucleoside reverse transcriptase inhibitors (e.g., stavudine, didanosine, zidovudine), statins (e.g., atorvastatin, cerivastatin, simvastatin) and valproic acid (Mitochondrial Biology and Experimental Therapeutics, (March 2018), pp 269 - 295, Massart).

[0258] In a particular embodiment, the composition according to any one of the above embodiments is for use in gene therapy, and the mitochondria contained in the composition according to any one of the above embodiments are transplanted into naturally occurring immune cells to produce mitochondria-enhanced immune cells. In another particular embodiment, the composition according to any one of the above embodiments is for use in gene therapy, and the mitochondria contained in the composition according to any one of the above embodiments are transplanted into modified immune cells including, but not limited to, chimeric antigen receptor (CAR) T cells, CAR-NK cells, CAR-macrophages, neutrophils, tumor-infiltrating lymphocytes (TIL), gamma delta T cells, etc. In some embodiments, the immune cells are T lymphocytes such as CD4 T cells, for example, Treg cells. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the immune cells are mucosa-associated invariant T cells. In some embodiments, the immune cells are gamma delta T cells. In some embodiments, the immune cells are monocytes or macrophages. In some embodiments, the immune cells are neutrophils. In some embodiments, the immune cells are B lymphocytes. In some embodiments, the immune cells are produced from stem cells, mesenchymal stem cells or induced pluripotent stem cells (iPSC). In some embodiments, the immune cells are allogeneic or autologous immune cells.

[0259] In a particular embodiment, the composition according to the above embodiments is for use in gene therapy, and the mitochondria contained in the composition according to any one of the above embodiments are first frozen and thawed according to any one of the above methods, or frozen, stored and thawed, and then transplanted into immune cells to produce mitochondria-enhanced immune cells, such as, but not limited to, chimeric antigen receptor (CAR) T cells, CAR-NK cells, CAR-macrophages, neutrophils, tumor-infiltrating lymphocytes (TIL), gamma delta T cells. In yet another embodiment, the mitochondria are transplanted into immune cells to produce mitochondria-enhanced immune cells before freezing.

[0260] In another aspect, the composition according to any one of the above embodiments is for use in gene therapy, and the mitochondria contained in the composition according to any one of the above embodiments are transplanted into stem cells. For example, the mitochondria of the composition are first frozen and thawed according to any one of the above methods, or frozen, stored, and thawed, and then transplanted into stem cells. In some aspects, the stem cells are embryonic stem cells. In some aspects, the stem cells are induced pluripotent stem cells. In some aspects, the immune cells are immune cells derived from pluripotent stem cells. In some aspects, the immune cells are T lymphocytes such as helper T cells, cytotoxic T cells, regulatory T cells, memory T cells, etc. In some aspects, the T lymphocyte is a CD8 T cell. In some aspects, the T lymphocyte is a CD4 T cell such as a Treg cell. In some aspects, the immune cells are natural killer (NK) cells. In some aspects, the immune cells are mucosa-associated invariant T cells. In some aspects, the immune cells are gamma delta T cells. In some aspects, the immune cells are monocytes or macrophages. In some aspects, the immune cells are neutrophils. In some aspects, the immune cells are B lymphocytes. In some aspects, the stem cells are allogeneic or autologous stem cells.

[0261] In certain aspects, the composition according to the above embodiments is for use in gene therapy, and the mitochondria contained in the composition according to any one of the above embodiments are first frozen and thawed according to any one of the above methods, or frozen, stored, and thawed, and then transplanted into stem cells to produce mitochondria-reinforced stem cells. In yet another aspect, mitochondria are transplanted into stem cells to produce mitochondria-reinforced stem cells prior to the freezing cycle.

[0262] In certain aspects, the composition according to any one of the above embodiments is for use in the treatment of acute or chronic graft-versus-host disease (GVHD).

[0263] The term Unless otherwise defined, all technical terms, notations, and other scientific terms used in this specification are intended to have the meanings commonly understood by those skilled in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined in this specification for clarity and / or ease of reference, and including such definitions in this specification should not necessarily be construed as representing a difference from what is commonly understood in the art. The techniques and procedures described or referenced in this specification are generally well understood and commonly employed by those skilled in the art using conventional methods. Procedures involving the use of commercially available kits and reagents, unless otherwise specified, are generally carried out according to the protocols and conditions defined by the manufacturer.

[0264] As used in this specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise.

[0265] Terms such as "include", "such as", etc. are intended to convey inclusion without limitation unless otherwise specified.

[0266] As used in this specification, the term "or" is generally used in its ordinary sense including "and / or" unless the context clearly indicates otherwise. The term "and / or" means one or all of the listed elements, or any combination of two or more of the listed elements.

[0267] As used in this specification, the term "comprising" also specifically includes embodiments "consisting of" and "consisting essentially of" the recited elements, unless otherwise specified.

[0268] As used herein, the term "about" refers to and encompasses the indicated value and the range above and below that value. In certain embodiments, the term "about" refers to the specified value ±10%, ±5%, or ±1%. In certain embodiments, where applicable, the term "about" refers to the specified value ± one standard deviation of that value.

[0269] As used herein, the term "isolated" means altered or removed from its natural state or environment. For example, nucleic acids, peptides, or intracellular particles such as mitochondria that are naturally present in a living animal or cell are not "isolated", but the same nucleic acids, peptides, or intracellular particles, such as mitochondria, that are partially or completely separated from their coexisting materials in their natural state are "isolated".

[0270] As used herein, "mitochondria" refers to viable mitochondria that (essentially) do not contain eukaryotic cell materials such as foreign eukaryotic cell materials isolated / purified from, for example, cells or cell cultures. Thus, only a minimal amount of cell components (other than mitochondria) are present in the mitochondria (composition) used herein. Preferably, cell components other than mitochondria are not present in the mitochondria (composition) used herein. In this sense, "mitochondria" as used herein is "isolated mitochondria", and the terms "mitochondria" and "isolated mitochondria" can be used interchangeably. For example, any currently known technique in the art, such as intracellular fractionation by repeated differential centrifugation (DC) or density gradient centrifugation (DGC), can be used for the isolation of mitochondria (Pallotti & Lenaz, 2007; Bharadwaj et al., 2015; Djafarzadeh & Jakob, 2017; Garcia-Cazarin, Snider, & Andrade, 2011; Lai et al., 2019; Alexander G.Bury et al. 2020). Preferred non-limiting examples of methods for obtaining "isolated mitochondria" are provided in Preble et al., JOVE (2014): "Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration" and International Publication No. WO 2015 / 192020 (McCully et al.), where the mitochondria are isolated by differential filtration, optionally followed by centrifugation. As used herein, the term "isolated mitochondria" refers to mitochondria separated from other cell components of a donor cell. As used herein, the term "donor cell" refers to the cell from which the mitochondria of the present invention are isolated. The terms "recipient cell", "acceptor cell" and "host cell" are used interchangeably herein to describe the cell that receives and encompasses the isolated mitochondria.

[0271] As used herein, the term "viable mitochondria" is used throughout this specification to describe viable mitochondria that are intact, active, functional, and respiration competent. According to some embodiments, "viable mitochondria" refers to mitochondria that exhibit biological functions such as respiration and ATP and / or protein synthesis.

[0272] As used herein, the term "intact mitochondria" is used throughout this specification to describe mitochondria that include an integral outer membrane and inner membrane, an integral intermembrane space, integral cristae (formed by the inner membrane), and an integral matrix. Alternatively, intact mitochondria are mitochondria that preserve their structure and ultrastructure. In another aspect, intact mitochondria include active respiratory chain complexes I-V embedded in the inner membrane and maintain the ability to synthesize membrane potential and ATP.

[0273] As used herein, the term "modified mitochondria" refers to mitochondria that have been modified by gene editing. Non-limiting examples of gene editing techniques for modifying mitochondrial DNA (mtDNA) are, for example, gene editing techniques by restriction endonuclease (RE) technology, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, CRISPR system, and pAgo-based system. Alternatively, mitochondria, for example, isolated viable mitochondria containing exogenous mtDNA, for example, mitochondria modified by transplanting exogenous mtDNA into self-mitochondria. Alternatively, the mitochondria are modified by gene editing TALED technology that can apply an enzyme that targets a specific mitochondrial DNA sequence depending on a TALE protein and performs editing from the desired adenine to guanine in addition to the reverse direction from cytosine to thymine.

[0274] As used herein, the term "mitochondrial-DNA (mtDNA)" refers to the DNA of mitochondria, which is a double-stranded circular molecule (outer heavy strand (H-strand) and inner light strand (L-strand)) containing 16,569 base pairs and 37 genes (Anderson et al., 1981). There are two non-coding regions in mtDNA. One is the origin of replication of the light strand (OL), and the other is the displacement loop (D-loop), also called the control region (CR), which contains the origin of replication of the heavy strand (OH), the heavy strand promoter, and the light strand promoter (Lott et al., 2013). The mtDNA of mammalian cells is relatively small and genetically compact, containing two overlapping genes and very little non-coding sequence (Ojala et al., 1981).

[0275] As used herein, the phrases "cryoprotective composition", "cryoprotecting composition", "cryopreservative composition", "cryoprotectant", "cryoprotective agent", "cryoprotecting agent", or "cryopreservative agent" refer to a chemical mixture, chemical solution, or chemical compound that promotes the process of cryoprotection by reducing damage to mitochondria, such as isolated viable mitochondria, during freezing and thawing (e.g., during freeze-thaw cycles). Cryoprotectants protect mitochondria from damage associated with storage and / or freezing at sub-zero temperatures, such as mitochondrial membrane damage caused by ice crystal formation.

