Compositions and methods for cryopreservation of mitochondria

Abstract

The present disclosure relates to compositions and methods for cryopreservation of mitochondria, cryopreserved compositions comprising mitochondria, and therapeutic uses thereof.

Description

Technical Field

[0001] The present application relates to compositions and methods for cryopreserving compositions comprising isolated viable mitochondria. The present application also relates to cryopreservation compositions for the methods described herein, and cryopreservation compositions comprising isolated viable mitochondria obtained using the methods described herein. These cryopreservation compositions comprising mitochondria exhibit advantageous and valuable long shelf lives and are ready-to-use for a variety of uses, which may include, but are not limited to, research, diagnostic, and therapeutic applications. Background Art

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

[0003] Mitochondria are composed of two concentric membranes with specific functions. The inner mitochondrial membrane contains proteins required for respiratory chain function and ATP synthesis. The outer mitochondrial membrane contains a large number of integral membrane proteins, encapsulating the entire organelle. The structure of mitochondria is strikingly similar to that of some modern prokaryotes. In fact, mitochondria are thought to have originated from ancient symbiotic relationships, when nucleated cells engulfed aerobic prokaryotes. In the symbiotic relationship, the host cell began to rely on the engulfed prokaryotic organism for energy production, and the prokaryotic cell began to rely on the protective environment provided by the host cell (The Origin of Mitochondria. Nature Education (2010), 3(9):58, Martin W. & Mentel M.)

[0004] As ubiquitous semi-autonomous cell organelles, mitochondria are separated from the cytoplasm by the mitochondrial outer membrane and the inner membrane. The outer membrane is porous, and ions and small uncharged molecules pass freely through pore-forming membrane proteins (porins), such as voltage-dependent anion channels (VDAC). Any larger molecules, especially proteins, must be imported through special translocases. Due to the porosity of the outer membrane, there is no membrane potential on the outer membrane. In contrast, the inner membrane is a strict diffusion barrier for all ions and molecules. These ions and molecules can only pass through with the help of specific membrane transporters, each of which is selective for specific ions or molecules. Due to its ion selectivity, an electrochemical membrane potential is potentially formed on the mitochondrial inner membrane. The inner membrane is where a series of membrane protein complexes undergo oxidative phosphorylation, which produces an electrochemical gradient on the inner membrane or is used for ATP synthesis. The outer membrane separates mitochondria from the cytoplasm. It surrounds the inner membrane, which separates the intermembrane space from the protein-dense central matrix. The inner membrane is differentiated into an inner boundary membrane and cristae. The two regions are continuous at the junction of the cristae. The cristae extend more or less deep into the matrix and are the main site of mitochondrial energy conversion. Due to the major role of mitochondria in cellular metabolism, damage and dysfunction of mitochondria can lead to a range of human diseases.

[0005] Mitochondria are subcellular organelles that are essential for the life of cells. Disruption of normal mitochondrial function is detrimental to cell viability. Mitochondrial disorders can be caused by mutations (acquired or inherited) in mitochondrial DNA (mtDNA) or in nuclear genes encoding mitochondrial components. Inherited 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, myoneurogenic gastrointestinal encephalopathy (MNGIE), Leber's hereditary optic neuropathy (LHON) and mitochondrial DNA depletion syndrome (Mol Syndromol (2016), 7:122–137, Nyazov D.M. et al.). Mitochondrial damage can also be caused by injury, toxicity, chemotherapy, infection and age-related changes. Reduced blood flow in tissues and organs can produce mitochondrial damage. In particular, ischemia / reperfusion injury can lead to mitochondrial damage, which will have a negative impact 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 ET). In general, mitochondria or mitochondrial-related diseases / disorders are chronic (long-term) diseases / disorders. The symptoms of mitochondrial diseases can be different. This depends on how many mitochondria are defective and their location in the body. Sometimes only one organ, tissue or cell type is affected. But the problem usually affects many of them. Muscles and nerve cells have particularly high energy requirements, so muscle and nerve problems are common. The disease ranges from mild to severe. Some types may 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 broken red fiber syndrome, as well as dysfunction or disease after stroke and traumatic brain or spinal cord injury (Mitochondrion (July 2017), 35: 70-79, Gollihue and Rabchevsky). Drugs can induce mitochondrial dysfunction through different mechanisms, including inhibition of fatty acid oxidation, impairment of oxidative phosphorylation and respiratory chain activity, and changes in mitochondrial membrane integrity. Some drugs also impair mitochondrial function by generating reactive oxygen species and generating reactive metabolites, which can covalently bind to key mitochondrial proteins. Drug-induced mitochondrial dysfunction plays an important role in the pathogenesis of adverse effects, such as liver damage, myopathy, and cardiotoxicity. Some examples of drugs that can induce mitochondrial dysfunction include, for example, acetaminophen, amiodarone, doxorubicin, nucleoside reverse transcriptase inhibitors (e.g., stavudine, zidovudine, didanosine), 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 treating mitochondrial or mitochondrial-related diseases and disorders, hereditary or acquired mitochondrial dysfunction, cancer, infectious diseases, inflammatory diseases, autoimmune diseases, ischemia, ischemia-related dysfunction, ischemia-reperfusion injury or thrombotic obstruction and other diseases and dysfunctions mentioned above. Particularly important are emerging treatments, such as therapeutic mitochondrial transplantation (J Transl Med, (May 17, 2021), 19 (1): 214, Hayashida K.; EMBO Rep. 2020 Sep 3; 21 (9): e50964, published online on August 2, 2020, RN Lightowlers et al.; J. et al.; Int J Mol Sci. 2021 May; 22 (9): 4793, published online on April 30, 2021, A. Park et al.).).

[0007] Cryopreservation of subcellular organisms / organelles is a process that allows the preservation of subcellular organisms (e.g., mitochondria) by cooling a sample to very low temperatures after the subcellular organisms (e.g., mitochondria) have been separated from cells. Mitochondria, which have great potential for application in basic research and many medical applications, cannot be preserved for long periods of time by simple cooling or freezing because ice crystal formation (e.g., intracellular ice formation and / or dendritic ice crystal formation), osmotic shock (e.g., solution effect damage described by Peter Mazur (1977)) and membrane damage during freezing and thawing can lead to mitochondrial damage or death. Many attempts have been made to cryopreserve mitochondria using cryoprotectants and temperature control equipment (Biochim. Biophys. Acta (1961) 50: 233-242, D. Greiff, M. Myers, CA Privitera; Nature (1961) 190: 1202-1204, Greiff, D. and M. Myers). However, cryopreservation known in the art causes mitochondrial damage during freezing, freezing-thawing and low-temperature storage. This degradation has a deleterious effect on the activity and viability of mitochondria, and therefore affects their potential use for research, diagnosis or therapeutic purposes. Being unable to store mitochondria over a long period of time severely limits the possible use of mitochondria, particularly in a clinical setting, where there is usually not enough time to prepare mitochondria freshly. In addition, mitochondrial separation is time-consuming, and is a process that is difficult to scale up in a single laboratory or hospital, resulting in high preparation costs. This is particularly problematic for therapeutic purposes, in which the preparation of products for patients must comply with good manufacturing practices (Good Manufacturing Practice). Therefore, there is still a strong need for compositions and methods for cryopreservation of mitochondria that can maintain mitochondrial structure and / or functional integrity (preferably both structural integrity and functional integrity of mitochondria).

[0008] It is extremely advantageous to have a composition comprising isolated mitochondria that can be stored for a long period of time and can be used at any time as needed for different purposes, such as basic research and / or medical applications. Having a ready-made (off-the-shelf) composition comprising healthy mitochondria will make it convenient and effective to administer viable mitochondria to patients (e.g., by transplanting mitochondria, for example, by replacing defective mitochondria in cells or tissues, or by enriching cells and tissues affected by diseases or dysfunctions with healthy mitochondria). Therefore, mitochondrial therapy will become a routine procedure, making it possible to prevent, improve or eliminate symptoms related to mitochondrial dysfunction or insufficient numbers. Therapeutic mitochondrial transplantation (TMT) has the potential to continuously affect mitochondrial function, revitalize or enhance cellular energy metabolism. For example, McCully et al. describes that mitochondrial transplantation can be effective in a variety of cell types and diseases. These include cardiac and skeletal muscle, lung and liver tissues and cells, and neural tissue. McCully foresees that mitochondrial transplantation will become a valuable treatment in the armamentarium of all clinicians and surgeons for treating a variety of ischemic conditions, mitochondrial diseases, and related conditions (Clin Trans Med (2016) 5:16, McCully et al.).

[0009] For such therapeutic use at the bedside of a patient in need of treatment, a ready-to-use composition comprising mitochondria would simplify the overall clinical procedure, making it, for example, safer, faster, more consistent, and easier to perform.

[0010] Since mitochondria have a double membrane structure and are more fragile than cells, although a variety of cryopreservatives or cryopreservative mixtures for cell preservation are known, these cryopreservatives are not suitable for freezing mitochondria. For this reason, the industry is actively trying to develop cryopreservatives specifically for mitochondria. However, during mitochondrial cryopreservation, mitochondria still very frequently swell, become damaged or even ruptured, preventing the mitochondria from functioning normally, such as after thawing. Therefore, protecting mitochondria from damage during cryopreservation remains an unresolved problem.

[0011] Mitochondria play an important role in the regulation of apoptosis. Permeabilization of the mitochondrial outer membrane is a key step in the apoptotic process. The outer membrane of isolated mitochondria tends to degenerate over time and rupture during freeze-thaw. By analyzing the retention of cytochrome c over time at different temperatures, Yamaguchi et al. showed that mitochondria frozen in a buffer containing trehalose maintained mitochondrial outer membrane integrity (Yamaguchi et al., Cell Death and Differentiation (2007), (14): 616-624). Yamaguchi et al. also attempted to prove that mitochondria frozen in trehalose are biologically similar to freshly prepared mitochondria. In fact, Yamaguchi et al. claimed that mitochondria frozen in a buffer containing trehalose allegedly retained many biological and bioenergetics functions, such as synthesizing ATP, responding to increases in calcium concentration by activating permeability transition pores (PTPs), retaining transmembrane potentials, and the ability to import and process proteins. However, the data shown by Yamaguchi et al. did not fully and effectively demonstrate that these mitochondrial functions were maintained, and therefore the results were uncertain. For example, a significant reduction in the rates of both phosphorylation and maximal uncoupled respiration indicated impaired bioenergetic function. In fact, Yamaguchi et al. acknowledge that upon closer inspection, almost all mitochondrial function was impaired.

[0012] US2019 / 0134088 A1 describes partially purified functional mitochondria, which are derived from cells or tissues and have undergone freeze-thaw cycles. The freezing buffer contains sugars, oligosaccharides or polysaccharides as cryoprotectants. In US2019 / 0134088 A1, it is disclosed that the concentration of sufficient sugars that play a role to maintain mitochondrial function is between 100mM and 400mM. It is also disclosed 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 1 month before thawing. In particular, the sugar used in US2019 / 013088 A1 is sucrose, and most importantly, sugar is always required not to be trehalose or mannitol. However, throughout the entire disclosure of US 2019 / 0134088A1, there is no sufficient evidence to support the fact that partially purified mitochondria are still intact and viable after freeze-thaw cycles or after restorage. Likewise, the finding that sucrose alone can be considered a sufficiently good cryoprotectant for mitochondria appears to be in clear conflict with the conclusions of Yamaguchi et al.

[0013] WO 2021 / 168764 A1 (Taiwan Mitochondrion Applied Tech Co Ltd (China)) discloses mitochondrial cryopreservatives and cryopreservation methods. It describes a first cryopreservation composition for cryopreserving mitochondria, which is composed of 300mM trehalose, 10mM 2-[4-(2-hydroxyethyl)-piperazine-1-yl]-ethanesulfonic acid (i.e., 10mM HEPES) and 0.1wt.% human serum albumin (human serum albumin, HSA). The cryopreservation composition does not contain KCl, NaCl, EDTA, EGTA and ethylene glycol. Comparative cryopreservation composition 1 consists of 300mM trehalose, 10mM HEPES, 0.1wt.% HSA, 10mM KCl, 1mM EDTA and 1mM EGTA. Comparative cryopreservation composition 2 consists of 300mM trehalose and 0.1wt.% HSA. The experimental data provided in WO 2021 / 168764 A1 attempt to demonstrate that the integrity of the mitochondrial outer membrane (MOM) of cryopreserved mitochondria is maintained when the first composition is used. However, the data shown in WO 2021 / 168764 A1 cannot prove that these frozen mitochondria maintain viability throughout the freeze-thaw cycle.

[0014] The technical problem is solved by the embodiments / projects presented herein and characterized by the claims.

[0015] Therefore, the present invention particularly relates to the following items:

[0016] 1. A composition comprising:

[0017] (i) an aqueous buffer having a pH of 5.5 to 8.5;

[0018] (ii) trehalose at a concentration of at least 150 mM;

[0019] (iii) a first cryoprotectant selected from one or more of (a) amino acids at a total concentration of at least 150 mM; and optionally

[0020] (iv) a second cryoprotectant selected from one or more of (b) sugars, (c) polymers, or combinations thereof, wherein:

[0021] (b) the total concentration of said sugars is at least 150 mM; and

[0022] (c) The total concentration of the polymer is 2.5% (w / v) to 30% (w / v).