[0276] As used herein, the term "permeating cryoprotectant" refers to a cryoprotectant designed to cross biological membranes and exert its effect, particularly inside biological structures or cells. "Permeating cryoprotectant" is also referred to as "intracellular cryoprotectant". Permeating cryoprotectants are small nonionic molecules that have high water solubility at low temperatures and high diffusibility across lipid membranes. This group includes propylene glycol, ethylene glycol, glycerol, and dimethyl sulfoxide (DMSO). These substances diffuse very efficiently through cell membranes and bind to the water inside the cells drawn from imagination.

[0277] As used herein, the term "cryopreserved mitochondrion" or "frozen mitochondrion" refers to the isolation of mitochondria, such as viable mitochondria preserved by cooling to sub-zero temperatures. "Cryopreserved mitochondria" include autologous (i.e., self or autogenic), allogeneic, or xenogeneic mitochondria. The term "cryopreserved mitochondria" refers to mitochondria isolated from and frozen in any type of organ, tissue, or cell, such as cultured cells, for example. In some particular embodiments, frozen mitochondria are stored for a certain period. In some particular embodiments, the mitochondria have undergone one or more freeze-thaw cycles. In some particular embodiments, the mitochondria that have undergone a freeze-thaw cycle are isolated viable mitochondria. In some specific embodiments, cryopreserved isolated viable mitochondria are contained (e.g., within) in a cryopreservation composition. In some embodiments, the mitochondria have undergone a spray-freezing process.

[0278] As used herein, the terms "cryopreservation amount" or "cryoprotective amount" refer to the amount of a cryopreservation or cryoprotective compound / agent that enables mitochondria to maintain their physical, chemical, functional, and structural stability and integrity during and after a freezing cycle or freeze-thaw cycle. The term "less than the cryopreservation amount" refers to an amount of a cryopreservation / cryoprotective compound / agent that is not sufficient to maintain the physical, chemical, functional, or structural stability and integrity of mitochondria during and after the freezing cycle of a freeze-thaw cycle.

[0279] As used herein, the terms "autologous," "autogenous," or "autogenic" refer to any material derived from the same individual that is later reintroduced into the individual.

[0280] As used herein, the term "allogeneic" refers to any material derived from the same or a different animal of a different patient of the same species into which the material is introduced. Two or more individuals are said to be allogeneic to each other if the genes at one or more loci are not identical. In some embodiments, allogeneic substances from individuals of the same species may be genetically different enough to interact antigenically.

[0281] As used herein, the term "xenogeneic" refers to a graft derived from an animal of a different species, i.e., "xenogeneic mitochondria" refers to mitochondria obtained from a species different from the subject being treated.

[0282] As used herein, the term "freeze-thaw cycle" refers to freezing the mitochondria of the present invention to a temperature below 0°C, maintaining the mitochondria at a temperature below 0°C for a defined period, and thawing the mitochondria to room temperature or body temperature or any temperature above 0°C, thereby enabling the administration of mitochondria to a person in need of treatment, such as a person suffering from a mitochondrial or mitochondrial-related disease or disorder, an inflammatory, infectious, autoimmune disease or disorder, cancer, ischemia or ischemia-related disorder and injury, thrombosis and blood flow insufficiency. Each possibility represents a separate embodiment of the present disclosure. As used herein, the term "room temperature" refers to a temperature of 18°C to 25°C. As used herein, the term "body temperature" refers to a temperature of 35.5°C to 37.5°C, preferably 37°C.

[0283] As used herein, the term "rapid freezing" or "snap freezing" refers to rapidly freezing mitochondria by exposing the mitochondria or a composition containing the mitochondria to extremely low temperatures.

[0284] As used herein, the terms "buffering agent", "buffer", "aqueous buffer", or "buffer system" should be understood to mean a substance that maintains the pH of an aqueous medium within a narrow range when a small amount of acid or base is added. The term "buffering agent" means a single substance or combination of substances that resists changes in hydrogen ion concentration upon addition of an acid or alkali.

[0285] As used herein, the term "chelating agent" or "chelating agent" refers to any organic or inorganic compound that binds to a metal ion having a valence greater than 1. Exemplary chelating agents or chelating agents are compounds that bind to calcium ions (i.e., calcium chelating agents or calcium chelating agents), such as EDTA and EGTA.

[0286] As used herein, the terms "sugar" or "sugar component" refer to any saccharide, such as common monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides. In some specific embodiments, the term "sugar" refers to a "modified sugar". The term "modified sugar" refers to a sugar analog that does not have the structure of a naturally occurring sugar, such as deoxyribose sugar or sucralose. In some specific embodiments, the term "sugar" refers to a "sugar derivative". The term "sugar derivative" refers to a compound derived from a sugar by a chemical reaction.

[0287] As used herein, the term "trehalose" refers to a sugar composed of two molecules of glucose. Trehalose is a disaccharide formed by a 1,1-glycosidic bond between two α-glucose units. Trehalose is also known as (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxane-3,4,5-triol. Other names are α,α-trehalose, α-d-glucopyranosyl-(1,1)-α-d-glucopyranoside, mycose or tremalose. In another embodiment, the term "trehalose" refers to the isomers α,β-trehalose, and β,β-trehalose (also called isotrehalose) known as neotrehalose.

[0288] As used herein, the term "amino acid" refers to natural or unnatural amino acids, as well as amino acid analogs and mimetics that function in a manner similar to natural amino acids. The term "amino acid" refers to proteinogenic or non-proteinogenic amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs are compounds having the same basic chemical structure as a naturally occurring amino acid, i.e., a compound having a hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as a naturally occurring amino acid. As used herein, the term "amino acid" may be referred to herein by either the commonly known three-letter symbol or the one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by the generally accepted one-letter code.

[0289] As used herein, the term "protein" refers to large biomolecules and macromolecules that contain one or more long chains of amino acid residues. These biomolecules can be products of either biosynthetic techniques or chemical (organic) synthetic techniques. The term "post-translationally modified protein" refers to a protein that has been modified according to "post-translational modification (PTM)". PTM refers to covalent and generally enzymatic post-translational modifications, and refers to any change in the amino acid sequence of the protein after its synthesis. PTM can include modification of amino acid side chains, terminal amino or carboxyl groups by covalent or enzymatic means after protein biosynthesis. Generally, these modifications affect the structure, stability, activity, cellular localization or substrate specificity of the protein. Post-translational modification provides complexity to the proteome for diverse functions through a limited number of genetic modifications of the protein after protein biosynthesis. Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, and then the polypeptide chains can undergo PTM to form mature protein products. PTM is an important component in cell signaling, for example when prohormones are converted to hormones. Post-translational modification can occur at amino acid side chains or at the C-terminus or N-terminus of the protein. They can expand the chemical repertoire of the 20 standard amino acids by modifying existing functional groups or, in particular, by introducing new ones, for example, by phosphorylation, glycosylation, sulfation, hydroxylation, methylation, SUMOylation (i.e., the functional group is SUMO, a 100 amino acid residue protein that binds to target proteins in the same way as ubiquitin), disulfide bond formation, lipidation, acetylation, prenylation, etc.

[0290] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be included in a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, this term refers to both short chains, which are generally also referred to in the art as peptides, oligopeptides, and oligomers, and long chains, which are generally referred to in the art as proteins, and there are many types thereof. "Polypeptide" includes, for example, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

[0291] As used herein, the term "polymer" refers to polymers suitable for clinical and medical applications. The polymer is biocompatible and, for example, suitable for exposure to the body and body fluids. The polymer can be synthetic or natural, hydrophilic or amphiphilic. The polymer can be biocompatible and thus suitable for medical and clinical applications. Non-limiting examples of polymers can be polyethylene glycol polymers (PEG) which can be, but are not limited to, PEG200, PEG400, PEG550, PEG600, PEG800, PEG1000, PEG3350, PEG4000, PEG8000, or PEG10000.

[0292] As used herein, the term "antibody" is used in its broadest sense and includes certain types of immunoglobulin molecules containing one or more antigen-binding domains that specifically bind to an antigen or epitope. This term also includes non-immunoglobulin antigen-binding protein molecules, so-called antibody mimetics. Antibodies specifically include intact antibodies (e.g., intact immunoglobulin G, IgG), antibody fragments (e.g., Fab fragments, single-chain Fv (scFv), single-domain antibodies, V H V L V HH NAR, tandem scFv, diabody, single-chain diabody, DART, tandAb, minibody, single-domain antibodies (e.g., camelid V HH ), other antibody fragments or formats known to those of skill in the art), and antibody mimetics (e.g., adnectin, affibody, affilin, anticalin, avimer, DARPins, knottin, etc.). Antibodies can be monospecific, bispecific, and multispecific.

[0293] As used herein, the term "antigen-binding domain" means a portion of an antibody or T cell receptor that can specifically bind to an antigen or epitope via a variable domain. An example of an antigen-binding domain is the antigen-binding domain formed by the interface of the variable domains V H and V L of the heavy and light chains of an antibody, respectively. Another example of an antigen-binding domain is the antigen-binding domain formed by the diversification of certain loops from an antibody mimetic. Another example of an antigen-binding domain is a variable domain of a TCR, e.g., CDR, e.g., a TCR domain containing αCDR1, αCDR2, αCDR3, βCDR1, βCDR2, and βCDR3.

[0294] As used herein, the term "variable domain" refers to a variable nucleotide sequence resulting from a recombination event and can include, for example, the V, J, and / or D regions of a T cell receptor (TCR) sequence derived from T cells such as activated T cells, or can include the V, J, and / or D regions of an antibody. The term "antigen-binding fragment" refers to an antigen-binding domain sufficient to confer recognition and specific binding of an antigen-binding fragment to a target such as an antigen and its defined epitope, i.e., at least a portion of an antibody or TCR or a recombinant variant thereof that includes the variable domain and hypervariable loops, so-called complementarity-determining regions (CDRs). Examples of antigen-binding fragments include Fab, Fab’, F(ab’)2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single-domain antibodies (abbreviated as “sdAb”) (V L or V H either), camelid V HH domains (nanobodies), multispecific antibodies generated from antibody fragments, and TCR fragments, but are not limited thereto. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), which is hereby incorporated by reference in its entirety for all that it teaches.

[0295] The term "scFv" includes a variable fragment of an antibody light chain (V L ) linked at the N-terminus and C-terminus of a variable fragment of an antibody heavy chain (V H ) via a flexible peptide linker and can be expressed as a single polypeptide chain, and scFv refers to a fusion protein that retains the specificity of the intact antibody from which it is derived.

[0296] As used herein, the term "antigen" or "Ag" refers to a molecule that can be specifically bound by an antibody or, alternatively, can elicit an immune response, for example when the antigen is processed by an antigen-presenting cell (APC). This immune response can include antibody production, activation of specific immunocompetent cells, or both.

[0297] One of ordinary skill in the art will understand that any macromolecule, including substantially all proteins or peptides, can function as an antigen. Further, an antigen can be derived from recombinant DNA or genomic DNA. Thus, one of ordinary skill in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that induces an immune response encodes an "antigen" as the term is used herein. Further, one of ordinary skill in the art will understand that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. The present disclosure includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and it is readily apparent that these nucleotide sequences can be arranged in various combinations to encode polypeptides that induce a desired immune response. Further, one of ordinary skill in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be produced by chemical synthesis, and it can also be derived from a biological sample or can be a macromolecule other than a polypeptide, such as a lipid or a carbohydrate. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or fluids having other biological components.

[0298] As used herein, the term "biocompatible" refers to substances, such as polymers, materials / excipients, within a composition / formulation that are non-toxic and meet, for example, the standards of the United States Pharmacopeia (USP) and the European Pharmacopeia (Eur.Ph). A biocompatible material is a material in biotechnology that comes into direct contact with living cells, tissues or organs and does not adversely affect their metabolism.

[0299] As used herein, the term "protease" refers to an enzyme that catalyzes (increases the reaction rate or "speeds up") proteolysis, the breakdown of a protein into smaller polypeptides or single amino acids. Examples of proteases are subtilisin, proteinase K, pepsin, trypsin, chymotrypsin, elastase, neutral protease, and other similar proteases.

[0300] As used herein, the term "complex mitochondrial factor" refers to isolated mitochondria artificially combined with a therapeutic agent, pharmaceutical agent, diagnostic agent, contrast agent, or any other agent. The agent is combined with the mitochondria in any manner, for example, linked to (e.g., chemically or electrostatically linked to), added to, embedded in the mitochondrial membrane, substantially encapsulated within, or completely encapsulated by the mitochondria as long as the mitochondria and the agent are in physical contact with each other. The combined mitochondrial agent is designed to act as a "carrier" such that the mitochondria can transport the agent to the patient's cells / tissues and release the agent (e.g., the cargo payload) to the cells / tissues, for example, after skin / subcutaneous administration, aerosolized administration, or direct injection. The complex mitochondrial agent can be prepared by a method comprising the step of isolating mitochondria from a cell or tissue and mixing the mitochondria with an effective amount of a therapeutic agent, diagnostic agent, or contrast agent under conditions sufficient to link the therapeutic agent, diagnostic agent, or contrast agent to the mitochondria. In some embodiments, the mitochondria are mixed with a contrast agent.

[0301] As used herein, the term "mitochondrial transplantation" or "mitochondrial grafting" or "mitochondrial implantation" or "mitochondrial importation" is used throughout this specification as a general term to describe the process of introducing into a recipient at least one mitochondrion, such as an isolated viable mitochondrion, e.g., an autologous, allogeneic, or xenogeneic isolated viable mitochondrion. This term includes, but is not limited to, all categories of mitochondrial transplantation known in the art, including direct (micro)injection of mitochondria into recipient cells or tissues, as well as intravascular injection, e.g., intravenous injection (i.e., iv injection), or intra-arterial injection or injection into the vasculature of a subject, where the vasculature is part of the subject's vascular system and carries blood to the target site.

[0302] As used herein, the term "transplantation" is used throughout this specification as a general term for describing the process of transplanting an organ, tissue, cell mass, individual cell, or organelle into a recipient. The term "cell transplantation" is used throughout this specification as a general term for representing the process of transferring at least one cell, for example, an enhanced immune cell as described herein, into a recipient. This term includes all categories of transplantation known in the art, including blood transfusions. Transplantations are classified by the site and genetic relationship between the donor and the recipient. This term includes, for example, autotransplantation (removal and transplantation of cells or tissues from one location of a patient to the same or another location of the same subject), allotransplantation (transplantation between members of the same species), and xenotransplantation (transplantation between members of different species).

[0303] As used herein, the term "stem cell" refers to an undifferentiated cell that can be induced to proliferate. Stem cells are capable of self-maintenance or self-renewal, which means that with each cell division, one daughter cell also becomes a stem cell. Stem cells can be obtained from embryonic tissue, postnatal tissue, juvenile tissue, or adult tissue. Stem cells can be pluripotent or multipotent. The term "progenitor cell" as used herein refers to an undifferentiated cell derived from a stem cell and is not itself a stem cell. Some progenitor cells can give rise to progeny that can differentiate into two or more cell types. Stem cells include pluripotent stem cells, which can form cells of any of the body's tissue lineages: mesoderm, endoderm, and ectoderm. Thus, for example, stem cells can be selected from human embryonic stem (ES) cells, human inner cell mass (ICM) / epiblast cells, human primitive ectoderm cells, human primitive endoderm cells, human primitive mesoderm cells, and human primordial germ (EG) cells. Stem cells also include, but are not limited to, pluripotent stem cells that can form multiple cell lineages that make up a tissue or an entire tissue, such as hematopoietic stem cells or neural progenitor cells. Stem cells also include totipotent stem cells, which can form an entire organism. In some embodiments, the stem cells are mesenchymal stem cells. The term "mesenchymal stem cell" or "MSC" is used interchangeably to refer to an adult cell that has not undergone terminal differentiation and can divide to give rise to cells that are stem cells or can irreversibly differentiate to give rise to cells of the mesenchymal cell lineage, such as adipose, bone, cartilage, elastic, and fibrous connective tissue, myoblasts, and adult cells that can give rise to tissues other than those derived from embryonic mesoderm (e.g., nerve cells) in response to various influences from bioactive factors such as cytokines. In some embodiments, the stem cells are partially differentiated cells or differentiated cells. In some embodiments, the stem cells are reprogrammed or dedifferentiated induced pluripotent stem cells (iPSCs). Stem cells can be obtained from embryonic tissue, fetal tissue, or adult tissue.

[0304] As used herein, the term "treating" (and its variations such as "treat" or "treatment") refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject who needs it. Treatment can be carried out both for prevention and during the course of clinical pathology. Desirable effects of treatment include prevention of the occurrence or recurrence of disease, alleviation of symptoms, reduction of the direct or indirect pathological consequences of the disease, prevention of metastasis, decrease in the rate of disease progression, improvement or alleviation of the disease state, and remission or improvement of the prognosis.

[0305] As used herein, a "therapeutically effective amount" is an amount of a composition or an active ingredient thereof sufficient to provide a beneficial effect to an individual to whom the composition is administered or otherwise to reduce an adverse, non-beneficial event. A "therapeutically effective dosage" as used herein means a dosage that produces one or more desired or desirable (e.g., beneficial) effects when administered, such administration being carried out one or more times over a given period. The exact dosage depends on the purpose of treatment and can be ascertained by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Pharmaceutical Dosage Forms Disperse Systems, A. Lieberman, Martin M. Rieger, Gilbert S. Banker, 2nd Edition, (2010); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)). Further, the term "therapeutically effective amount" means an amount of mitochondria or a composition containing mitochondria, in any amount, that results in an improved treatment, cure, prevention or amelioration of a disease, disorder or side effect, or a decrease in the rate of progression of a disease or disorder, as compared to a corresponding subject not receiving such amount. The term also includes, within its scope, an amount effective to enhance normal physiological function.

[0306] As used herein, the term "therapeutic effect" refers to the effect of a therapeutic agent, substance, biological particle or composition, such as an isolated viable mitochondrion or a composition containing isolated mitochondria, obtained by reduction, suppression, remission or eradication of a disease state.

[0307] As used herein, the term "prevention" means the prevention or prophylactic treatment of a disease or disease state.

[0308] As used herein, the term "subject" means a mammalian subject. Exemplary subjects include humans or mammals such as monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits and sheep. In certain embodiments, the subject is a human. The terms "subject in need", "subject requiring the same", "subject in need of treatment" or "patient" are intended to include an organism, i.e., a subject, that has or is at risk of a disease, disorder, or condition and that requires the compositions and methods provided herein.

[0309] As used herein, "preventing" refers to the prevention of a disease or condition in a patient, such as tumorigenesis. For example, if an individual at risk of developing a tumor or other form of cancer is treated by the methods of the present invention and later does not develop a tumor or other form of cancer, the disease is prevented in that individual for at least a certain period of time.