[0023] 2. A composition comprising:

[0024] (i) an aqueous buffer having a pH of 5.5 to 8.5

[0025] (ii) trehalose at a concentration of at least 150 mM;

[0026] (iii) a first cryoprotectant selected from one or more (b) sugars at a total concentration of at least 160 mM; and optionally

[0027] (iv) a second cryoprotectant selected from (a) an amino acid, (c) a polymer, or a combination thereof, wherein:

[0028] (a) the total concentration of the amino acids is at least 150 mM; and

[0029] (c) The total concentration of the polymer is 2.5% (w / v) to 30% (w / v).

[0030] 3. A composition comprising:

[0031] (i) an aqueous buffer having a pH of 5.5 to 8.5

[0032] (ii) trehalose at a concentration of at least 150 mM; and

[0033] (iii) a cryoprotectant selected from one or more (c) polymers, wherein the total concentration of the polymers is 16% (w / v) to 30% (w / v).

[0034] 4. The composition of 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.

[0035] 5. The composition of item 4, wherein (i) the pH of the aqueous buffer is 6.8 to 8.0, for example 7.2.

[0036] 6. The composition of any one of items 1 to 5, wherein (i) the aqueous buffer comprises a buffering agent, preferably, wherein the buffering agent is selected from 2-[4-(2-hydroxyethyl)-piperazine-1-yl]-ethane-sulfonic acid (HEPES), piperazine-N,N'-bis(2-ethane-sulfonic 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 dimethylcacoate, tris-(hydroxymethyl)aminomethane hydrochloride) (Tris), 4-morpholinepropanesulfonic acid (MOPS), 1,3-bis-[tris-(hydroxymethyl)-methylamino]-propane (Bis-Tris propane), sodium acetate, or a combination thereof.

[0037] 7. The composition of any one of items 1 to 6, wherein the concentration of the buffer in the aqueous buffer is 0.5 mM to 50 mM.

[0038] 8. The composition of any one of items 1 to 7, wherein the concentration of the buffer in the aqueous buffer is 2 mM to 35 mM, such as 5 mM to 30 mM.

[0039] 9. The composition of any one of items 1 to 8, wherein the concentration of the buffer in the aqueous buffer is 10 mM to 25 mM, such as 15 mM.

[0040] 10. The composition of any one of items 1 to 9, wherein (i) the aqueous buffer has a pH of 7.2 and comprises 10 mM 2-[4-(2-hydroxyethyl)-piperazin-1-yl]-ethanesulfonic acid (HEPES).

[0041] 11. The composition of any one of items 1 to 10, further comprising a calcium chelator, wherein the calcium chelator is selected from ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2,2',2",2"'-(ethane-1,2-diacid dinitrile)-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.

[0042] 12. The composition of item 11, wherein the concentration of the calcium chelator is 0.1 mM to 10 mM, for example 0.2 mM to 5 mM.

[0043] 13. The composition of item 12, wherein the concentration of the calcium chelator is 0.5 to 2 mM, for example 1 mM.

[0044] 14. The composition of any one of items 11 to 13, wherein the calcium chelator is ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 2,2',2',2"'-(ethane-1,2-diacid dinitrile)-tetraacetic acid (EDTA).

[0045] 15. The composition of any one of items 1 to 14, further comprising an ionic component.

[0046] 16. The composition of claim 15, wherein the concentration of the ionic component is 0.01% (w / v) to 10% (w / v), for example 0.5% (w / v) to 10% (w / v), more particularly 1% (w / v) to 5% (w / v).

[0047] 17. The composition of item 15, wherein the concentration of the ionic component is 0.1 mM to 100 mM, for example 1 mM to 30 mM, more particularly 5 mM to 15 mM.

[0048] 18. The composition of any one of items 15 to 17, wherein the ionic component is selected from: comprising Mg 2+ 、Na + , K + , Cl - , HCO 3 - salts, acids or bases, or combinations thereof.

[0049] 19. The composition of any one of items 15 to 17, wherein the ionic component is selected from: MgCl 2 MgSO 4 , KCl, KH 2 PO 4 、NaHCO 3 、Na 2 HPO 4 , C 2 H 2 MgO 4 (magnesium formate), C 3 H 3 NaO 3 (sodium pyruvate), C 2 H 3 NaO 2 (sodium acetate), or a combination thereof.

[0050] 20. The composition of any one of items 15 to 17, wherein the ionic component is an organic anion selected from the group consisting of citrate, pyruvate, malate, oxaloacetate, formate, glutamate, α-ketoglutarate, succinate, acetate anions, or a combination thereof.

[0051] 21. The composition of 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).

[0052] 22. The composition of any one of items 1 to 21, wherein the albumin is bovine serum albumin (BSA), human serum albumin (HSA), or a combination thereof.

[0053] 23. The composition of 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).

[0054] 24. The composition of any one of items 1 to 3, wherein (i) the buffer has a pH of 7.4 and comprises 20 mM tris-(hydroxymethyl)aminomethane hydrochloride) (Tris), 2 mM 2,2',2",2"'-(ethane-1,2-diacetonitrile)-tetraacetic acid (EDTA) and 10 mM MgCl 2 .

[0055] 25. The composition of any one of items 1 to 3, wherein (i) the buffer has a pH of 7.25 and comprises 5 mM 4-morpholinepropanesulfonic acid (MOPS), 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and 5 mM sodium pyruvate.

[0056] 26. The composition of any one of items 1 to 3, wherein (i) the buffer has a pH of 7.2 and comprises 10 mM 2-[4-(2-hydroxyethyl)-piperazin-1-yl]-ethanesulfonic acid (HEPES) and 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0057] 27. The composition of any one of items 1 to 26, wherein the composition comprises less than a cryopreservable amount of propylene glycol, ethylene glycol, glycerol and dimethyl sulfoxide (DMSO), or does not comprise propylene glycol, ethylene glycol, glycerol and dimethyl sulfoxide (DMSO).

[0058] 28. The composition of any one of items 1 to 26, wherein the composition comprises less than a cryopreservable amount of dimethyl sulfoxide (DMSO) or does not comprise dimethyl sulfoxide (DMSO).

[0059] 29. The composition of any one of items 1 to 28, wherein (ii) the concentration of trehalose does not exceed 1500 mM, such as does not exceed 1300 mM.

[0060] 30. The composition of item 29, wherein the concentration of (ii) trehalose does not exceed 500 mM, preferably does not exceed 450 mM.

[0061] 31. The composition of any one of items 1 to 29, wherein (ii) the concentration of trehalose is at least 250 mM, such as 300 mM.

[0062] 32. The composition of any one of items 1 to 29, wherein the concentration of (ii) trehalose is 150 mM to 1000 mM, for example, 200 mM to 600 mM.

[0063] 33. The composition of any one of items 1, 2 or 4 to 32, wherein the amino acid (a) 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, thiproline, valine (e.g., L-valine), alanine (e.g., L-alanine), glycine, asparagine (e.g., L-asparagine), aspartic acid (e.g., L-aspartic acid), glutamine 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.

[0064] 34. The composition of claim 33, wherein the amino acid in (a) 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.

[0065] 35. The composition of item 33, wherein the amino acid in (a) is selected from methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof.

[0066] 36. The composition of claim 33, wherein the amino acid in (a) is proline, such as L-proline.

[0067] 37. The composition of any one of items 1, 2 or 4 to 36, wherein the concentration of (a) the amino acid is at least 160 mM, such as at least 180 mM.

[0068] 38. The composition of any one of items 1, 2 or 4 to 37, wherein the concentration of (a) the amino acid is at least 200 mM, such as at least 250 mM.

[0069] 39. The composition of any one of items 1, 2 or 4 to 38, wherein the concentration of (a) the amino acid is at least 300 mM, such as at least 500 mM.

[0070] 40. The composition of any one of items 1, 2 or 4 to 39, wherein the concentration of (a) the amino acid is at least 800 mM, such as at least 1000 mM.

[0071] 41. The composition of any one of items 1, 2 or 4 to 40, wherein the concentration of (a) the amino acid is at least 1100 mM, such as at least 1200 mM.

[0072] 42. The composition of any one of items 1, 2 or 4 to 41, wherein the concentration of (a) the amino acid is at least 1300 mM, such as at least 1500 mM.

[0073] 43. The composition of any one of items 1, 2 or 4 to 42, wherein the concentration of (a) the amino acid is at least 1800 mM, such as 2000 mM.

[0074] 44. The composition of any one of items 1, 2 or 4 to 43, wherein the sugar (b) is selected from: a monosaccharide, a disaccharide or a trisaccharide, or a combination thereof.

[0075] 45. The composition of any one of items 1, 2 or 4 to 44, wherein the sugar (b) is selected from maltose, lactose, fructose, sucrose, glucose, dextran, melezitose, raffinose, nigerose, maltotriose, maltotrione, kestose, cellobiose, chitobiose, lactulose, or a combination thereof.

[0076] 46. ​​The composition of any one of items 1, 2, or 4 to 45, wherein the sugar (b) is selected from sucrose, glucose, or a combination thereof.

[0077] 47. The composition of item 46, wherein the sugar (b) is a combination of glucose and sucrose.

[0078] 48. The composition of claim 46, wherein the sugar (b) is glucose.

[0079] 49. The composition of claim 46, wherein the sugar in (b) is sucrose.

[0080] 50. The composition of any one of items 1, 2 or 4 to 49, wherein the concentration of the sugar (b) is at least 160 mM, such as at least 180 mM.

[0081] 51. The composition of claim 50, wherein the concentration of the sugar (b) is at least 200 mM, such as at least 250 mM.

[0082] 52. The composition of any one of items 50 or 51, wherein the concentration of the sugar (b) is at least 300 mM, such as at least 330 mM.

[0083] 53. The composition of any one of items 1, 2 or 4 to 52, wherein the concentration of the sugar (b) does not exceed 2000 mM, such as does not exceed 1800 mM, more particularly does not exceed 1500 mM.

[0084] 54. The composition of item 53, wherein the concentration of the sugar (b) does not exceed 1400 mM, such as not exceeding 1200 mM, more particularly not exceeding 1000 mM.

[0085] 55. The composition of any one of items 1 to 54, wherein the polymer (c) is a biocompatible polymer, a hydrophilic polymer, an amphiphilic polymer, or a combination thereof.

[0086] 56. A composition according to any one of items 1 to 55, wherein the polymer (c) 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, polyvinyl pyrrolidone, or a combination thereof.

[0087] 57. The composition of any one of items 1 to 56, wherein the polymer (c) is polyethylene glycol (PEG).

[0088] 58. The composition of any one of items 1 or 2, wherein the concentration of (c) the polymer is 2.5% (w / v) to 25% (w / v), such as 5% (w / v) to 25% (w / v).

[0089] 59. The composition of any one of items 1, 2 or 55 to 58, wherein the concentration of (c) the polymer is 10% (w / v) to 25% (w / v), such as 15% (w / v) to 20% (w / v).

[0090] 60. The composition of any one of items 1, 4 to 33 or 36 to 59, wherein:

[0091] (iii) the first cryoprotectant is (a) an amino acid, wherein the amino acid is proline, such as L-proline, at a concentration of at least 150 mM, such as at least 200 mM, preferably at least 300 mM; and

[0092] (iv) the second cryoprotectant is selected from one or more of (b) sugars, (c) polymers, or a combination thereof, wherein:

[0093] (b) the sugar is glucose, sucrose, or a combination thereof, and the total concentration is at least 150 mM, such as at least 200 mM, more particularly at least 300 mM, such as at least 500 mM; and

[0094] (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 particularly 10% (w / v) to 20% (w / v), such as 15% (w / v).

[0095] 61. The composition of any one of items 1, 4 to 33, 35 or 37 to 59, wherein:

[0096] (iii) the first cryoprotectant is one or more (a) amino acids, wherein the amino acid is a proline derivative selected from the group consisting of methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof, at a concentration of at least 150 mM, such as at least 200 mM, preferably at least 300 mM; and

[0097] (iv) the second cryoprotectant is selected from one or more of (b) sugars, (c) polymers, or a combination thereof, wherein:

[0098] (b) the sugar is glucose, sucrose, or a combination thereof, and the total concentration is at least 150 mM, such as at least 200 mM, more particularly at least 300 mM, such as at least 500 mM; and

[0099] (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 particularly 10% (w / v) to 20% (w / v), such as 15% (w / v).

[0100] 62. The composition of any one of items 2 or 4 to 59, wherein:

[0101] (iii) the first cryoprotectant is one or more (b) sugars, wherein the sugar is glucose, sucrose, or a combination thereof, in a total concentration of at least 160 mM, such as at least 200 mM, more particularly at least 300 mM, such as at least 500 mM; and

[0102] (iv) the second cryoprotectant is selected from one or more of (a) amino acids, (c) polymers, or a combination thereof, wherein:

[0103] (a) the amino acid is selected from the amino acids according to any one of items 33 to 36, and the concentration is at least 150 mM, such as at least 200 mM, preferably at least 300 mM;

[0104] (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 particularly 10% (w / v) to 20% (w / v), such as 15% (w / v).