[0310] The term "pharmaceutical composition" refers to a preparation that enables the biological activity of an active ingredient and / or maintains or improves the viability of biological entities (e.g., cells) contained therein and is effective for the treatment of a subject, and that does not contain additional ingredients that are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0311] The term "pharmaceutically acceptable carrier" includes physiological saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, etc., which are suitable for pharmaceutical administration. In some embodiments, the pharmaceutically acceptable carrier is phosphate buffered saline, physiological saline, Krebs buffer, Tyrode's solution, contrast agent, or Omnipaque, or a mixture thereof. The term "pharmaceutically acceptable carrier" also includes sterile mitochondrial buffer (300 mM sucrose, 10 mM K + -HEPES (potassium buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2), 1 mM K+-EGTA, (potassium buffer ethylene glycol tetraacetic acid, pH 8.0)). This term further includes respiratory buffer (250 mM sucrose, 2 mM KH2PO4, 10 mM MgCl2, 20 mM K-15 HEPES buffer (pH 7.2) and 0.5 mM K + -EGTA (pH 8.0). This term further includes T cell medium, for example, RPMI 1640 medium GlutaMAX(™) Supplement 500 ml (ThermoFisher, 61870010).

[0312] As used herein, the term "ischemia-related disease" is a disease associated with ischemia. Ischemia as used herein is a decrease in blood flow to an organ and / or tissue. The decrease in blood flow can be caused by any suitable mechanism such as partial or complete occlusion (blockage), stenosis (narrowing), and / or leakage / rupture of one or more blood vessels supplying blood to the organ and / or tissue.

[0313] The term "tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder", and "tumor" are not mutually exclusive when referred to herein. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer. In some aspects, the tumor is a solid tumor. In some situations, the tumor is a hematological malignancy (blood tumor).

[0314] The phrase "disease associated with the expression of [target]" includes, but is not limited to, diseases associated with the expression of [target], or conditions associated with cells expressing [target], such as proliferative diseases or precancerous conditions such as cancer or malignant tumors. In one aspect, the cancer is mesothelioma. In one aspect, the cancer is pancreatic cancer. In one aspect, the cancer is ovarian cancer. In one aspect, the cancer is gastric cancer. In one aspect, the cancer is lung cancer. In one aspect, the cancer is endometrial cancer. Indications not related to cancer associated with the expression of [target] include, but are not limited to, autoimmune diseases (e.g., lupus, rheumatoid arthritis, ulcerative colitis), inflammatory disorders (allergies and asthma), and transplantation.

[0315] The term "endogenous" refers to any material produced from or within an organism, cell, tissue, or system.

[0316] The term "exogenous" refers to any material introduced into or produced outside of an organism, cell, tissue, or system. In the case of a patient, the term "exogenous" may refer to materials derived from the patient, donor, or cell culture. For example, mitochondria isolated from a patient's muscle tissue and subsequently introduced into a population of immune cells that may be autologous or autologous to the patient are considered exogenous. The term "exogenous mitochondria" refers to any mitochondria isolated from an autologous source, allogeneic source, and / or xenogeneic source, and the nature of the source may be that of tissue, blood, or cultured cells.

[0317] In the context of the present invention, the following abbreviations are used for cryoprotectants. "CRYO" refers to a commercially available cryoprotectant identified by a code number. For example, CRYO33 represents 70% w / v D-(+)-glucose monohydrate. Examples of commercially available cryoprotectants or cryoprotectants are provided in Table 1b of this specification.

[0318] As used herein, the term "parenteral" administration of a composition, such as a composition containing mitochondria, refers to administration via different routes, for example, subcutaneous (s.c.), transdermal (with a systemic effect), intradermal, intraocular, intravitreal, intranasal, transmucosal, intravenous (i.v.), intramuscular (i.m.), perivascular, intra-articular, intraosseous, epidural, intratracheal, intracerebral, intraventricular, extra-amniotic, intrauterine, intravaginal, intracavitary, intracardiac, intravesical, intraperitoneal, intrasternal, intratumoral, or intralesional (into a skin lesion) injection / infusion.

[0319] As used herein, the term "local" administration of a composition, such as a composition containing mitochondria, refers to administration of the composition or drug to a local area or surface of the body, which has a local effect.

[0320] As used herein, the term "cancer" refers to various types of malignant neoplasms that can mostly infiltrate surrounding cells, tissues, or organs and can metastasize to different sites, as defined by Stedman’s Medical Dictionary 25’ edition (Hen syl ed. 1990). Examples of cancers that can be treated by the present invention include, but are not limited to, brain cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer, bladder cancer, breast cancer, lung cancer, oral cancer, and skin cancer. Other examples of cancers are mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian cancer, mixed Müllerian ovarian cancer, endometrial mucinous ovarian cancer, malignant pleural disease, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct cancer, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, or breast cancer.

[0321] Scope: Throughout the present disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is for mere convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Thus, a description of a range should be considered to specifically disclose all possible sub-ranges as well as the individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95 to 99% identity includes those having 95%, 96%, 97%, 98%, or 99% identity and includes sub-ranges such as 96 to 99%, 96 to 98%, 96 to 97%, 97 to 99%, 97 to 98%, and 98 to 99% identity. This applies regardless of the width of the range.

Examples

[0322] The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments can be implemented in view of the general description provided herein.

[0323] Abbreviations

Table 1b

[0324] Examples of commercially available cryoprotectants are shown in Table 1b above.

[0325] The cryoprotectants described in Table 1b are used to prepare the compositions by diluting solutions of 5%, 10% and 20% (v / v) (see CRYO1 - CRYO47 in FIGS. 3A and 3B and Table 1b), 5%, 10%, 15%, 20%, 25% and 30% (v / v) (see FIG. 3C for CRYO6, CRYO12, CRYO15, CRYO22, CRYO25, and CRYO33) and 10%, 20% and 30% (v / v) (FIGS. 4A - D for CRYO6+CRYO12, CRYO6+CRYO25, CYRO6+CRYO33 and CRYO33+CRYO25) with the following mitochondrial "trehalose - based" isolation buffer (1).

[0326] Preferred cryoprotectants are CRYO13, CRYO14, CRYO15, CRYO16, CRYO17, CRYO18, CRYO22, CRYO23, CRYO25, CRYO38, CRYO46, and / or CRYO47. More preferably, the composition comprises a cryoprotectant selected from the group consisting of CRYO6, CRYO12, CRYO25 and / or CRYO33.

[0327] Isolation buffer (1) "Trehalose - based" aqueous buffer: 10 mM HEPES, 1 mM EGTA, 300 mM trehalose, or (2) "Sucrose - based" aqueous buffer: 10 mM HEPES, 1 mM EGTA, 300 mM sucrose

[0328] Example 1a: Isolation of mitochondria from tissue samples or cultured cells Experiments were conducted to isolate mitochondria from tissue samples.

[0329] Preparation The following solutions were prepared to isolate intact, viable, respiration-competent mitochondria. To successfully isolate mitochondria using the method of the present invention, it is necessary to keep the solutions and tissue samples on ice to preserve mitochondrial viability. Even when maintained on ice, isolated mitochondria will show a decline in functional activity over time (Olson et al., J Biol Chem 242:325-332, 1967). If possible, the following solutions should be prepared in advance. - 1 M K-HEPES stock solution (adjusted to pH 7.2 with KOH). - 0.5 M K-EGTA stock solution (adjusted to pH 8.0 with KOH). - 1 M MgCl2 stock solution (used when ionic components are present in the composition). - 1x PBS (ThermoFisher, 10010031) - The subtilisin A stock was prepared by weighing 2 mg of subtilisin A into a 1.5 mL microcentrifuge tube. Store at -20 °C until use. Prepared at 2 mg / ml in isolation buffer.

[0330] Using the above stock solutions, an isolation buffer with a pH of 7.2 consisting of 300 mM sucrose, 10 mM K-HEPES and 1 mM K-EGTA was prepared and stored at 4 °C.

[0331] Isolation of Mitochondria from Tissue Two 6 mm biopsy fresh sample punches taken from skeletal muscle were transferred to 5 mL of isolation buffer in a gentleMACS C Tube (Miltenyi Biotec, Somerville, Massachusetts), and the samples were homogenized using a 1-minute homogenization program of the gentleMACS™ Dissociator (Miltenyi Biotec). Subtilisin A stock solution (250 μL) was added to the homogenate in the gentleMACS C tube and incubated on ice for 10 minutes. The homogenate was centrifuged at 750 x g for 4 minutes (as an optional step). Thereafter, the homogenate was filtered through a pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge tube on ice. The filtrate was re-filtered through a new pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge on ice. The filtrate was re-filtered again through a new pre-wetted 10 μm mesh filter in a 50 mL conical centrifuge tube on ice. The filtrate was re-filtered through a new pre-wetted 6 μm mesh filter in a 50 mL conical centrifuge tube on ice. The resulting filtrate was transferred to a 1.5 mL microcentrifuge tube and centrifuged at 9000 x g for 10 minutes at 4°C. The supernatant was removed, and the pellet containing mitochondria was resuspended and combined in 1 mL of isolation buffer.

[0332] Example 1b: Isolation of Mitochondria from Cultured Cells An experiment was conducted to isolate mitochondria from cultured cells.

[0333] Preparation The following solutions were prepared to isolate intact, viable, respiration-competent mitochondria. To successfully isolate mitochondria using the methods of the present invention, it is necessary to keep the solutions and tissue samples on ice to preserve mitochondrial viability. Even when maintained on ice, isolated mitochondria will show a decline in functional activity over time (Olson et al., J Biol Chem 242:325-332, 1967). If possible, the following solutions should be prepared in advance. - 1 M K-HEPES stock solution (adjusted to pH 7.2 with KOH). - 0.5 M K-EGTA stock solution (pH adjusted to 8.0 with KOH). - 1x PBS (ThermoFisher, 10010031) - Subtilisin A stock was prepared by weighing 2 mg of subtilisin A into a 1.5 mL microcentrifuge tube. Store at -20 °C until use. Prepared at 2 mg / ml in isolation buffer.

[0334] Using the above stock solutions, an isolation buffer (pH 7.2) was prepared: 300 mM sucrose, 10 mM K-HEPES and 1 mM K-EGTA. Store at 4 °C. - Trypsin (Catalog No. 0103, ScienCell Research Laboratories, Carlsbad, CA).

[0335] Using the above stock solutions, an isolation buffer at pH 7.2 consisting of 300 mM sucrose, 10 mM K-HEPES and 1 mM K-EGTA was prepared and stored at 4 °C.

[0336] Isolation of mitochondria from cultured cells Mitochondria were also isolated from cultured cells, such as the human cardiac fibroblast (HCF) cell line (obtained from ScienCell Research Laboratories, Carlsbad, CA).

[0337] Culture of human cardiac fibroblast (HCF) cells Human cardiac fibroblasts (HCF) were maintained in Fibroblast Medium-2 containing fetal bovine serum, fibroblast growth supplement-2 and antibiotics (penicillin / streptomycin) according to the supplier's instructions (ScienCell). Cells were maintained as a monolayer at 37 °C in a humidified atmosphere of 5% CO2 and passaged when 80% confluence was reached.

[0338] Preparation of human cardiac fibroblast (HCF) cells HCF cells from two flasks (T150) with 80% confluence density were washed once with PBS. The cells were then detached using trypsin according to the supplier's instructions (ScienCell Research Laboratories, Carlsbad, CA). The reaction was stopped by adding trypsin neutralization solution according to the supplier's instructions (ScienCell Research Laboratories, Carlsbad, CA). The cells were collected in 50 mL centrifuge tubes and centrifuged at 1000 rpm (190 xg) for 5 minutes. The supernatant was discarded and a total of three washes with 1x PBS were performed.

[0339] The preparation of culture cells different from HCF should be performed according to the manufacturer's instructions. Notably, the cells used as a source of mitochondria can be adherent, semi - adherent or in suspension.

[0340] Isolation of Mitochondria from HCF Cultured Cells HCF cells from each flask were transferred to 5 mL of isolation buffer (i.e., a "sucrose-based" isolation buffer) in a gentleMACS C Tube (Miltenyi Biotec, Somerville, Massachusetts), and the samples were homogenized using a 1-minute homogenization program on a gentleMACS™ Dissociator (Miltenyi Biotec). Subtilisin A stock solution (250 μL) was added to the homogenate in the gentleMACS C tube and incubated on ice for 10 minutes. The homogenate was filtered through a pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge tube on ice. The filtrate was re-filtered through a new pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge on ice. The filtrate was re-filtered again through a new pre-wetted 10 μm mesh filter in a 50 mL conical centrifuge tube on ice. Optionally, the filtrate was re-filtered again through a new pre-wetted Precellys 5 μm mesh filter in a 50 mL conical centrifuge tube on ice. The resulting filtrate was concentrated by centrifugation. After centrifugation, the filtrate was transferred to 1.5 mL microcentrifuge tubes and centrifuged at 9500 x g for 5 minutes at 4 °C. Three washes were performed at the same centrifugation speed.

[0341] Quantification of Isolated Mitochondria The isolated mitochondria were suspended in the isolation buffer of Example 1b and kept on ice until use. The amount of mitochondria was measured using a Qubit™ Fluorometer (ThermoFisher Scientific / Invitrogen) using the Qubit™ Protein Assay kit according to the manufacturer's instructions in preparation for administration at various doses. For protein concentration measurement, the mitochondria were resuspended in PBS (ThermoFisher, 10010031). The mitochondrial dose was estimated with respect to the protein content expressed in μg.

[0342] Example 2: Survival Rate of Frozen-Thawed Mitochondria Experiments were performed using isolated mitochondria to evaluate their functional state after freezing and thawing under different conditions.

[0343] 2.1 Mitochondrial Isolation Mitochondria were isolated in the same manner as in Example 1b with the slight modifications described below. HCF cells were cultured in FM-2 medium (ScienCell) in a T150 flask (Sarstedt) at 37 °C and 5% CO2. Cells were harvested by trypsinization from two T150 flasks at 90% confluence, washed twice with Gibco PBS, pH 7.4 (Fisher Scientific, catalog number 10010023), homogenized using the 1-minute homogenization program of the gentleMACS™ Dissociator (Miltenyi Biotec), and incubated on ice for 10 minutes with 0.1 mg / ml subtilisin A (a protease from Bacillus licheniformis, Sigma-Aldrich) in isolation buffer containing either sucrose (10 mM HEPES pH 7.2, 1 mM EGTA, 300 mM sucrose) or trehalose (10 mM HEPES, pH 7.2, 1 mM EGTA, 300 mM trehalose). The resulting cell lysate was filtered through two 40-μm filters (Falcon® 40-μm Cell Strainer, Corning, catalog number 352340) and one 10-μm filter (pluriStrainer® 10-μm (Cell Strainer), PluriSelect, catalog number 43-50010-03), and the mitochondria were pelleted at 9500 g for 5 minutes. To remove unbroken cells, it was spun at 500 g for 5 minutes at 4 °C. The protein concentration in the mitochondrial suspension was determined by Qubit Protein assay (Thermo Fisher) as described in Example 1b. The mitochondria were pelleted again and washed twice with isolation buffer.

[0344] 2.2 Freezing of Isolated Mitochondria The isolated mitochondria were frozen under different conditions (i.e., liquid nitrogen or dry ice) and then thawed for subsequent functional analysis.

[0345] Procedure: Isolated mitochondria in pellet form were resuspended in two different isolation buffers containing either 300 mM sucrose or 300 mM trehalose, respectively, and then frozen in 1.5 ml Eppendorf tubes by placing them in liquid nitrogen for about 1 minute or on dry ice for about 3 minutes. For thawing, the tubes were placed in a 37 °C water bath until the entire sample was thawed and then immediately transferred onto ice. Subsequently, the thawed isolated mitochondria were compared to non-frozen controls using a mitochondrial membrane potential assay, an ATP assay, or by assessing cytochrome c release (Figure 1).

[0346] 2.3 Mitochondrial membrane potential assay To evaluate the mitochondrial membrane potential, isolated mitochondria were resuspended in either isolation buffer containing 1 mg / ml sucrose or isolation buffer containing trehalose, and 15 μg of the mitochondrial suspension was used for each sample. Both non-frozen mitochondria and mitochondria frozen and thawed according to the procedure described in Section 2.2 were stored on ice until use. For example, they were stored for 5 minutes to a maximum of 60 minutes, preferably for several minutes (e.g., up to 15 minutes, e.g., 5 minutes or 10 minutes). For Mitotracker Red CMXRos / Mitotracker Green FM staining, a labeling solution was prepared by adding 200 nM MitoTracker Red CMxROS and 200 nM MitoTracker Green FM (Thermo Fisher) into the isolation buffer. Then, the isolated mitochondria were suspended in 1 ml of the labeling solution, vortexed, and incubated in an incubator at 37 °C for 15 minutes. The mitochondria were sedimented by centrifuging at 9500 g for 5 minutes at 4 °C, washed once with each isolation buffer, and resuspended in the isolation buffer at a concentration of 1 mg / ml. Subsequently, 15 μl of the mitochondrial suspension was mixed with 15 μl of 40% PEG8000, and MitoTracker Red (579 / 599 nm) and MitoTracker green (490 / 516 nm) labeled mitochondria were imaged by taking three images / sample at different positions using an automated microscope Keyence BZ-8000. The MitoTracker Red or MitoTracker Green signal was quantified using Keyence BZ-800 Analyzer software, the integrated luminance per image was obtained for calculation, and averaged between the images acquired from one sample.

[0347] Results: This experiment showed that mitochondria frozen with both sucrose-containing isolation buffer and trehalose-containing isolation buffer did not maintain the inner membrane potential (Figure 1A, Figure 1B, Figure 1C, and Figure 1D).

[0348] 2.4 ATP assay ATP measurement was performed using the ATPlite Luminescence Assay System (Catalog No.: 6016941, Perkin Elmer) according to the manufacturer's instructions. 10 μg of mitochondria suspended in isolation buffer (i.e., either "sucrose-based" buffer or "trehalose-based" buffer) was used per sample. Non-frozen mitochondria (control sample) were kept on ice. Mitochondria were frozen by placing them on dry ice, thawed on a 37 °C water bath, pelleted at 4 °C for 5 minutes at 9500 g, resuspended in 50 μl of isolation buffer, and transferred to an OptiPlate-96, White Opaque 96-well Microplate (Catalog No.: 6005290, Perkin Elmer). 25 μl of mammalian cell lysis solution (i.e., Luminescence Assay, Perkin Elmer) was added, and the plate was shaken at 700 rpm for 5 minutes. Subsequently, 25 μl of the lytic substrate solution dissolved in ATPlite buffer (provided in ATPlite Luminescence Assay, Perkin Elmer) was added, followed by further shaking at 700 rpm for 5 minutes. The plate was kept in the dark, and luminescence was measured 10 minutes after mixing. Values were normalized against the control, and the results were shown as the ATP content relative to the control.