[0105] 63. The composition of item 62, wherein the amino acid in (a) is proline, such as L-proline.

[0106] 64. The composition of any one of items 3 to 32 or 55 to 57, wherein:

[0107] (iii) the cryoprotectant is (c) a polymer, wherein the polymer is polyethylene glycol (PEG) and has a concentration of 16% (w / v) to 30% (w / v), such as 20% (w / v) to 30% (w / v), such as 25% (w / v).

[0108] 65. The composition according to any one of items 60, 62 or 63, wherein:

[0109] (a) The amino acid is proline, such as L-proline, at a concentration of at least 500 mM, such as at least 600 mM, in particular at least 800 mM.

[0110] 66. The composition of any one of items 60, 62 or 63, wherein:

[0111] The amino acid is proline, such as L-proline, at a concentration of at least 1000 mM, such as 1200 mM.

[0112] 67. The composition of any one of items 60, 62 or 63, wherein:

[0113] The amino acid is proline, such as L-proline, at a concentration of at least 1300 mM, such as at least 1500 mM.

[0114] 68. The composition of any one of items 60, 62 or 63, wherein:

[0115] The amino acid is proline, such as L-proline, at a concentration of at least 1600 mM, such as at least 1800 mM, in particular at least 2000 mM.

[0116] 69. The composition of any one of items 60, 62, 63 or 65 to 68, wherein:

[0117] The amino acid is proline, such as L-proline; and

[0118] The sugar is glucose at a concentration of 200 mM to 1300 mM, such as 300 mM to 1200 mM, in particular 350 mM to 1100 mM.

[0119] 70. The composition of any one of items 60, 62, 63 or 65 to 68, wherein:

[0120] The amino acid is proline, such as L-proline; and

[0121] The sugar is sucrose at a concentration of 160 mM to 900 mM, such as 200 mM to 800 mM, in particular 250 mM to 650 mM.

[0122] 71. The composition of any one of items 60, 62, 63 or 65 to 68, wherein:

[0123] The amino acid is proline, such as L-proline; and

[0124] The polymer is polyethylene glycol (PEG) at a concentration of 5% (w / v) to 30% (w / v), 10% (w / v) to 20% (w / v), such as 15% (w / v).

[0125] 72. The composition of any one of items 1, 4 to 33, 36 or 60, wherein the cryoprotectant consists of proline (eg, L-proline) at a concentration according to any one of items 37 to 43.

[0126] 73. The composition of claim 72, wherein the concentration of proline (eg, L-proline) is 600 mM or 1200 mM.

[0127] 74. The composition of any one of items 1, 4 to 32, 35 or 61, wherein the cryoprotectant consists of one or more (a) amino acids, wherein (a) the amino acid is a proline derivative selected from the following: methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof, with a total concentration of at least 200mM, preferably at least 300mM, and more preferably at least 500mM.

[0128] 75. The composition of any one of items 1, 4 to 32, 35, 61 or 74, wherein the cryoprotectant consists of one or more (a) amino acids, wherein:

[0129] The amino acid is a proline derivative selected from the group consisting of 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.

[0130] 76. The composition of any one of items 1, 4 to 32, 35, 61, 74 or 75, wherein the cryoprotectant consists of one or more (a) amino acids, wherein:

[0131] The amino acid is a proline derivative selected from the group consisting of 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, such as at least 1200 mM, in particular at least 1500 mM, such as 2000 mM.

[0132] 77. The composition of 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, for example at least 300 mM, more particularly at least 500 mM, and not more than 2000 mM, for example not more than 1700 mM.

[0133] 78. The composition of 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, for example at least 650 mM, more particularly at least 800 mM, and not more than 2000 mM, for example not more than 1650 mM.

[0134] 79. The composition of 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, for example at least 1200 mM, in particular at least 1350 mM, and not more than 2000 mM, for example not more than 1650 mM.

[0135] 80. The composition of any one of items 2, 4 to 32 or 62, wherein the cryoprotectant consists of glucose in a total concentration of at least 300 mM, such as at least 500 mM, in particular at least 800 mM, but not more than 1200 mM, such as not more than 1000 mM.

[0136] 81. A composition according to any one of items 2, 4 to 32 or 62, wherein the cryoprotectant consists of sucrose in a total concentration of at least 200 mM, such as at least 250 mM, in particular at least 300 mM, but not more than 800 mM, such as not more than 650 mM.

[0137] 82. The composition of any one of items 1 to 81, further comprising isolated mitochondria, such as isolated viable mitochondria.

[0138] 83. The composition of item 82, wherein the isolated mitochondria are mammalian isolated mitochondria, such as mammalian isolated viable mitochondria.

[0139] 84. The composition of item 82, wherein the isolated mitochondria are human isolated mitochondria, such as human isolated viable mitochondria.

[0140] 85. The composition of any one of items 82 to 84, wherein the mitochondria have been isolated from cells, tissues or organs.

[0141] 86. The composition of claim 85, wherein the tissue is liver tissue, skeletal muscle, heart, brain, kidney, placenta, lung tissue or adipose tissue.

[0142] 87. The composition of item 86, wherein the mitochondria have been isolated from cultured cells (eg, cultured human cells).

[0143] 88. The composition of any one of items 85 or 87, wherein mitochondria have been 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.

[0144] 89. The composition of any one of items 82 to 88, wherein the concentration of the isolated mitochondria is at least 0.02 μg / μL, such as at least 0.05 μg / μL, in particular at least 0.1 μg / μL.

[0145] 90. The composition of item 89, wherein the concentration of the isolated mitochondria is at least 0.2 μg / μL, such as at least 0.5 μg / μL, in particular at least 0.75 μg / μL.

[0146] 91. The composition of any one of items 89 or 90, wherein the concentration of the isolated mitochondria is at least 1.0 μg / μL, such as at least 1.5 μg / μL, in particular at least 2 μg / μL, such as 2.5 μg / μL.

[0147] 92. The composition of any one of items 89 to 91, wherein the concentration of the isolated mitochondria is at least 5 μg / μL, such as at least 10 μg / μL, such as 20 μg / μL or 30 μg / μL.

[0148] 93. The composition of any one of items 82 to 92, wherein the concentration of the isolated mitochondria does not exceed 100 μg / μL, such as does not exceed 80 μg / μL, in particular does not exceed 50 μg / μL.

[0149] 94. The composition of any one of items 82 to 93, wherein the concentration of the isolated mitochondria is no more than 25 μg / μL, such as no more than 15 μg / μL, in particular no more than 10 μg / μL, such as no more than 5 μg / μL.

[0150] 95. The composition of any one of items 82 to 94, wherein the isolated mitochondria are linked to a pharmaceutical agent, a diagnostic agent, an imaging agent, a therapeutic agent or any other biocompatible substance.

[0151] 96. The composition of any one of items 82 to 94, wherein the mitochondria are linked to an antibody.

[0152] 97. The composition of any one of items 82 to 94, wherein the mitochondria are linked to an antigen binding fragment, wherein the antigen binding fragment is at least a portion of an antibody or TCR or a recombinant variant thereof.

[0153] 98. The composition of any one of items 95 to 97, wherein the mitochondria is linked to the agent, antibody or antigen-binding fragment via a covalent bond.

[0154] 99. The composition of any one of items 95 to 97, wherein the mitochondria are linked to the agent, antibody or antigen via a non-covalent bond (eg, an electrostatic bond).

[0155] 100. The composition of any one of items 95 to 97, wherein the agent, antibody or antigen is embedded in the mitochondria, embedded in the mitochondrial membrane, substantially enclosed within the mitochondria or completely encapsulated by the mitochondria.

[0156] 101. The composition of any one of items 95 to 99, wherein the agent, antibody or antigen is attached to the outer mitochondrial membrane via a covalent bond or a non-covalent bond (eg, an electrostatic bond).

[0157] 102. The composition of any one of items 82 to 101, wherein the isolated mitochondria are mitochondria modified by gene editing.

[0158] 103. The composition of any one of items 82 to 101, wherein the isolated mitochondria comprise exogenous mtDNA.

[0159] 104. A method for cryopreservation of a composition comprising isolated mitochondria, such as isolated viable mitochondria, according to any one of the preceding items, the method comprising the following steps:

[0160] (a) freezing the composition at a temperature below 0°C; and

[0161] (b) storing the frozen composition obtained according to step (a) at a temperature below 0°C, such as -4°C, preferably -20°C.

[0162] 105. The method described in item 104, wherein the method further comprises an additional step (c) after step (b), wherein step (c) comprises thawing the frozen composition at a temperature above 0°C.

[0163] 106. The method of any one of items 104 or 105, wherein:

[0164] (a) Freezing at a temperature below -20°C, such as below -50°C, preferably below 70°C.

[0165] 107. The method of any one of items 104 or 105, wherein:

[0166] (a) Freezing at a temperature below -100°C, such as below -150°C, in particular below -180°C.

[0167] 108. The method of any one of items 104 or 105, wherein:

[0168] (a) Freezing 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.

[0169] 109. The method of any one of items 104 or 105, wherein:

[0170] (a) Freezing is rapid freezing.

[0171] 110. The method of any one of items 104 or 105, wherein:

[0172] (a) Freezing is gradual freezing at a rate of at least 5°C / min, such as at least 10°C / min, such as 20°C / min or 30°C / min.

[0173] 111. The method of any one of items 104 to 110, wherein:

[0174] (b) The duration of storage is at least 24 hours before thawing, such as 120 hours, preferably, wherein:

[0175] (b) The duration of storage is at least 1 week, such as 4 weeks.

[0176] 112. The method of any one of items 104 to 111, wherein:

[0177] (c) Thawing is performed at a temperature above 4°C and below 40°C, for example, at a temperature between 20°C and 38°C.

[0178] 113. A composition for treating a disease in a subject in need thereof, e.g., by therapeutic mitochondrial transplantation (TMT), comprising a therapeutically effective amount of isolated mitochondria according to any one of the preceding items, e.g., isolated viable mitochondria, or a composition prepared by the method of any one of items 104 to 112.

[0179] 114. A composition according to item 113, for use in treating mitochondrial disease or mitochondrial-related disease in a subject in need thereof.

[0180] 115. A composition according to item 113, for use in treating cancer or tumors in a subject in need thereof.

[0181] 116. The composition according to item 113, which is used for treating autoimmune diseases.

[0182] 117. The composition according to item 113, for use in treating ischemia-related injuries, such as lung, kidney, heart or brain ischemia-reperfusion injuries.

[0183] 120. The composition according to item 114, which is used for removing blockages in blood vessels, such as blood clots.

[0184] 121. The composition according to item 114, for use in gene therapy, in particular for gene therapy for the treatment of cancer, infectious diseases or autoimmune diseases.

[0185] 122. A composition according to item 114, which is used to transplant mitochondria, such as isolated viable mitochondria, into immune cells to produce mitochondria-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), and γ-δ T cells.

[0186] 123. A composition according to item 114 for use in treating a disease in a subject in need thereof, wherein the composition is to be administered to the subject in need thereof by topical or parenteral administration.

[0187] 124. A composition according to item 114, for use in treating a disease in a subject in need thereof, wherein the composition is administered to the subject in need thereof in the form of an aerosol.

[0188] 125. A composition according to item 114 for treating a disease in a subject in need thereof, wherein the composition is administered to the subject in need thereof by direct injection into a blood vessel, tissue or organ.

[0189] 126. A composition according to any one of the preceding items for use in treating a disease in a subject in need thereof, wherein the composition is delivered in vitro to a target cell of a subject in need thereof or a donor.

[0190] 127. The composition according to any of the preceding items, wherein the viable mitochondria contained in the composition are autologous, allogeneic or xenogeneic.

[0191] 128. A composition according to any one of items 114 to 127, wherein the composition has been subjected to a freeze-thaw cycle, such as a freeze-thaw cycle according to the method of any one of items 106 to 114, prior to use in treating a disease.

[0192] 129. Use of the composition of any of the preceding items for cryopreservation of viable mitochondria, such as isolated viable mitochondria.

[0193] 130. Use of a composition prepared by the method of any one of items 105 to 113 for cryopreservation of viable mitochondria, such as isolated viable mitochondria.

[0194] The present invention provides new and efficient compositions and methods for cryopreserving isolated mitochondria (e.g., isolated viable mitochondria). The present invention also provides cryopreservation compositions comprising isolated mitochondria (e.g., isolated viable mitochondria). The cryopreserved mitochondria of the present invention can be used for different types of treatments, such as for cell and gene therapy after thawing. These treatments require consistent and reproducible results under high mitochondrial viability and functionality for therapeutic effects. Mitochondrial damage during cryopreservation and thawing may result in ineffective treatment. Mitochondria are particularly sensitive to the stress of freezing and thawing, and the lack of optimized cryopreservation methods has hindered research and medical applications. As of today, there is a strong need for cell and gene therapy and therapeutic mitochondrial transplantation (TMT) that can be relied on for cryopreservation methods and compositions to safely and effectively preserve mitochondria during preparation, storage and transportation. Until now, in basic research and medical applications (e.g., in treatment, diagnosis and imaging), it is still desirable to obtain ready-to-use isolated mitochondria that maintain their integrity and viability during the freezing process, low temperature storage, and during the thawing process before use. Therefore, the technical problem underlying the present invention is to provide new and innovative methods and compositions suitable for cryopreservation of isolated viable mitochondria, which allow the structural integrity and / or functional properties of isolated viable mitochondria to be maintained. The present invention provides cryopreservation compositions comprising isolated viable mitochondria, which remain viable, respiratory active and / or intact during low temperature storage and freeze-thaw cycles, preferably, the mitochondria remain viable, respiratory active and intact. The cryopreserved mitochondria of the present invention exhibit comparable structural and functional properties 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. Summary of the invention

[0195] The present invention relates to a 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; (iii) a first cryoprotectant selected from one or more (a) amino acids having a total concentration of at least 150 mM; and optionally (iv) a second cryoprotectant selected from one or more (b) sugars, (c) polymers, or a combination thereof, wherein the total concentration of the (b) sugars is at least 150 mM; and the total concentration of the (c) polymers is 2.5% (w / v) to 30% (w / v). In the composition of the present invention, it may be that: (iii) the first cryoprotectant is (a) an amino acid, wherein the amino acid is proline, such as L-proline, and the concentration is 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 (b) sugars, (c) polymers, or a combination thereof, wherein the (b) sugar is glucose, sucrose, or a combination thereof, and the total concentration is at least 150 mM, such as at least 200 mM, more particularly at least 300 mM, such as at least 500 mM; and the (c) polymer is polyethylene glycol (PEG) and the concentration is 2.5% (w / v) to 30% (w / v), such as 5% (w / v) to 25% (w / v), more particularly 10% (w / v) to 20% (w / v), such as 15% (w / v). It may also be that: (iii) the first cryoprotectant is one or more (a) amino acids, wherein the amino acid is a proline derivative selected from the following: methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridine carboxylic acid, azetidine carboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof, 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 (b) sugars, (c) a polymer, or a combination thereof, wherein the (b) sugar is glucose, sucrose, or a combination thereof and is at a total concentration of at least 150 mM, such as at least 200 mM, more particularly at least 300 mM, such as at least 500 mM; and the (c) 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 particularly 10% (w / v) to 20% (w / v), such as 15% (w / v).

[0196] In another embodiment, the present invention relates to a 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; (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 a combination thereof, wherein the total concentration of (a) amino acids is at least 150 mM; and the total concentration of (c) polymers is 2.5% (w / v) to 30% (w / v). It may be that in the composition, (iii) the first cryoprotectant is one or more (b) sugars, wherein the sugar is glucose, sucrose, or a combination thereof, in a total concentration of at least 160 mM, such as at least 200 mM, more particularly at least 300 mM, such as at least 500 mM; and (iv) the second cryoprotectant is selected from one or more (a) amino acids, (c) polymers, or a combination thereof, wherein (a) the amino acid is selected from the amino acids according to any one of items 33 to 36, in 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), in a concentration of 2.5% (w / v) to 30% (w / v), such as 5% (w / v) to 25% (w / v), more particularly 10% (w / v) to 20% (w / v), such as 15% (w / v). It may also be that: (a) the amino acid is proline, such as L-proline.

[0197] In another embodiment, the present invention relates to a 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 one or more (c) polymers, wherein the total concentration of the polymers is 16% (w / v) to 30% (w / v). It may be that: (iii) the cryoprotectant is (c) a polymer, wherein the polymer 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).

[0198] The present invention relates to 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; (iii) a cryoprotectant selected from (a) an amino acid, (b) a sugar, (c) a polymer, or a combination thereof, wherein: the concentration of the amino acid is at least 150 mM; the concentration of the sugar is at least 150 mM, for example, at least 160 mM; and the concentration of the polymer is 2.5% (w / v) to 30% (w / v), for example, 16% (w / v) to 30% (w / v).

[0199] 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; (iii) a cryoprotectant selected from (a) an amino acid, (b) a sugar, (c) a polymer, or a combination thereof, wherein: the amino acid is proline, such as L-proline, at a concentration of at least 150 mM; the sugar is glucose, sucrose, or a combination thereof, and the total concentration is at least 150 mM, such as at least 160 mM; and the polymer is polyethylene glycol (PEG) at a concentration of 2.5% (w / v) to 30% (w / v), such as 16% (w / v) to 30% (w / v).

[0200] The cryopreservation compositions of the invention also comprise a therapeutically effective amount of isolated mitochondria, such as isolated viable mitochondria, for use in treating a condition that benefits from enhanced mitochondrial function in a subject in need thereof.

[0201] The present invention also provides a method suitable for cryopreserving a composition comprising isolated mitochondria, such as isolated viable mitochondria, the method comprising the following steps: (a) freezing the composition at a temperature below 0°C; and (b) storing the frozen composition obtained according to step (a) at a temperature below 0°C.

[0202] In the present invention, the mitochondria may also be freeze-dried and / or spray-frozen, such as spray-freeze-drying or spray-freeze-freeze-drying. BRIEF DESCRIPTION OF THE DRAWINGS

[0203] Figure 1. Mitochondria frozen in sucrose-based buffers do not maintain inner and outer membrane integrity.

[0204] Will Figure 1A , Figure 1B , Figure 1C and Figure 1D The isolated mitochondria were suspended in an aqueous buffer with a pH of 7.2 (10 mM HEPES, adjusted to pH 7.2 with KOH), 1 mM EGTA, and 300 mM sucrose. Figure 1E , Figure 1F and Figure 1G The isolated mitochondria were suspended in two different buffers (pH 7.2): "trehalose-based buffer" (10 mM HEPES, 1 mM EGTA, 300 mM trehalose) and "sucrose-based buffer" (10 mM HEPES, 1 mM EGTA, 300 mM sucrose).

[0205] Figure 1AIsolated mitochondria were frozen in liquid nitrogen (LN), thawed in a 37°C water bath, stained with MitoTracker Red and MitoTracker Green, and imaged using fluorescence microscopy. Figure 1B .Follow and target Figure 1A The same procedure was followed, but the mitochondria were frozen on dry ice rather than in liquid nitrogen. Figure 1C . Figure 1A Quantification of MitoTracker Red signal of stained mitochondria (n=3). Figure 1D . Figure 1B Quantification of MitoTracker Red signal of stained mitochondria (n=3). Figure 1E Mitochondria were isolated in sucrose-based or trehalose-based isolation buffer, snap-frozen in liquid nitrogen (LN) or kept on ice (not frozen), thawed in a 37°C water bath, and ATP content was measured (n=3). Figure 1F .Follow and target Figure 1E The same procedure was followed, but mitochondria were frozen on dry ice (n=3). Figure 1G Mitochondria isolated in sucrose-based or trehalose-based isolation buffer were frozen on dry ice, thawed in a 37°C water bath, incubated at 37°C for 15 minutes after thawing, and precipitated to release cytochrome C ( Figure 1G Cytochrome C (S) associated with mitochondrial sedimentation Figure 1G The samples were further analyzed by acrylamide gel electrophoresis and western blotting using anti-cytochrome C antibody. All data are expressed as mean ± SD (standard deviation).

[0206] Figure 2. Freezing of mitochondria in a trehalose-based buffer is not sufficient to maintain their membrane potential and ATP content.

[0207] Figure 2A Mitochondria were isolated in a trehalose-based isolation buffer and freeze-thawed in a dry ice / 37°C water bath for as long as Figure 2A The number of cycles indicated in (e.g., 1 to 5 cycles) was kept on ice only at about 4°C. Figures 2A to 2G Mitochondria were imaged using automated fluorescence microscopy after staining with MitoTracker Red and MitoTracker Green. Figure 2B . Figure 2A Quantification of MitoTracker Red signal for samples shown in (n=3). Figure 2C .like Figure 2AATP content of frozen mitochondria as described in (n=3). Figure 2D Mitochondria were frozen on dry ice, in liquid nitrogen (LN), or first frozen on dry ice and then placed in liquid nitrogen (dry ice + LN). Membrane potential was determined by staining with MitoTracker Red and MitoTracker Green, as described for Figure 1A Overlay images from MitoTracker Red and MitoTracker Green staining are shown. Figure 2E .From Figure 2D Quantification of MitoTracker Red signal (MTR) of stained mitochondria of samples (n=3). Figure 2F .If targeted Figure 2D ATP content of the treated mitochondria (n=3). Figure 2G Isolated mitochondria were frozen on dry ice and thawed in a water bath (37°C), at room temperature (20°C), or on ice (4°C). ATP content was measured and compared with unfrozen mitochondria incubated on ice. All data are presented as mean ± SD.

[0208] Figure 3. Addition of certain cryoprotectants to the freezing buffer prevents mitochondrial damage in a concentration-dependent manner.

[0209] Figure 3A The isolated mitochondria were suspended in an aqueous buffer containing 10 mM HEPES (adjusted to pH 7.2 with KOH), 300 mM trehalose, and 1 mM EGTA. Each sample containing a cryoprotectant was prepared by adding one of six different cryoprotectants (i.e., CRYO6, CRYO12, CRYO15, CRYO22, CRYO25, or CRYO33; detailed information for each cryoprotectant is provided in Table 1b) to the mitochondrial suspension at different concentrations, the concentrations being 5%, 10%, 15%, 20%, 25%, or 30% by volume of the cryoprotectant relative to the final total volume of the suspension (i.e., % (v CRYO / v tot)). For example, a suspension (e.g., a composition) containing 20% ​​(v / v) CRYO6 is prepared by adding 10 μL of a 6M proline solution (i.e., CRYO6) to a 40 μL suspension containing 300 mM trehalose, 1 mM EGTA, 10 mM HEPES (pH 7.2) and mitochondria. The mitochondrial concentration of the samples used in the ATP assay measurement was 10 μg / 50 μL, and the mitochondrial concentration of the samples used for microscopy analysis was 15 μg / 20 μL. One of the samples did not contain a cryoprotectant (i.e., 0% of the final volume of the suspension). As a control, a suspension of unfrozen freshly isolated mitochondria was used ("fresh"). The samples were first (1) frozen; then (2) thawed, and then (3) stained with MitoTracker Red and MitoTracker Green and imaged with an automated fluorescence microscope. The MitoTracker Red signal was calculated relative to unfrozen mitochondria stored on ice (n=3). The average values ​​are presented as a heat map. Figure 3B Representative images of isolated mitochondria frozen with the indicated cryoprotectants and stained with MitoTracker Red and MitoTracker Green, as well as corresponding controls (unfrozen, freshly isolated mitochondria, and frozen mitochondria without cryoprotectants). Figure 3C ATP content of isolated mitochondria frozen in the presence of the indicated concentrations of selected cryoprotectants relative to the ATP content of unfrozen (fresh) isolated mitochondria.

[0210] FIG4. Viability of frozen-thawed mitochondria contained in a composition comprising two cryoprotectants

[0211] Figure 4A Freshly isolated mitochondria were kept on ice (indicated by "fresh" in the figures), frozen in the absence of cryoprotectants (indicated by "frozen" in the figures), or frozen in the presence of one or two cryoprotectants at the indicated concentrations (indicated in the figures by their respective concentrations (volume of cryoprotectant / % of the total volume of the suspension) and the type of cryoprotectant used). The mitochondrial ATP content was measured after thawing and normalized to that of unfrozen mitochondria.

[0212] FIG5 . Freezing rate affects the viability of mitochondria contained in compositions containing cryoprotectants

[0213] Figure 5AMitochondria were frozen at controlled flow rates of 2K / min, 6K / min, and 10K / min, stored at -80°C for 1 month, and compared to freshly isolated mitochondria by MitoTracker Red / MitoTracker Green staining. The MitoTracker Red / MitoTracker green ratio was calculated and shown relative to freshly isolated mitochondria. Error bars represent standard deviations of technical replicates (n=3). Figure 5B .like Figure 5A Mitochondria were frozen in 5% PBS and their ATP content was determined after storage at -80°C for one month. Values ​​were normalized to control samples from the same mitochondrial isolate frozen at -80°C to compensate for possible protein assay errors and biological variability between isolates. Data are shown relative to 2K / min. Error bars represent standard deviations of technical replicates (n=3).

[0214] Figure 6 In vivo coronary blood flow experiment

[0215] Freshly isolated mitochondria from the animal's own skeletal muscle tissue ("fresh autologous") or cultured human cardiac fibroblasts ("fresh HCF"), as well as frozen mitochondria isolated from cultured HCF cells, frozen in a buffer supplemented with cryoprotectants (7 mM HEPES, pH 7.2, 0.7 mM EGTA, 210 mM trehalose, 1.2 M proline, and 353 mM glucose) and stored at -80°C for up to 1 week, were injected into the coronary arteries of anesthetized healthy pigs, and coronary blood flow (CBF) was measured before and after injection. N = 1, 1000 μg of mitochondria for each condition were diluted in 5 mL of sucrose-based isolation buffer per injection. DETAILED DESCRIPTION

[0216] The present invention relates to cryopreservation compositions and methods 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 low temperatures, such as cryopreserved for weeks, months or years, and can be used for example to treat mitochondrial diseases or mitochondrial-related diseases, ischemic diseases / injuries or ischemia-reperfusion-related diseases / injuries after thawing, such as by promoting the repair process of 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, SM, Del Nido, PJ, McCully JD et al.; Biochimica et Biophysica Acta (BBA) – Bioenergetics Vol. 1847, No. 11, (November 2015), pp. 1387-1400, RK Lane et al.), infectious, inflammatory or autoimmune diseases; for coronary intervention (WO 2017 / 124037, McCully et al.); or for diagnosis or research purposes. The composition of the present invention is also used for gene therapy. An example of gene therapy for the treatment of cancer, infectious diseases or autoimmune diseases is to transplant mitochondria into immune cells to produce mitochondrial 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), γ-δ T cells, particularly mitochondrial enhanced chimeric antigen receptor (CAR) T cells or mitochondrial enhanced tumor infiltrating lymphocytes (TIL) (WO2021203046A1, Schueller A. et al.).