[0349] Results: This experiment showed that mitochondria frozen with both sucrose-containing and trehalose-containing isolation buffers did not maintain their ATP content when compared to freshly isolated mitochondria (Figures 1E and 1F).

[0350] 2.5 Cytochrome c release assay Cytochrome c release was evaluated by separating cytochrome c bound to mitochondria from released cytochrome c and analyzed by Western blotting. Mitochondria isolated in either sucrose-based buffer or trehalose-based buffer were frozen / thawed on dry ice / 37 °C water bath, incubated at 37 °C for 15 minutes, and pelleted at 20,000 g for 20 minutes. The supernatant was separated from the pellet and transferred to a clean tube. Both pellet samples and supernatant samples were dissolved in 1x NuPage loading buffer (Thermo Fisher, catalog number NP0008) with a final volume of 20 μl and analyzed using a NuPAGE system containing Bolt™ 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 15 wells (Thermo Fisher, NW00125BOX) and NuPAGE™ MES SDS Running Buffer (20X) (Thermo Fisher, NP0002). Proteins were blocked with 5% non-fat milk dissolved in phosphate buffered saline containing 0.1% Tween-20 (Thermo Fisher catalog number 14190144) using a wet blotting technique with a Mini-PROTEAN® Tetra system (BioRad catalog number 1703930). Lower Blotter Pre-cut Membranes and Filters, PVDF, normal size (Thermo Fisher PB9320) were transferred and cytochrome c was detected using a primary anti-cytochrome c antibody (Proteintech, catalog number 10993-1-AP) and a secondary (SA00001-2) antibody, including SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher catalog number 34577) and CL-XPosure™ Film, 5x7 inch (Thermo Fisher catalog number 34090) film. (13x18 cm).

[0351] Results: Mitochondria frozen and thawed in sucrose-containing isolation buffer do not maintain the integrity of the outer membrane, while mitochondria frozen and thawed in trehalose-containing isolation buffer maintain the integrity of the outer membrane (Figure 1G).

[0352] Example 3: Effect of Freezing and Thawing Rates on Mitochondrial Survival Mitochondria were isolated as described in Example 2.1 and treated as described in Example 2.2, except that only trehalose-based isolation buffer (10 mM HEPES-KOH, pH 7.2, 1 mM EGTA, 300 mM trehalose) was used in all experiments. Mitochondrial membrane potential was evaluated by MitoTracker staining, and ATP content was determined as described in Examples 2.3 and 2.4.

[0353] First, mitochondria were repeatedly frozen / thawed on dry ice / 37 °C water bath and tested by MitoTracker staining and ATP assay as described in Example 2. Next, mitochondria were frozen under different conditions (i.e., either placing them on dry ice, in liquid nitrogen, or first on dry ice and then transferring them to liquid nitrogen). Mitochondrial survival was estimated using MitoTracker staining and ATP assay as in Examples 2.3 and 2.4. Finally, isolated mitochondria were frozen on dry ice and thawed in a 37 °C water bath or at room temperature, or by placing them on ice, and ATP content was determined using an ATP assay.

[0354] Results: Mitochondria frozen and thawed in trehalose-containing isolation buffer were damaged functionally and structurally to different extents depending on the freezing temperature rate and thawing temperature (Figures 3A - 3G).

[0355] Example 4: Survival of Frozen-Thawed Mitochondria Contained in a Composition Containing One or More Cryoprotectants 4.1 Integrity of the Inner Mitochondrial Membrane of Frozen-Thawed Mitochondria. The mitochondria isolated according to Example 2.1 were resuspended in a "trehalose-based" isolation buffer. As described in Example 2.3, the effect of cryoprotectant addition on maintaining the integrity of the inner mitochondrial membrane was studied using a MitoTracker staining assay. The mitochondria were added to the mitochondria of a single cryoprotectant selected from the list of cryoprotectants provided in Table 1b and then frozen on dry ice. The cryoprotectants had three different concentrations of 5% (v / v), 10% (v / v), and 20% (v / v). The mitochondria of the control sample were frozen on dry ice without cryoprotectant, and the unfrozen mitochondria were kept on ice (4 °C). All frozen samples were thawed in a 37 °C water bath and analyzed by MitoTracker staining. The MitoTracker Red signal was quantified and normalized to the signal of the non-frozen control, and the results are shown in Figure 3A in the form of a heat map. The Mito Tracker Red / Green staining and MitoTracker Red / Green signal assay of four selected cryoprotectants (i.e., CRYO12, CRYO06, CRYO33, and CRYO25) are shown in Figure 3B.

[0356] Results: The Mito Tracker Red staining assay shows that the cryoprotectants CRYO12, CRYO06, CRYO33, and CRYO25 maintain the integrity of the inner mitochondrial membrane.

[0357] 4.2 ATP content of freeze-thawed mitochondria. 4.2.1 Use of a single cryoprotectant. Different cryopreservation compositions were prepared by increasing the concentration of a single cryoprotectant selected from the group consisting of proline, sucrose, glucose, PEG400, PEG1000, and pentaerythritol propoxylate by adding CRYO6, CRYO12, CRYO15, CRYO22, CRYO25, and CRYO33 to the mitochondria resuspended in a trehalose-based isolation buffer. The resulting samples were then frozen on dry ice (-78 °C approximately) and thawed in a 37 °C water bath. The ATP content was determined in comparison to a non-frozen control (maintained on ice at approximately 4 °C).

[0358] Result: It has been shown that the mitochondrial inner membrane potential and ATP content depend on the type and concentration of cryoprotectant (Figure 3C).

[0359] 4.2.2 Use of two cryoprotectants. Different cryopreservation compositions were prepared by increasing the concentration of one of the two cryoprotectants contained in each composition. The cryoprotectant was selected from the group consisting of proline, sucrose, glucose, or PEG400.

[0360] Each composition contained isolated viable mitochondria, a buffer consisting of 10 mM HEPES-KOH (pH 7.2), a calcium chelator consisting of 1 mM EGTA, trehalose at a concentration of 300 mM, and one or two cryoprotectants at specific concentrations described in Table 2.

Table 2

[0361] These compositions were then added to isolated mitochondria (10 μg per sample in triplicate as described in Example 2.4) pre-suspended in a trehalose-containing isolation buffer as described in Example 2. The resulting samples were then frozen in dry ice (-78 °C approximately) and thawed in a water bath at 37 °C. The ATP content was determined by comparison with non-frozen controls (maintained on ice (4 °C approximately)). The ATP assay was performed according to the procedure provided in Example 2.

[0362] Result: It has been shown that the mitochondrial inner membrane potential and ATP content depend on the type and concentration of cryoprotectant (Figures 4A - 4D).

[0363] Example 5: Effect of Controlled Freezing Rate on Mitochondrial Viability Mitochondria were isolated from HCF cells as described in Example 2 and resuspended in a trehalose-based isolation buffer supplemented with a cryoprotectant (10 mM HEPES, pH 7.2, 1 mM EGTA, 300 mM trehalose, 1.2 M proline, 353 mM D-glucose). The protein content was determined by Qubit assay as described in Example 1b. 100 μg of mitochondria were transferred to a 1.5 ml Eppendorf tube and frozen in an -80 °C freezer. These samples were later used for normalizing the mitochondrial content between different mitochondrial isolations. The remaining mitochondria were diluted to a concentration of 0.166 μg / μl in a trehalose-based isolation buffer supplemented with a cryoprotectant (10 mM HEPES, pH 7.2, 1 mM EGTA, 300 mM trehalose, 1.2 M proline, 353 mM D-glucose). 50 μl of this mitochondrial suspension was loaded into 12 wells of a Greiner 96 well μFT flat bottom microplate (i.e., 100 μg of mitochondrial suspension in the plate). The microplate was attached to an ILK multi-well rack heat exchanger and frozen in a SY-LAB IceCube 15M controlled rate freezer using a sample-specific temperature program. After pre-cooling to -5 °C within 17 minutes, ice nucleation was mechanically induced and freezing was carried out at a sample-specific cooling rate of 2 ± 0.1 K / min, 6 ± 0.3 K / min or 10 ± 0.5 K / min (i.e., Kelvin / min). The sample-specific temperature program was developed by iterative adjustment of the IceCube temperature envelope for the desired sample freezing rate monitored at different positions of the microplate using 20 Omega TJC2-NNIN-IM050U-800 0.50 mm type N thermocouples operated by a Delphin ExpertKey 200 measurement system. The frozen plates were stored at -80 °C for 1 month and then analyzed using ATP assay and MitoTracker staining as described in Example 2. To normalize for differences in mitochondrial amount caused by variation between mitochondrial isolations, the ATP content was also measured in samples frozen in Eppendorf tubes at -80 °C.For MitoTracker Red / MitoTracker green staining, fresh mitochondria were isolated, and the mitochondrial staining intensity was compared between fresh mitochondria and mitochondria frozen at a controlled rate, and stored for one month.

[0364] Results: The results demonstrate that mitochondria frozen at a rate of 10K / min have a higher ATP content and a higher membrane potential compared to mitochondria frozen at slower freezing rates (2K / min and 6K / min). Furthermore, this experiment shows that a composition containing isolated viable mitochondria can be frozen by the method described in Example 5 and stored for at least one month.