[0217] The compositions and methods of the present invention provide isolated mitochondria, such as isolated viable mitochondria with respiratory activity, which can be successfully cryopreserved, stored, transported, and subsequently recovered for later use. The shelf life of the cryopreservation compositions of the present invention is at least 3 months, such as at least 6 months, such as 12 months or 24 months.

[0218] The cryopreserved isolated mitochondria, e.g., cryopreserved isolated viable mitochondria, contained in the composition of the present invention are ready-to-use and have advantages in terms of ease of application in research and clinical settings and in terms of efficacy. In fact, the cryopreserved compositions of the present invention and the isolated mitochondria, e.g., isolated viable mitochondria contained therein, can be easily used for application in a variety of biomedical technologies, the purpose of which is to improve or rescue mitochondrial function in cells and tissues, in particular mitochondrial function in damaged cells and tissues.

[0219] Composition

[0220] Embodiment A1: Cryopreservation composition

[0221] In one aspect, the present invention relates to a composition comprising:

[0222] (i) an aqueous buffer having a pH of 5.5 to 8.5;

[0223] (ii) trehalose at a concentration of at least 150 mM; and

[0224] (iii) a cryoprotectant selected from an amino acid, a sugar, a polymer, or a combination thereof, wherein the concentration of the amino acid (i.e. the total concentration of the amino acid in the composition) is at least 150 mM; the concentration of the sugar (i.e. the total concentration of the sugar in the composition) is at least 150 mM, such as at least 160 mM; and the concentration of the polymer (in the composition) is from 2.5% (w / v) to 30% (w / v), such as from 16% (w / v) to 30% (w / v). The composition is preferably a cryopreservation composition. The composition is preferably (suitable for) cryopreservation of mitochondria as defined herein. Preferably, the mitochondria are viable and / or isolated mitochondria.

[0225] Exemplary cryoprotectants (alone or in combination) included in the composition of the above embodiments are listed in Table 1a below:

[0226] Table 1a

[0227] CRYO6 6.0M L-Proline CRYO11 100% Polyethylene glycol 200 CRYO12 100% Polyethylene glycol 400 CRYO13 100% Polyethylene glycol monomethyl ether 550 CRYO14 80% v / v Polyethylene glycol 600 CRYO15 50% w / v polyethylene glycol 1,000 CRYO16 50% w / v polyethylene glycol 3,350 CRYO17 50% w / v polyethylene glycol 4,000 CRYO18 50% w / v Polyethylene glycol monomethyl ether 5,000 CRYO19 50% w / v polyethylene glycol 8,000 CRYO20 50% w / v polyethylene glycol 10,000 CRYO21 50% w / v Polyvinylpyrrolidone K15 CRYO22 50% v / v Pentaerythritol propoxylate (5 / 4PO / OH) CRYO23 100% Polypropylene glycol P 400 CRYO25 70%w / v D-(+)-sucrose CRYO26 70% w / v D-sorbitol CRYO27 30% w / v D-(+)-Maltose Monohydrate CRYO28 35% w / v erythritol CRYO29 70% w / v Xylitol CRYO30 15% w / v Inositol CRYO31 20% w / v D-(+)-Raffinose pentahydrate CRYO32 50% w / v D-(+)-Trehalose dihydrate CRYO33 70%w / v D-(+)-Glucose Monohydrate CRYO47 30% w / v polyethylene glycol 3,350, 20% v / v glycerol

[0228] All weight / volume or volume / volume percentages (% (w / v) or % (v / v)) and molar concentrations (M) of each cryoprotectant in Table 1a above or Table 1c and Table 1b below are provided for aqueous solutions or suspensions, i.e., for solutions or suspensions in which the solvent is water.

[0229] The cryoprotectant is added to the (i) aqueous buffer contained in the cryopreservation composition in such an amount that the desired cryoprotectant concentration is obtained in the final total volume of the composition. For example, the cryoprotectant CRYO25 is a 70% w / v D-(+)-sucrose solution containing 700 g of sucrose per 1 liter of water. Since the molecular weight of sucrose is equal to 342.3 g / mol, the 70% w / v D-(+)-sucrose solution contains a measured concentration of sucrose of about 2.0 M molar concentration. This 2.0 M sucrose aqueous solution has been added to the composition in such an amount that, for example, a concentration of 150 mM is obtained in the final total volume of the composition. For example, the cryoprotectant CRYO33 is a 70% w / v D-(+)-glucose monohydrate solution containing 700 g of glucose monohydrate per 1 liter of water. Since the molecular weight of glucose monohydrate is equal to 198.17 g / mol, a 70% w / v D-(+)-glucose monohydrate solution contains glucose at a measured concentration of about 3.5 M molar concentration. This 3.5 M aqueous glucose solution is added to the composition in such an amount that a concentration of, for example, 150 mM is obtained. For example, a composition (e.g., a suspension) containing 150 mM CRYO6 (i.e., 150 mM L-proline) is 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 trehalose, 1 mM EGTA, 10 mM HEPES (pH 7.2).

[0230] In light of the above, exemplary cryoprotectants that may be included in the above composition alone or in combination are given below:

[0231] Table 1c

[0232]

[0233]

[0234] Examples of commercially available cryoprotectants are provided in Table 1c above.

[0235] The solution has been prepared by mixing at 5%, 10% and 20% (v / v) (see Figure 3A and Figure 3B and CRYO1-CRYO47 of Table 1b), at 5%, 10%, 15%, 20%, 25% and 30% (v / v) (for CRYO6, CRYO12, CRYO15, CRYO22, CRYO25 and CRYO33 see Figure 3C ) and at 10%, 20% and 30% (v / v) (for CRYO6+CRYO12, CRYO6+CRYO25, CYRO6+CRYO33 and CRYO33+CRYO25, FIG. 4A to FIG. 4D) were diluted in "trehalose-based" mitochondrial isolation buffer (1) and the cryoprotectants described in Table 1c above were used to prepare the composition.

[0236] Some 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 CRYO6, CRYO12, CRYO25 and / or CRYO33.

[0237] In one aspect, the pH of the aqueous buffer included in the composition of the above-mentioned embodiment is 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 pH of the aqueous buffer is 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 pH of the aqueous buffer is 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 composition provided herein is preferably an aqueous composition. Preferably, the (aqueous) composition has the same pH as the aqueous buffer included therein. The limitations and descriptions of the pH of the aqueous buffer provided herein are applicable to the (aqueous) composition after necessary modifications (mutatis mutandis). Preferably, deionized water and / or water without RNase / DNase are used to prepare the aqueous composition and / or aqueous buffer. Furthermore, in one aspect, the (only) aqueous component of the (aqueous) composition is an aqueous buffer as described herein. The amino acids, sugars and / or polymers may be (directly) dissolved in the aqueous buffer. Alternatively, the amino acids, sugars and / or polymers may first be dissolved in a non-aqueous solution and subsequently added to the aqueous buffer or aqueous composition.

[0238] In one aspect, the aqueous buffer contained in the composition of any of the above embodiments comprises a buffer, preferably a pH buffer. The buffer can be selected from, but is not limited to, the group comprising: 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-Tris propane), sodium acetate, or a combination thereof. In certain embodiments, the concentration of the pH buffer is 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 concentration of the pH buffer is 10 mM to 35 mM, such as 15 mM, 20 mM, 25 mM or 30 mM. In certain preferred embodiments, the aqueous buffer comprises HEPES, such as 10 mM HEPES (adjusted to pH 7.2 with KOH).

[0239] In another aspect, the composition of any of the above embodiments further comprises a calcium chelator. In certain embodiments, the calcium chelator is ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2,2',2",2"'-(ethane-1,2-diacid dinitrile)-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 chelator is EDTA or EGTA. In certain embodiments, the concentration of the calcium chelator is 0.1 mM to 10 mM, for example, such as 0.5 mM to 5 mM, for example, 1 mM or 1.5 mM. In a preferred embodiment, the calcium chelator is EGTA at a concentration of 0.5 mM to 2 mM, such as 1 mM. In another preferred embodiment, the calcium chelator is EDTA at a concentration of 0.5 mM to 2 mM, such as 1 mM.

[0240] In another aspect, the composition of any of the above embodiments further comprises an ionic component. In certain embodiments, the ionic component comprises an ion such as Mg 2+ 、Na + , K + , Cl -, HCO 3 - In other embodiments, the ionic component is, for example, a suitable salt, including but not limited to MgCl 2 MgSO 4 , KCl, KH 2 PO 4 、NaHCO 3 、Na 2 HPO 4 , C 2 H 2 MgO 4 (magnesium formate), C 3 H 3 NaO 3 (sodium pyruvate), C 2 H 3 NaO 2 (sodium acetate) etc., or a combination thereof. Alternatively, the ionic component is an organic anion derived from an organic acid. In certain embodiments, the organic anion includes but is not limited to citrate, pyruvate, malate, oxaloacetate, formate, glutamate, α-ketoglutarate, succinate and acetate anions. In certain embodiments, the ionic component is selected from 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 certain embodiments, the scope of the ionic component is 0.01% (w / v) to 10% (w / v), such as 0.1% (w / v) to 10% (w / v), such as 0.5% (w / v), 1.0% (w / v), 1.5% (w / v), 2.5% (w / v), 3% (w / v) or 5% (w / v). In certain embodiments, the range of the ionic component is 0.5% (w / v) to 9% (w / v), 1% (w / v) to 8% (w / v), 2% (w / v) to 6% (w / v), such as 2.5% (w / v), 3% (w / v) or 5% (w / v). In other embodiments, the concentration of the ionic component (e.g., the concentration expressed in molar concentration) ranges from, for example, 0.01 mM to 200 mM, such as 0.1 mM to 180 mM, 0.5 mM to 150 mM, 1 mM to 120 mM, 1 mM to 100 mM, such as 3 mM, 5 mM, 10 mM, 20 mM, 40 mM, 50 mM, 70 mM or 90 mM. In certain preferred embodiments, the concentration of the ionic component is from 1 mM to 30 mM, such as 15 mM. In other preferred embodiments, the concentration of the ionic component is 2 to 20 mM, such as, for example, 4 mM, 6 mM, 8 mM, 12 mM, 14 mM, 16 mM or 18 mM.

[0241] In another aspect, the composition of any 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 concentration of the albumin is 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 concentration of the albumin is 0.1% (w / v), such as BSA 0.1% (w / v).

[0242] Serum albumin mainly regulates osmotic pressure or colloid osmotic pressure. In addition, it helps in the saturation of proteases and the binding of fatty acids.

[0243] In another aspect, the composition of any of the above embodiments comprises 20 mM Tris (adjusted to pH 7.4 with HCl), 2 mM EDTA and 10 mM MgCl 2 In another embodiment, the composition of any of the above embodiments comprises 5 mM MOPS (adjusted to pH 7.25 with KOH), 10 mM BAPTA and 5 mM sodium pyruvate. In yet another embodiment, the composition of any of the above embodiments comprises 10 mM HEPES (adjusted to pH 7.2 with KOH) and 1 mM EGTA.

[0244] In another aspect, the composition of any of the above embodiments comprises a permeable cryoprotectant less than the cryopreservation amount or does not comprise a permeable cryoprotectant. For example, the permeable cryoprotectant is selected from propylene glycol, ethylene glycol, glycerol and dimethyl sulfoxide (DMSO). In a specific embodiment, the composition of any of the above embodiments comprises dimethyl sulfoxide (DMSO) less than the cryopreservation amount or does not comprise dimethyl sulfoxide (DMSO).

[0245] In one aspect, the composition of any of the above embodiments comprises trehalose at a concentration of at least 160 mM, such as 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, such as 260 mM, at least 270 mM, at least 280 mM, at least 290 mM or at least 300 mM.

[0246] In another aspect, the composition according to any of the above embodiments comprises trehalose at a concentration of at least 150mM and no more than 1500mM. In some specific embodiments, the composition according to any of the above embodiments comprises trehalose at a concentration of no more than 1400mM, no more than 1300mM or no more than 1200mM. In some specific embodiments, the composition according to any of the above embodiments comprises trehalose at a concentration of at least 150mM and no more than 1100mM, such as no more than 1000mM, no more than 900mM, no more than 800mM or no more than 700mM. Preferably, the composition according to any of the above embodiments comprises trehalose at a concentration of at least 150mM and no more than 650mM, such as no more than 640mM, no more than 630mM, no more than 620mM or no more than 610mM. In another preferred embodiment, the composition according to any of the above embodiments comprises trehalose at a concentration of at least 150mM and no more than 600mM, such as no more than 590mM, no more than 580mM, no more than 570mM or no more than 560mM. In a more preferred embodiment, the composition according to any of the above embodiments comprises trehalose at a concentration of at least 150mM and no more than 550mM, such as no more than 540mM, no more than 530mM, no more than 520mM or no more than 510mM. In another more preferred embodiment, the composition according to any of the above embodiments comprises trehalose at a concentration of at least 150mM and no more than 500mM, such as no more than 490mM, no more than 480mM, no more than 470mM or no more than 460mM. Even more preferably, the composition according to any of the above embodiments comprises trehalose at a concentration of at least 150mM and no more than 450mM. In some particularly preferred embodiments, the composition according to any of the above embodiments comprises trehalose at a concentration of 150mM to 450mM, such as 160mM to 440mM, 170mM to 430mM, 180mM to 420mM or 190mM to 410mM. In some further particularly preferred embodiments, the composition according to any of the above embodiments comprises trehalose at a concentration of 200mM to 400mM, such as 220mM to 380mM, such as 240mM, 260mM, 280mM, 320mM, 340mM or 360mM. In some further particularly even more preferred embodiments, the concentration of trehalose is 250mM to 350mM, such as 300mM.

[0247] In one aspect, the sugar contained in the composition of any of the above embodiments, such as the sugar of (iii) cryoprotectant contained in the composition, is selected from any suitable monosaccharide, disaccharide, trisaccharide, oligosaccharide, polysaccharide, or a combination thereof. In a preferred aspect, the sugar contained in the composition of any of the above embodiments, such as the sugar of (iii) cryoprotectant contained in the composition, is selected from any suitable monosaccharide, disaccharide or trisaccharide, or a combination thereof. In a specific embodiment, the sugar is all sugars except trehalose. In certain embodiments, the sugar is a sugar derivative, such as a sugar alcohol. Exemplary sugar alcohols to be used herein are sorbitol, erythritol xylitol (e.g., D-sorbitol, middle erythritol and / or xylitol, particularly 70% w / v D-sorbitol, 35% w / v middle erythritol and / or 70% w / v xylitol), inositol, mannitol, arabitol and / or ribitol. In some other embodiments, the sugar is a modified sugar.

[0248] Preferably, the term "sugar" used herein refers to sugar in a narrow sense, i.e., monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, more preferably monosaccharides and / or disaccharides, as further defined and described herein. In a specific embodiment, sugar is maltose, lactose, fructose, sucrose, glucose, dextran, melezitose, raffinose, niger triose, maltotriose, maltotrione, kestose, cellobiose, chitobiose, lactulose, or a combination thereof. In one embodiment, sugar is preferably sucrose, glucose, or a combination thereof. In one embodiment, the sugar of (iii) cryoprotectant is not trehalose. In one aspect, the concentration of the sugar included in the composition according to any of the above-mentioned embodiments is at least 150mM. In another aspect, the concentration of the sugar included in the composition according to any of the above-mentioned embodiments is at least 160mM. In another aspect, the concentration of the sugar contained in the composition according to any of the above embodiments is at least 170mM, for example, at least 180mM, at least 190mM, at least 200mM, at least 210mM, at least 220mM, at least 230mM or at least 240mM. In other embodiments, the concentration of the sugar is at least 250mM, for example, at least 260mM, at least 270mM, at least 280mM or at least 290mM. In other embodiments, the concentration of the sugar is at least 300mM, for example, at least 310mM, at least 320mM, at least 330mM, at least 340mM, at least 350mM, at least 360mM, at least 370mM, at least 380mM, at least 390mM, at least 400mM, at least 410mM, at least 420mM, at least 430mM, at least 440mM or at least 450mM. In certain preferred embodiments, the concentration of sugar is 300mM to 800mM, for example 320mM, 350mM, 400mM, 410mM, 420mM, 430mM, 440mM, 450mM, 460mM, 470mM, 480mM, 490mM, 500mM, 510mM, 520mM, 530mM, 540mM, 550mM, 560mM, 570mM, 580mM, 590mM, 600mM, 650mM, 700mM or 750mM. The maximum concentration of sugar (e.g., the total amount of sugar) does not exceed 2000mM, for example, such as not exceeding 1900mM, or not exceeding 1800mM. In one embodiment, the maximum concentration of sugars is no more than 1700 mM, for example no more than 1600 mM, no more than 1500 mM, no more than 1400, no more than 1300 mM or no more than 1200 mM. In some preferred embodiments, the maximum concentration of sugars is no more than 1500 mM, for example no more than 1300 mM, no more than 1000 mM, no more than 950 mM, no more than 900 mM, no more than 850 mM, no more than 800 mM, no more than 750 mM or no more than 700 mM.Sugar is provided with such concentration: it is within the range of the endpoints limited by any minimum concentration listed above and any maximum concentration greater than the minimum concentration listed above. When there is more than one sugar in the composition of any of the above-mentioned embodiments, the concentration of sugar reflects the total concentration of all sugars in the composition. In other preferred embodiments, the concentration of sugar is 250mM to 650mM, for example 280mM to 600mM, for example such as 300mM, 400mM or 500mM. Therefore, in some embodiments, sugar is present with a concentration of 150mM to 1600mM, for example 200mM to 1500mM, 250mM to 1200mM or 300mM to 1100mM. In some embodiments, the concentration of sugar is 250mM to 650mM, for example 280mM to 600mM, for example such as 300mM, 400mM or 500mM. In certain embodiments, the sugar included in the composition of any of the above embodiments is sucrose at a concentration of 150mM to 700mM, for example, 160mM to 675mM, for example, 200mM to 650mM, for example, 250mM to 620mM, for example, 300mM to 600mM, for example, 400mM or 500mM. In other embodiments, the composition comprises glucose at a concentration of 300mM to 1200mM, for example, 450mM to 1100, for example, 500mM to 1000mM, for example, 700mM or 800mM. In some specific embodiments, the composition of any of the above embodiments comprises more than one sugar, for example, fructose together with glucose or sucrose, or a combination of fructose, glucose and sucrose. In some preferred embodiments, the composition of any of the above embodiments comprises sucrose and glucose at a concentration (e.g., total concentration) of at least 150mM and no more than 2000mM. In a preferred embodiment, sugar is sucrose, glucose, or a combination thereof, and the total concentration of sucrose and glucose is at least 160mM, such as 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM or 1500mM. In another preferred embodiment, the total concentration of sucrose and glucose is 500mM to 1800mM, more preferably 600mM to 1700mM, such as 800mM to 1450mM, such as 900mM, 1120mM or 1320mM. In one aspect, the total concentration of sugar included in the composition without amino acids is preferably at least 160mM, such as at least 180mM or at least 120.

[0249] The sugar is provided at a concentration within a range having endpoints defined by any minimum concentration listed above and any maximum concentration greater than the minimum concentration listed above. When more than one sugar is present in the composition of any of the above embodiments, the concentration of the sugar reflects the total concentration of all sugars in the composition.

[0250] In one aspect, the amino acid contained in the composition of any of the above embodiments, such as the amino acid (iii) of the cryoprotectant contained in the composition, is any suitable amino acid, amino acid derivative, oligopeptide, peptide, or a combination 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 a combination thereof. In certain preferred embodiments, 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 methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid (e.g., homoproline), oxaproline, thiaproline, or a combination thereof. In an even more preferred embodiment, the amino acid is proline (e.g., L-proline). The concentration of the amino acid included in the composition of any of the above embodiments is at least 160mM, such as at least 180mM, at least 200mM, at least 250mM, at least 300mM, at least 350mM, at least 400mM, at least 450mM, at least 500mM, at least 550mM, at least 600mM, at least 650mM, at least 700mM, at least 750mM, at least 800mM, at least 850mM or at least 900mM. Preferably, the concentration of the amino acid is at least 1000mM, such as at least 1100mM, at least 1200mM, at least 1300mM, at least 1400mM, at least 1500mM, at least 1600mM, at least 1700mM, at least 1800mM, at least 1900mM or at least 2000mM.The amino acid component can be provided at a maximum concentration of no more than 3000 mM, such as no more than 2900 mM, no more than 2850 mM, no more than 2800 mM, no more than 2750 mM, no more than 2700 mM, no more than 2650 mM, no more than 2600 mM, no more than 2550 mM, no more than 2500 mM, no more than 2450 mM, no more than 2400 mM, no more than 2350 mM, no more than 2300 mM, no more than 2250 mM, no more than 2200 mM, no more than 2150 mM, no more than 2100 mM or no more than 2050 mM. The amino acid is provided at a concentration that is within the range of endpoints defined by any of the minimum concentrations listed above and any of the maximum concentrations listed above that are greater than the minimum concentration. When there is more than one amino acid in the composition, the concentration of the amino acid reflects the total concentration of all amino acids in the composition. Thus, in some embodiments, the amino acid is present at a concentration of 500 mM to 3000 mM, such as 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, such as 900 mM to 1900 mM, such as 1500 mM or 1800 mM. In a preferred embodiment, the amino acid is proline at a concentration of 1000 mM to 1700 mM, such as 1500 mM. In another preferred embodiment, the amino acid is proline at a concentration of 1200 mM. In some additional preferred embodiments, the amino acid is a proline derivative, such as but not limited to methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid (e.g., homoproline), oxaproline, thiaproline, at a concentration of 150 mM to 3000 mM, such as 200 mM to 2800 mM or 300 mM to 2500 mM. In a preferred embodiment, the concentration of the proline derivative is 500 mM to 2000 mM, such as 600 mM to 1800 mM, such as 800 mM, 1000 mM, 1300 mM or 1500 mM.

[0251] In one aspect, the polymer included in the composition of any of the above-mentioned embodiments, such as the polymer of (iii) cryoprotectant of composition, is one or more of the polymers with the following characteristics: it is biocompatible, hydrophilic or amphiphilic. For example, polymer component comprises 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, polyvinyl pyrrolidone, or its combination. In some preferred embodiments, polymer is alginate or polyethylene glycol (PEG). In some preferred embodiments, polymer is polyethylene glycol (PEG). The concentration of the polymer included in the composition of any of the above embodiments is 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 concentration of the polymer included in the composition of any of the above embodiments is at least 16% (w / v), for example, at least 18% (w / v). The polymer is provided at a maximum concentration of no more than 30% (w / v), no more than 25% (w / v), no more than 22% (w / v), no more than 21% (w / v), no more than 20% (w / v) or no more than 18% (w / v). Polymer is provided with such concentration: it is within the range of endpoints defined by any minimum concentration listed above and any maximum concentration greater than the minimum concentration listed above. When there is more than one polymer in the composition, the concentration of the polymer reflects the total concentration of all polymers in the composition. Therefore, in some embodiments, the polymer component is provided with 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 concentration of the polymer is 8% (w / v) to 18% (w / v). In other specific embodiments, the concentration of the polymer is 14% (w / v) to 16% (w / v). For example, in a preferred embodiment, the polymer is polyethylene glycol (PEG) and its concentration is 2.5% (w / v) to 30% (w / v), more preferably a concentration of 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) and its concentration is 16% (w / v) to 30% (w / v), more preferably a concentration of 18% (w / v) to 25% (w / v), for example 20% (w / v), 21% (w / v), 23% (w / v) or 24% (w / v).

[0252] In one aspect, the polymer included in the composition is the only cryoprotectant and is present in 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 included in the composition without amino acids and sugars (except trehalose) is polyethylene glycol (PEG) at a concentration of 16% (w / v) to 30% (w / v), such as at a concentration of 18% (w / v) to 28% (w / v), such as 20% (w / v) to 25% (w / v).

[0253] In one aspect, the composition of any of the above embodiments comprises:

[0254] (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;

[0255] (ii) trehalose at a concentration of at least 150 mM; and

[0256] (iii) a cryoprotectant selected from proline, sucrose, glucose, polyethylene glycol (PEG), or a combination thereof, wherein the concentration of proline is at least 150 mM, such as at least 500 mM, preferably at least 1000 mM, such as 1200 mM; the total concentration of sucrose and glucose is at least 150 mM, such as at least 200 mM, such as 300 mM or 500 mM; and the concentration of polyethylene glycol (PEG) is 2.5% (w / v) to 30% (w / v), such as 5% (w / v) to 25% (w / v), such as 10% (w / v), 15% (w / v) or 20% (w / v).

[0257] In another aspect, the composition of any of the above embodiments comprises an aqueous buffer as described in any of the above embodiments.

[0258] In another aspect, the composition of any of the above embodiments further comprises a calcium chelator as described in any of the above embodiments.

[0259] In another aspect, the composition of any of the above embodiments further comprises an ionic component as described in any of the above embodiments.

[0260] In another aspect, the composition of any of the above embodiments further comprises albumin as described in any of the above embodiments.

[0261] In another aspect, the composition of any of the above embodiments comprises 20 mM Tris (adjusted to pH 7.4 with HCl), 2 mM EDTA and 10 mM MgCl 2 In another embodiment, the composition of any of the above embodiments comprises 5 mM MOPS (adjusted to pH 7.25 with KOH), 10 mM BAPTA and 5 mM sodium pyruvate. In yet another embodiment, the composition of any of the above embodiments comprises 10 mM HEPES (adjusted to pH 7.2 with KOH) and 1 mM EGTA.