[0365] Example 6: In Vivo Coronary Blood Flow Experiment Mitochondria were isolated as described in Example 2 and resuspended at a concentration of 10 mg / ml in a freezing preservation buffer (7 mM HEPES, pH 7.2, 0.7 mM EGTA, 210 mM trehalose, 1.2 M proline, and 353 mM D - glucose). Tubes were placed on dry ice and subsequently stored at -80° for up to one week, frozen in 1.5 ml Eppendorf tubes with a 500 μg aliquot. For each in vivo experiment, the desired number of aliquots (as shown in the examples) were thawed in a 37°C water bath and immediately diluted with 6.5 ml of a sucrose - based isolation buffer. The mitochondria were injected intracoronarily into the hearts of healthy anesthetized pigs (Sus scrofa) within 5 minutes of thawing. In certain experiments (as shown in the examples), mitochondria were freshly isolated from HCF cells (described in Example 2) or skeletal muscle tissue of animals (described in Example 1a), stored on ice, and subsequently injected exactly as described above for the frozen mitochondria.

[0366] Results: In pre - clinical trials in pigs, a composition containing isolated mitochondria frozen under the conditions described in Example 6 has the same effect on coronary blood flow as freshly isolated mitochondria.

[0367] Example 7: Mitochondrial translocation increases the killing ability of CAR-T cells Transplant mitochondria suspended in a solution containing 1.2 M proline, 353 mM glucose, 210 mM trehalose, 0.7 mM K-EGTA, and 7 mM K-HEPES (pH 7.2) into CAR-T cells and subject them to a freeze-thaw cycle in advance.

[0368] To evaluate the effect of mitochondrial transplantation on CAR-T cell killing ability, a FACS-based killing assay is performed 24 hours after transplantation. CAR-T cells (CD19-41BB-CD3z, PMC746) are purchased from ProMab Biotechnologies (Richmond, CA 94806). The proportion of target cells expressing early markers of apoptosis is evaluated by the proportion of Annexin V+, Annexin V+ / PI+, or PI+ to measure apoptosis or translocation to apoptosis, respectively. Transplanted or non-transplanted CAR-T cells are plated at an effector-to-target ratio of 5 to 1. CAR-T cells are co-incubated with target cells Daudi (ATCC CCL-213), a B-lymphoblastoid cell line expressing CD19, for 4 hours. After co-incubation, the cells are harvested and stained with anti-human CD8 PerCP-Cy5.5 (Stemcell, 60022PS) and anti-human CD3 APC (Stemcell, 60011AZ) for 20 minutes at 4°C. After washing with FACS buffer, the cells are stained with Annexin V FITC (Biolegend, 640914) and Propidium Iodide (PI, Biolegend, 640914) for 15 minutes at room temperature according to the supplier's instructions and acquired on a FACSLyric (BD Biosciences).

[0369] Procedure (i) CAR-T cell culture is performed in the presence of 100 U / ml of recombinant human IL-2 (Peprotech, 200-02). The CAR-T cells are cultured in RPMI 1640 Medium GlutaMAX™ Supplement 500 ml (ThermoFisher, 61870010) supplemented with 1% L-glutamine (ThermoFisher, 25030024), 1% penicillin-streptomycin (10,000 U / mL, Gibco, 15140122), 1% non-essential amino acids (NEAA, ThermoFisher, 11140050), 1% sodium pyruvate (ThermoFisher, 11360070), 10% fetal bovine serum and 0.1% 2β-mercaptoethanol (Gibco, 31350-010). (ii) Mitochondrial isolation: Mitochondria are isolated from human cardiac fibroblasts (HCF) according to the procedure described in Example 1b. (iii) Quantification of isolated mitochondria: The mitochondrial dosage is estimated with respect to the protein content expressed in μg according to the procedure described in Example 1b.

[0370] Mitochondrial transplantation enhances the killing ability of CAR-T cells. The percentage of target cells expressing early markers of apoptosis, markers during the transition to apoptosis, or late markers of apoptosis increases upon co-incubation with the transplanted CAR-T cells.

[0371] Example 8: Therapeutic Mitochondrial Transplantation (TMT) - Preclinical Study in Pigs The cryopreserved mitochondria are tested in an animal model of cardiac ischemia-reperfusion injury. The experiment is conducted in an established porcine model of regional ischemia-reperfusion injury. The mitochondria are isolated from human cardiac fibroblasts as described in Example 1b. The isolated mitochondria are resuspended in 35 μl of trehalose-based mitochondrial isolation buffer (300 mM trehalose, 1 mM EGTA, 10 mM HEPES, pH 7.2). 10 μl of 3.53 M D-glucose and 5 μl of 6 M L-proline are added to the mitochondrial suspension, incubated for 2 minutes, and the tube is placed on dry ice for freezing. After 10 minutes, the tube is transferred to a -80°C storage box and kept frozen until use. For the animal experiment, myocardial ischemia is induced in healthy female pigs (Sus scrofa) by occluding the coronary artery (LAD) for 90 minutes. The cryopreserved mitochondria (stored at -80°C for no more than 2 weeks) are rapidly thawed by placing them in a metal block heated to 37°C and immediately resuspended in 5 ml of sucrose-based mitochondrial isolation buffer (300 mM sucrose, 1 mM EGTA, 10 mM HEPES, pH 7.2) and injected into the porcine coronary artery within 15 minutes after reperfusion. Blood samples are collected on days 0, 3, 30, and 90, and infarct biomarkers (such as creatine kinase-myocardial band (CKMB) or troponin I) in the serum are measured. Cardiac function is evaluated by magnetic resonance imaging (MRI) on days 3, 30, and 90 from the start of treatment by determining functional parameters such as end-systolic volume, end-diastolic volume, and ejection fraction. The pigs are sacrificed and histological samples from the heart and other organs are collected for analysis. The therapeutic effect is evaluated by a decrease in serum infarct biomarkers, a decrease in infarct size, and an increase in ejection fraction compared to the control group.

[0372] Results: Interim analyses on days 3 and 28 showed the following: In animals treated with the composition stored at -80°C for up to 2 weeks and thawed before use (n = 2) and animals receiving an equal volume of vehicle, the ejection fraction was measured using magnetic resonance imaging (MRI). MRI was performed on the 3rd and 28th days after ischemia. Preliminary results showed that the ejection fraction decreased between the 3rd and 28th days in 2 out of 3 animals (67%) administered the vehicle, indicating impairment of cardiac function, while in both animals treated with mitochondria, the ejection fraction increased, indicating improvement.

[0373] Example 9: Spray Freezing of a Cryopreserved Composition Containing Isolated Mitochondria Mitochondria are isolated from an HEPG2 cell line stably expressing mitochondrial-targeted green fluorescent protein (GFP) in the same manner as in Example 1. The isolated mitochondria are resuspended in 350 μl of trehalose-based mitochondrial isolation buffer (300 mM trehalose, 1 mM EGTA, 10 mM HEPES, pH 7.2). Then, 100 μl of 3.53 M D-glucose and 50 μl of 6 M L-proline are added to the mitochondrial suspension. The composition formed after the addition of glucose and L-proline is then spray frozen, i.e., the composition is sprayed in the form of droplets directly into liquid nitrogen using a spraying device. Then, the liquid nitrogen is evaporated at room temperature, and the remaining frozen composition is collected in an Eppendorf tube pre-cooled with dry ice. Thawing of the frozen composition is performed by placing the tube in a metal rack pre-heated to 37°C. The thawed composition is diluted to 1.5 ml with trehalose-based mitochondrial isolation buffer (300 mM trehalose, 1 mM EGTA, 10 mM HEPES, pH 7.2), precipitated by centrifugation at 9500 g for 5 minutes, and resuspended in 20 μl of trehalose-based mitochondrial isolation buffer.

[0374] After thawing, the suspension containing mitochondria is added to the wells of a 96-well plate containing cultured human cardiac fibroblasts and incubated for 24 hours. The internalization of the freeze-thawed GFP-labeled mitochondria is observed using a fluorescence microscope after washing the cultured cells four times with warm cell culture medium. This experiment demonstrates that mitochondria contained in the composition spray-frozen in liquid nitrogen (i.e., the cryopreservation composition containing trehalose, glucose, and proline) maintain the ability to be internalized by living cells.

[0375] Other embodiments The above disclosure may encompass a plurality of distinct inventions having independent utility. Each of these inventions is disclosed in its preferred form, but the specific embodiments thereof disclosed and illustrated herein should not be considered in a limiting sense, as many variations are possible. The subject matter of the present invention includes all novel and non-obvious combinations and sub-combinations of the various elements, features, functions, and / or characteristics disclosed herein. The following claims particularly point out certain combinations and sub-combinations that are considered novel and non-obvious. Inventions embodied in other combinations and sub-combinations of features, functions, elements, and / or characteristics may be claimed in this application, an application claiming priority from this application, or a related application. Such claims are considered to be within the scope of the subject matter of the inventions disclosed herein, regardless of whether they are directed to different inventions or the same invention, and regardless of whether the scope is broader, narrower, equal to, or different from the original claims.

Claims

1. Isolated human mitochondria, (i) an aqueous buffer having a pH of 5.5 to 8.5, (ii) Trehalose at a concentration of at least 150 mM, (iii) A first cryoprotective agent selected from one or more (a) amino acids in a total concentration of at least 160 mM, A composition containing the following:

2. (iv) A second cryoprotectant selected from one or more (b) sugars, (c) polymers, or combinations thereof, This further includes, and here, (b) The sugar has a total concentration of at least 150 mM, and (c) The polymer having a total concentration of 2.5% (w / v) to 30% (w / v) The composition according to claim 1.

3. (i) The aqueous buffer contains a buffer selected from 2-[4-(2-hydroxyethyl)-piperazine-1-yl]-ethanesulfonic acid (HEPES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 4-morpholineethanesulfonic acid (MES), bis-(2-hydroxyethyl)amino-tris-(hydroxymethyl)-methane (bis-tris), 2-(N-cyclohexylamino)-ethanesulfonic acid (CHES), N,N-bis-(2-hydroxyethyl)-glycine (bicine), potassium phosphate, sodium cacodylate, tris-(hydroxymethyl)aminomethane hydrochloride) (tris), 4-morpholinepropanesulfonic acid (MOPS), 1,3-bis-[tris-(hydroxymethyl)-methylamino]-propane (bis-trispropane), sodium acetate, or a combination thereof, The composition according to claim 1, wherein the buffer in the aqueous buffer has a concentration of 0.5 mM to 50 mM.

4. It further contains a calcium chelating agent, and the calcium chelating agent is Selected from the group consisting of ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2,2',2'',2''-(ethane-1,2-diyldinitrilo)-tetraacetic acid (EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis-(acetoxymethyl ester) (BAPTA-AM) or combinations thereof, and / or The composition according to claim 1, having a concentration of 0.1 mM to 10 mM.

5. It further contains an ionic component, and the ionic component is At concentrations of 0.1 mM to 100 mM, and / or Mg ２＋ 、 Na ＋ 、 K ＋ , Cl － 、 HCO ３ － selected from salts, acids, or bases, or MgCl ２ . MgSO ４ 、 KCl、 KH ２ PO ４ 、 NaHCO ３ 、 Na ２ HPO ４ 、 formate anion, C ２ H ２ MgO ４ (magnesium formate), pyruvate anion, C ３ H ３ NaO ３ (sodium pyruvate), acetate anion, C ２ H ３ NaO ２ (sodium acetate), malate anion, oxaloacetate anion, glutamate anion, α-ketoglutarate anion, succinate anion, or a combination thereof, the composition according to claim 1.

6. The composition according to claim 1, further comprising albumin in a concentration of 0.01% (w / v) to 10% (w / v), or comprising albumin in a concentration of 0.01% (w / v) to 10% (w / v), wherein the albumin is bovine serum albumin (BSA), human serum albumin (HSA), or a combination thereof.

7. (i) The buffering agent is The pH is 7.4, and the solution contains 20 mM Tris, 2 mM EDTA, and 10 mM MgCl. ２ including, It has a pH of 7.25 and contains 5 mM MOPS, 10 mM BAPTA, and 5 mM sodium pyruvate, or It has a pH of 7.2 and contains 10 mM HEPES and 1 mM EGTA. The composition according to claim 1.

8. The composition according to claim 1, comprising less than the amount required for freezing, propylene glycol, ethylene glycol, glycerol, and dimethyl sulfoxide (DMSO), or not comprising propylene glycol, ethylene glycol, glycerol, and dimethyl sulfoxide (DMSO).

9. (a) The amino acid is selected from leucine, isoleucine, proline, methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, valine, alanine, glycine, asparagine, aspartic acid, glutamic acid, serine, histidine, cysteine, tryptophan, tyrosine, arginine, glutamine, lysine, threonine, selenocysteine, methionine, phenylalanine, creatine, taurine, betaine, ectoin, dimethylglycine, ethylmethylglycine, RGD peptide, or a combination thereof, or (a) The amino acid is selected from methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof, (a) The composition according to claim 1, wherein the amino acid is proline.

10. (a) The amino acid has a concentration of at least 180 mM or at least 200 mM, (a) The composition according to claim 1, wherein the amino acid has a concentration of at least 1000 mM.

11. (b) The composition according to claim 2, wherein the sugar is selected from monosaccharides, disaccharides, trisaccharides, or combinations thereof.

12. (b) The sugar is selected from maltose, lactose, fructose, sucrose, glucose, dextran, meletitose, raffinose, nigerotriose, maltotriose, maltotriulose, kestose, cellobiose, chitobiose, lactulose, or a combination thereof, (b) The composition according to claim 2, wherein the sugar is selected from sucrose, glucose, or a combination thereof.

13. (c) The polymer is A biocompatible, hydrophilic, amphiphilic polymer, or a combination thereof, Poloxamers, for example, poloxamer 142, poloxamer 188, poloxamer 331 or poloxamer 407, arginate, polyethylene glycol (PEG), polyglutamic acid, polyvinyl alcohol, polyvinylpyrrolidone, or combinations thereof, The composition according to claim 2.

14. (iii) The first cryoprotectant is one or more (a) amino acids selected from proline, methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or combinations thereof, and (iv) The second cryoprotectant is selected from one or more (b) sugars, (c) polymers, or a combination thereof. (b) The sugar is glucose, sucrose, or a combination thereof, (c) The polymer is polyethylene glycol (PEG), The composition according to claim 2.

15. (a) The amino acid is proline at a concentration of at least 500 mM or at least 1000 mM. The composition according to claim 14.

16. The amino acid is proline at a concentration of at least 1300 mM or at least 1600 mM. The composition according to claim 14.

17. The aforementioned amino acid is proline, The aforementioned sugar is either glucose at a concentration of 200 mM to 1300 mM, or sucrose at a concentration of 160 mM to 900 mM. The composition according to claim 14.

18. The aforementioned amino acid is proline, The polymer is polyethylene glycol (PEG) at a concentration of 5% (w / v) to 30% (w / v). The composition according to claim 1.

19. The composition according to claim 1, wherein the cryoprotectant comprises proline and is at the concentration specified in claim 15.

20. The composition according to claim 19, wherein the concentration of proline is 600 mM or 1200 mM.

21. The composition according to claim 1, wherein the cryoprotectant comprises one or more (a) amino acids selected from methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or combinations thereof, in a total concentration of at least 200 mM.

22. The aforementioned cryoprotectant consists of one or more (a) amino acids, The amino acid is selected from methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof, and is at a concentration of at least 300 mM or at least 500 mM. The composition according to claim 1.

23. The aforementioned cryoprotectant consists of one or more (a) amino acids, The composition according to claim 1, wherein the amino acid is selected from methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof, and is at a concentration of at least 1000 mM or at least 1500 mM.

24. The composition according to claim 1, wherein the mitochondria are isolated from a cell, tissue, or organ.

25. The composition according to claim 1, wherein the isolated mitochondria have a concentration of at least 0.02 μg / μL.

26. The composition according to claim 1, wherein the isolated mitochondria have a concentration of 100 μg / μL or less.

27. The isolated mitochondria described above (i) Pharmaceutical agents, diagnostic agents, imaging agents, therapeutic agents, or any other biocompatible agents, (ii) Antibodies, or (iii) Antigen-binding fragment which is at least a part of an antibody or TCR or its recombinant variant, The composition according to claim 1, which is connected to the

28. The composition according to claim 27, wherein the mitochondria are linked to the drug, antibody, or antigen-binding fragment by covalent or non-covalent bonds.

29. The drug, the antibody, or the antigen (i) embedded in the mitochondria, embedded in the mitochondrial membrane, substantially encapsulated within the mitochondria, or completely encapsulated by the mitochondria, (ii) The composition according to claim 27, which is connected to the mitochondrial outer membrane by covalent, non-covalent, or electrostatic bonds.

30. The isolated mitochondria described above (i) Mitochondria modified by gene editing, or (ii) The composition according to claim 1, comprising exogenous mtDNA.

31. A method for cryopreserving a composition comprising isolated mitochondria as described in claim 1, wherein the method is: (a) A step of freezing the composition at a temperature below 0°C, (b) A step of storing the frozen composition obtained in step (a) at a temperature below 0°C, Methods that include...

32. (c) A step of thawing the frozen composition at a temperature above 0°C. The method according to claim 31, further comprising:

33. (a) Freezing is performed at a temperature below -20°C or below -100°C. The method according to claim 31.

34. (a) Freezing is carried out in liquid nitrogen at a temperature of -196°C or in dry ice at a temperature of -78.5°C. The method according to claim 31.

35. (a) Freezing is rapid freezing, The method according to claim 31.

36. (a) Freezing is a stepwise freezing at a rate of at least 5°C / min. The method according to claim 31.

37. (b) Storage period is at least 24 hours or at least 1 week before thawing. The method according to claim 31.

38. (c) Thawing is performed at a temperature higher than 4°C but lower than 40°C. The method according to claim 31.

39. The composition according to claim 1, in a therapeutically effective amount for use in the treatment of a disease.

40. In the object that is required (i) Mitochondrial or mitochondrial-related diseases, (ii) Cancer or tumor, (iii) autoimmune disease; (iv) Ischemia-related injury, ischemia-reperfusion injury of the lungs, kidneys, heart, or brain, or (v) occlusion of blood vessels, The composition according to claim 39 for use in the treatment of [unspecified condition].

41. The composition according to claim 39, for use in gene therapy, or in gene therapy for the treatment of cancer, infectious diseases, or autoimmune diseases.

42. The composition is (i) By local administration or parenteral administration, (ii) By direct injection into blood vessels, tissues, or organs, (iii) In the form of an aerosol, It is administered to those who need it. The composition according to claim 39 for use in treating a disease in the aforementioned target.

43. The composition according to claim 1, wherein the mitochondria contained in the composition are self, homogeneous, or heterogeneous.

44. The composition according to claim 39, wherein the composition is subjected to at least a freeze-thaw cycle before being used to treat the disease.

45. A method for cryopreserving viable mitochondria using the composition described in Claim 1.

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