[0262] In another aspect, the composition of any of the above embodiments comprises a permeable cryoprotectant less than the cryopreservation amount or does not comprise a permeable cryoprotectant. The permeable cryoprotectant is, for example, selected from propylene glycol, ethylene glycol, glycerol and dimethyl sulfoxide (DMSO). In a specific aspect, the composition of any of the above embodiments comprises dimethyl sulfoxide (DMSO) less than the cryopreservation amount or does not comprise dimethyl sulfoxide (DMSO).

[0263] In one aspect, the composition according to any of the above embodiments comprises trehalose in a concentration as defined in any of the above embodiments.

[0264] In one aspect, the composition according to any of the above embodiments comprises sucrose and glucose at a total concentration of at least 150mM and no more than 2000mM, for example 150mM to 1800mM, for example 200mM to 1750mM. In certain embodiments, the composition comprises sucrose and glucose at a total concentration of at least 250mM, for example 250mM to 1650mM. In a specific embodiment, the total concentration of sucrose and glucose is 500mM to 1500mM, for example 600mM to 1400mM, for example 800mM to 1320mM, for example 900mM or 1120mM.

[0265] In one aspect, the concentration of proline included in the composition of any of the above embodiments is at least 200mM, such as 300mM or 400mM. In certain embodiments, the concentration of proline is at least 500mM, such as 600mM, 700mM, 800mM or 900mM. In a more preferred embodiment, the concentration of proline is 600mM. In other preferred embodiments, the concentration of proline included in any of the above compositions of the present invention is at least 1000mM. In a more preferred embodiment, the concentration of proline is 1200mM. In other preferred embodiments, the concentration of proline is at least 1300mM, such as at least 1500mM, 1600mM, 1800mM, 1900mM, 2000mM or 2200mM.

[0266] In another aspect, the cryoprotectant of the composition according to any of the above embodiments is preferably a combination of proline and sucrose. In a specific embodiment, the cryoprotectant is a combination of proline and sucrose, wherein preferably the concentration of proline is at least 500mM, such as 600mM, and the concentration of sucrose is 150mM to 650mM, such as 200mM, 300mM, 400mM, 500mM or 600mM. In a specific embodiment, the cryoprotectant is a combination of proline and sucrose, wherein preferably the concentration of proline is at least 1000mM, such as 1200mM, and the concentration of sucrose is 150mM to 650mM, such as 200mM, 300mM, 400mM, 500mM or 600mM.

[0267] In a preferred embodiment, the cryoprotectant of the composition according to any of the above embodiments consists of proline and sucrose, wherein the concentration of proline is 10% by volume proline (i.e. 600 mM proline) relative to the final total volume of the suspension (i.e. % (v CRYO / v tot )), and the concentration of sucrose is 5%, 10%, 15%, 20%, 25% or 30% by volume sucrose relative to the final total volume of the suspension (i.e., % (v CRYO / v tot )), i.e. the concentration of sucrose is about 100mM, 200mM, 300mM, 400mM, 500mM or 600mM, respectively. In another preferred embodiment, the (iii) cryoprotectant of the composition according to any one of the above embodiments consists of proline and sucrose, wherein the concentration of proline is 20% by volume proline (i.e. 1200mM proline) relative to the final total volume of the suspension (i.e. % (v CRYO / v tot )), and the concentration of sucrose is about 100 mM, 200 mM, 300 mM, 400 mM, 500 mM or 600 mM.

[0268] In another aspect, the cryoprotectant of the composition according to any 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, wherein the concentration of proline is at least 500 mM, such as 600 mM, and the concentration of glucose is 180 mM to 1500 mM, preferably 350 mM 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, wherein the concentration of proline is at least 1000 mM, for example 1200 mM, and the concentration of glucose is 180 mM to 1500 mM, preferably 350 mM to 1200 mM, such as, for example, 450 mM, 550 mM, 700 mM, 850 mM, 900 mM, 1000 mM or 1100 mM.

[0269] In a preferred embodiment, the cryoprotectant of the composition according to any of the above embodiments consists of proline and glucose, wherein the concentration of proline is 10% by volume of proline (i.e. 600 mM proline) relative to the final total volume of the suspension (i.e. % (v CRYO / v tot )), and the concentration of glucose is 5%, 10%, 15%, 20%, 25% or 30% by volume of glucose relative to the final total volume of the suspension (i.e., % (v CRYO / v tot )), i.e., the concentration of glucose is about 175mM, 350mM, 525mM, 700mM, 875mM or 1050mM, respectively. In another preferred embodiment, the cryoprotectant (iii) of the composition according to any of the above embodiments consists of proline and glucose, wherein the concentration of proline is 20% by volume of proline (i.e., 1200mM proline) relative to the final total volume of the suspension (i.e., % (v CRYO / v tot )), and the concentration of glucose is about 175mM, 350mM, 525mM, 700mM, 875mM or 1050mM.

[0270] However, in another aspect, the cryoprotectant of the composition according to any 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, for example 1200 mM, in combination with polyethylene glycol (PEG) at a concentration of 2.5% (w / v) to 30% (w / v), preferably 5% (w / v) to 25% (w / v), for example 10% (w / v), 15% (w / v) or 20% (w / v).

[0271] In one aspect of the invention, the cryoprotectant included in the composition of any of the above embodiments is sucrose, glucose or a combination thereof, and the concentration is (i.e., the total concentration of sucrose and glucose is) at least 200mM, such as at least 300mM, such as 400mM or 500mM. In certain embodiments, the concentration of sucrose and glucose is (i.e., the total concentration of sucrose and glucose is) at least 600mM, such as 850mM to 1550mM, such as 900mM to 1400mM, such as 1000mM or 1350mM. In one embodiment, the cryoprotectant is sucrose alone, and its concentration is at least 250mM and no more than 1200mM, such as at least 300mM and no more than 650mM, such as 400mM and 500mM. In another preferred embodiment, the cryoprotectant is glucose alone, and its concentration is at least 450mM and no more than 1550mM, for example at least 500mM and no more than 1400mM, for example at least 700mM to 1200mM, for example 850mM or 950mM. In another preferred embodiment, the cryoprotectant included in the composition of any of the above embodiments is a combination of sucrose and glucose, and the total concentration is 550mM to 1850mM, for example 800mM to 1700mM, for example a concentration of 900mM, 1150mM, 1400mM or 1500mM. For example, the combination of sucrose and glucose comprises a concentration of 200mM to 650mM, for example 400mM or 500mM sucrose, and a concentration of 350mM to 1350mM, for example 530mM, 880mM or 1100mM glucose.

[0272] In one aspect of the invention, the cryoprotectant contained in the composition according to any of the above embodiments is polyethylene glycol (PEG), such as PEG400 or PEG1000, in a concentration of 2.5% (w / v) to 25% (w / v), such as 5% (w / v) to 25% (w / v), for example, at a concentration of 10% (w / v), 15% (w / v) or 20% (w / v).

[0273] In another aspect of the invention, the only cryoprotectant contained in the composition according to any of the above embodiments is a polymer, such as polyethylene glycol (PEG), such as PEG400 or PEG1000, and its concentration is preferably 16% (w / v) to 30% (w / v), such as 18% (w / v) to 28% (w / v), such as 20% (w / v), 22% (w / v), 24% (w / v), 25% (w / v) or 26% (w / v).

[0274] In one aspect of the invention, the cryoprotectant contained in the cryopreservation composition according to any of the above embodiments is proline at a concentration of at least 150 mM. In certain embodiments, the concentration of proline is at least 300 mM, such as at least 500 mM, such as 600 mM. In a preferred embodiment, the concentration of proline is at least 600 mM, such as 700 mM, 800 mM or 900 mM. In another preferred embodiment, the concentration of proline is at least 1000 mM, such as 1200 mM. In other specific embodiments, the concentration of proline is at least 1300 mM, such as 1500 mM or 1800 mM. In certain preferred embodiments, the concentration of proline is at least 600 mM and no more than 3000 mM, for example at least 1000 mM and no more than 2500 mM, for example such as, 1300 mM to 2200 mM, for example 1500 mM, 1600 mM, 1700 mM, 1800 mM, 1900 mM or 2000 mM.

[0275] In one aspect of the invention, the cryopreservation composition comprises:

[0276] (i) an aqueous buffer having a pH of 6.0 to 8.0, for example pH 7.2;

[0277] (ii) trehalose, at a concentration of 300 mM;

[0278] (iii) a cryoprotectant comprising a combination of proline and sucrose or glucose,

[0279] in:

[0280] - The concentration of proline is 1200 mM;

[0281] - the concentration of sucrose is 200 mM to 1700 mM, such as for example 300 mM to 1500 mM, for example 400 mM to 1300 mM, for example 500 mM or 600 mM; and

[0282] - The concentration of glucose is 300 mM to 1700 mM, such as for example 450 mM to 1500 mM, such as for example 600 mM to 1300 mM, for example 700 mM or 800 mM.

[0283] In one aspect of the invention, the cryopreservation composition comprises:

[0284] (i) an aqueous buffer having a pH of 6.0 to 8.0, for example 7.2;

[0285] (ii) trehalose, at a concentration of 300 mM;

[0286] (iii) a cryoprotectant comprising a combination of proline and polyethylene glycol (PEG),

[0287] in:

[0288] - The concentration of proline is 1200 mM;

[0289] - The concentration of polyethylene glycol (PEG) is 5% (w / v) to 20% (w / v), such as 10% (w / v) or 15% (w / v).

[0290] In one aspect of the invention, the cryopreservation composition comprises:

[0291] (i) an aqueous buffer having a pH of 6.0 to 8.0, for example pH 7.2;

[0292] (ii) trehalose, at a concentration of 300 mM;

[0293] (iii) a cryoprotectant comprising a combination of proline and sucrose or glucose,

[0294] in:

[0295] - The concentration of proline is 600 mM;

[0296] - the concentration of sucrose is 200 mM to 1700 mM, such as for example 300 mM to 1500 mM, for example 400 mM to 1300 mM, for example 500 mM or 600 mM; and

[0297] - The concentration of glucose is 300 mM to 1700 mM, such as for example 450 mM to 1500 mM, such as for example 600 mM to 1300 mM, for example 700 mM or 800 mM.

[0298] In one aspect of the invention, the cryopreservation composition comprises:

[0299] (i) an aqueous buffer having a pH of 6.0 to 8.0, for example 7.2;

[0300] (ii) trehalose, at a concentration of 300 mM;

[0301] (iii) a cryoprotectant comprising a combination of proline and polyethylene glycol (PEG),

[0302] in:

[0303] - The concentration of proline is 600 mM;

[0304] - The concentration of polyethylene glycol (PEG) is 5% (w / v) to 20% (w / v), such as 10% (w / v) or 15% (w / v).

[0305] In another aspect of the invention, the composition of any one of the above embodiments comprises HEPES, such as 10 mM HEPES (adjusted to pH 7.2 to 7.5, such as pH 7.2, with KOH).

[0306] In another embodiment, the composition of any of the above embodiments comprises a calcium chelator, such as EGTA or MEDTA, such as 1 mM EGTA or 1 mM EDTA.

[0307] In another embodiment, the composition of any of the above embodiments further comprises an ionic component, such as Mg 2+ 、Na + , K + , Cl - , HCO 3 - , HPO 4 2- , C 3 H 3 O 3 - (pyruvate anion), C 2 H 2 O 4 2 (formate anion), C 2 H 3 O 2 - (acetate anion) or a combination thereof.

[0308] In another aspect, the composition of any of the above embodiments further comprises albumin, such as, for example, BSA or HSA.

[0309] Embodiment A2: Cryopreservation Compositions Comprising Isolated Viable Mitochondria

[0310] In another aspect of the invention, the composition of any of the above embodiments further comprises isolated mitochondria, such as isolated viable mitochondria. The composition is obtained, for example, by adding isolated mitochondria, such as isolated viable mitochondria, to the composition of any 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.

[0311] In another aspect, the composition of the above-mentioned embodiment comprises 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 HEPG2, A549, MCF7 or RKO, yeast, etc., 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 cells and tissues is yeast or animals, preferably mammals, more preferably people.

[0312] In another aspect, the composition of any of the above embodiments comprises isolated mitochondria, such as isolated viable mitochondria, at a concentration of at least 0.02 μg / μL, such as 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 concentration of isolated mitochondria, such as isolated viable mitochondria, is at least 5 μg / μL, such as at least 10 μg / μL. In another embodiment, the concentration of isolated mitochondria, such as isolated viable mitochondria, contained in the composition of any of the above embodiments is at least 20 μg / μL, such as 25 μg / μL, 30 μg / μL, 35 μg / μL, 40 μg / μL, 45 μg / μL or 50 μg / μL. The isolated mitochondria, e.g., isolated viable mitochondria, are provided at a maximum concentration of no more than 100 μg / μL, e.g., no more than 80 μg / μL, no more than 70 μg / μL, or no more than 60 μg / μL. In one embodiment, the isolated mitochondria, e.g., isolated viable mitochondria, contained in the composition are present at a concentration of 0.02 μg / μL to 10 μg / μL, e.g., such as 0.05 μg / μL to 8 μg / μL, e.g., such as 0.1 μg / μL to 5 μg / μL. In another embodiment, the isolated mitochondria, e.g., isolated viable mitochondria, contained in the composition are present at a concentration of 0.1 μg / μL to 1 μg / μL, e.g., such as 0.2 μg / μL to 0.75 μg / μL.

[0313] In one aspect, the composition of any of the above embodiments comprises isolated mitochondria, such as isolated viable mitochondria, which are connected to a pharmaceutical agent, a diagnostic agent, an imaging agent, a therapeutic agent, or any other biocompatible agent. In some other embodiments, the mitochondria are connected to an antibody or an antigen, such as an antibody or an antigen binding fragment. In another embodiment, the mitochondria are connected 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-helix nucleic acid molecules. In some cases, the nucleic acid polymer is DNA, interfering RNA (siRNA), and microRNA. In a specific embodiment, the reagent and the mitochondria are in physical contact with each other, for example, the reagent is connected to the mitochondria, embedded in the mitochondria, attached to the mitochondria, embedded in the mitochondria membrane, substantially wrapped in the mitochondria, or completely encapsulated by the mitochondria. In another specific embodiment, the reagent and the mitochondria are electrostatically connected. In another specific embodiment, the reagent is covalently connected to the outer membrane of the mitochondria, such as chemically connected, or non-covalently connected, such as electrostatically connected. Mitochondria connected to the reagent described herein are referred to as "combined mitochondrial reagents".

[0314] In one aspect, the composition of the above embodiments comprises mitochondria, for example, isolated viable mitochondria, which are connected to a therapeutic agent, which is selected from but not limited to: inorganic or organic compounds; small molecules (less than 500 Daltons) or macromolecules; protein 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-helix nucleic acid molecules. In some embodiments, the therapeutic agent is a natural product derived from any known organism (e.g., from animals, plants, bacteria, fungi, protists or viruses) or from a synthetic molecule library. In some embodiments, the therapeutic agent is a monomer or polymer compound. Some exemplary therapeutic agents include cytotoxic agents, DNA vectors, small interfering RNA (siRNA), microRNA (miRNA), reactive peptides, nanoparticles, microspheres and fluorescent molecules. In another aspect, the composition of any of the above embodiments comprises mitochondria linked to an imaging agent, which is a radioactive agent, a fluorescent agent, or any agent detectable by any imaging technique, such as but not limited to X-ray, magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), microcomputed tomography (μCT), PET / CT, PET / MRI, fluorescent molecular tomography (FMT), FMT / CT, scintigraphy, or ultrasound. Exemplary imaging agents include but are not limited to Mito tracer fluorophores (ThermoFisher Scientific Inc.), RFP, BacMam 2.0 (Thermo Fisher Scientific Inc.), pH-sensitive pHrodo fluorescent dye (Thermo Fisher Scientific Inc.). In another aspect, the composition of any of the above embodiments comprises mitochondria linked to a diagnostic agent, such as a fluorescent dye or a molecule containing an elemental radioisotope, designed to, for example, determine intracellular conditions, such as intracellular pH and oxidative stress.

[0315] In one aspect, the composition of any of the above embodiments comprises isolated mitochondria, such as isolated viable mitochondria, which have been modified by gene editing. Some non-limiting examples of gene editing techniques for modifying mitochondrial DNA (mtDNA) are, for example, gene editing techniques performed by: restriction endonuclease (RE) technology, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, CRISPR system and pAgo-based system. Alternatively, the composition of any 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.).

[0316] In another aspect, the viable mitochondria contained in the composition according to any of the above embodiments are isolated according to any isolation method known in the art. In a specific embodiment, viable mitochondria are isolated by density gradient centrifugation or ultracentrifugation, differential centrifugation, cell-specific mitochondria 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, HT et al., (2009), Isolation of functional pure mitochondria by superparamagnetic microbeads. Anal. Biochem. 389: 1–5), or differential filtration (e.g., as Preble et al., JOVE (2014): "Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration" and WO 2015 / 192020A1, McCully et al.). Preferably, mitochondria, e.g., viable mitochondria, are isolated by differential filtration, e.g., as performed by differential filtration in a filtration device comprising two or more filters, e.g., differential filtration in a cylindrical filtration device comprising three filters, wherein the filter at the upper end of the device has pores with a pore size of 30 μm to 50 μm, the filter at the lower end of the device has pores with a pore size of 5 μm to 20 μm, and the filter placed between the upper filter and the lower filter has pores with a pore size of 15 μm to 50 μm. In a specific embodiment, the mitochondria contained in the composition of any of the above embodiments have been freshly isolated from cells or tissues before being incorporated (i.e., suspended) into the composition of the present invention.

[0317] method

[0318] Embodiment B: Method for cryopreservation of mitochondria

[0319] The isolated mitochondria of the present invention, such as isolated viable mitochondria, undergo freeze-thaw cycles. Freeze-thaw cycles include freezing the isolated mitochondria in (i.e., contained in) any of the above-mentioned cryopreservation compositions. Without wishing to be bound by any theory or mechanism, the feasibility of freezing the mitochondria contained in any of the compositions disclosed herein and subsequently thawing them without compromising their functionality and structural integrity during freeze-thaw cycles makes the mitochondria easy to store and use, even after long-term storage, with reproducible results.

[0320] In one aspect, the present invention provides a method for cryopreservation of a composition according to any of the above embodiments, the composition comprising isolated mitochondria, such as isolated viable mitochondria, the method comprising the following steps:

[0321] (a) freezing the composition at a temperature below 0°C; and

[0322] (b) storing the frozen composition obtained according to step (a) at a temperature below 0°C.

[0323] In another aspect, the method according to any of the above embodiments comprises a further step (c) followed by step (b) consisting of thawing the frozen composition at a temperature above 0°C.

[0324] In another aspect, the present invention provides a method for cryopreserving a composition, comprising the steps of:

[0325] (a.1) adding isolated mitochondria, such as isolated viable mitochondria, to a cryopreservation composition according to any of the above embodiments;

[0326] (a.2) freezing the composition at a temperature below 0°C; and

[0327] (b) storing the frozen composition obtained according to step (a.2) at a temperature below 0°C;

[0328] (c) optionally thawing the frozen composition at a temperature above 0°C.

[0329] In another aspect, the freezing step (a) or (a.2) of the method according to any of the above-mentioned embodiments is completed in less than 10 hours, for example, in 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, such as in 2 hours or 1.5 hours. In another embodiment, the freezing step (a) or (a.2) of the method according to any of the above-mentioned embodiments is completed in less than 60 minutes, for example, such as in 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, such as in 25, 20 or 15 minutes. In another preferred embodiment, the freezing step (a) or (a.2) of the method according to any of the above-mentioned embodiments is completed in less than 10 minutes, more preferably in less than 5 minutes, for example, in 3 or 2 minutes.

[0330] In another aspect, the freezing step (a) or (a.2) of the method according to any of the above embodiments is carried out at a temperature of at least -200°C (i.e. at a temperature of -200°C or below). In some specific embodiments, the freezing step (a) or (a.2) of the method according to any of the above embodiments is carried out at a temperature of at least (i.e. below) -170°C, for example in liquid nitrogen (e.g. 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 (i.e. below) -80°C, for example at a temperature of at least -90°C or -100°C. In some specific embodiments, the freezing step (a) or (a.2) of the method according to any of the above embodiments is carried out at a temperature of at least (i.e. below) -70°C, for example at a temperature of dry ice (e.g., 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 (i.e. below) -4°C. In certain embodiments, the freezing step (a) of the method of any of the above embodiments is performed at a temperature of at least 0° C. (e.g., 0° C. or below 0° C.). Preferably, the freezing step (a) or (a.2) of the method according to any of the above embodiments is performed in dry ice or in liquid nitrogen, more preferably in dry ice.

[0331] In another aspect, according to the method of any of the above embodiments, the freezing of the composition comprising isolated mitochondria (e.g., isolated viable mitochondria) performed in step (a) or (a.2) is rapid freezing. An example of rapid freezing can be spray freezing as described herein. In another embodiment, the freezing of step (a) or (a.2) of the method of any of the above embodiments is gradual. In another embodiment, the freezing of step (a) or (a.2) is gradually performed at a rate of at least 5°C / minute, such as 8°C / minute or 10°C / minute. In a preferred embodiment, the freezing rate of freezing step (a) or (a.2) is at least 15°C / minute, such as 20°C / minute, 25°C / minute, 30°C / minute, 35°C / minute or 40°C / minute.

[0332] In a specific embodiment, the storage of step (b) of the method for cryopreservation of isolated mitochondria (e.g., isolated viable mitochondria) is carried out 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 of step (b) of the method of any of the above embodiments is at least below -10°C, such as -20°C or -30°C. In one embodiment, the storage temperature of the storage step (b) of the method of any of the above embodiments is the temperature of dry ice (-78.5°C). In another embodiment, the storage temperature of the storage step (b) is the temperature of liquid nitrogen (e.g., -196°C).

[0333] In another aspect, the duration of storage in step (b) of the method according to any of the above embodiments (e.g., storage of a frozen composition comprising isolated mitochondria (e.g., isolated viable mitochondria) at low temperature) is at least 2 hours, such as 3, 4, 5, 6, 7, 8, 10, 12, 15 or 18 hours before thawing (i.e., the storage time of the frozen composition is at least 2 hours, such as 3, 4, 5, 6, 7, 8, 10, 12, 15 or 18 hours before thawing). In some embodiments, the duration of storage in step (b) is 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 other embodiments, the storage period of step (b) is at least one week (i.e., 7 days), such as 2, 3 or 4 weeks before thawing. In other embodiments, the duration of storage of the composition of step (b) of the method according to any of the above embodiments is at least 1 month before thawing, such as 3 months, 6 months, 12 months, 18 months, 24 months or longer. Each possibility represents a separate embodiment of the present invention.

[0334] In another aspect, according to the method of any of the above embodiments, step (c) consists of thawing the frozen composition at a temperature higher than 4°C, such as higher than 10°C. In another embodiment, the thawing of step (c) is carried out at a temperature higher than 18°C, such as 20°C or 22°C. In a specific embodiment, step (c) consists of thawing the frozen composition at a temperature not exceeding 40°C, such as 39°C or 38.5°C. In a specific embodiment, the thawing of step (c) is preferably carried out at a temperature between 20°C and 38°C, such as at a temperature of 37°C (e.g., 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 of step (c) is carried out at a temperature that enables the mitochondria to be administered to patients in need of treatment. In another specific embodiment, the thawing of step (c) is carried out gradually. For example, in some embodiments, the thawing is carried out at a temperature that increases by at least 1°C per minute (i.e., 1°C / minute), such as 2°C / minute, 3°C / minute, or 4°C / minute. In a preferred embodiment, thawing is carried out when the temperature increases at a rate of at least 5°C / min, such as 6°C / min, 7°C / min, 8°C / min, or 9°C / min, more preferably when the temperature increases at a rate of at least 10°C / min, such as 15°C / min, 20°C / min, or 30°C / min.

[0335] In another aspect, the thawing step (c) according to the method for any of the above-mentioned embodiments is carried out in a time period of up to 6 hours, for example, 5,4 or 3 hours. In one embodiment, the duration of thawing step (c) is up to 2.5 hours, for example, 2 hours or 1.5 hours. In a specific embodiment, thawing is carried out for up to 60 minutes, for example, 50,45,40 or 35 minutes. In a preferred embodiment, the duration of thawing step (c) is up to 30 minutes, for example, 25 minutes or 20 minutes. In another preferred embodiment, the duration of thawing step (c) is up to 15 minutes, for example, 10 minutes. In a more preferred embodiment, the duration of thawing step (c) is up to 5 minutes, for example, 3 minutes.

[0336] Another method for cryopreserving mitochondria contained in a composition according to any of the above embodiments comprises a freezing step, in particular a spray freezing (SF) step, optionally followed by a drying step (i.e., spray freeze drying (SFD)). Spray freezing (SF) and spray freeze drying (SFD) directly address the challenge of preserving thermolabile mitochondria (including mitochondrial subparticles). Spray freezing is applied by first spraying a composition comprising mitochondria into a liquid freezing medium (e.g., liquid nitrogen contained in a chamber or container), and then by maintaining the composition at a temperature below 0°C, such as the temperature of the freezing medium itself. The composition can be sprayed by any suitable spraying device, such as a spray gun, which is optionally pre-cooled appropriately before use. In the SFD method, the composition is first sprayed into a freezing medium, such as liquid nitrogen, and frozen therein. The shortest time between isolating fresh mitochondria and spray freezing a composition comprising these isolated mitochondria (i.e., a cryopreservation composition of the present invention comprising isolated mitochondria) is preferably no more than 120 minutes after isolating fresh mitochondria, more preferably no more than 60 minutes, and even more preferably no more than 30 minutes. According to this method, the freezing step requires a very short time: it is rapid, almost instant (e.g., flash freezing). The droplets of the spray composition comprising mitochondria according to any of the above embodiments, once frozen, can be preserved / stored in a freezing medium such as liquid nitrogen, or in any other suitable cryogen such as dry ice for a period of at least 2 minutes, 5 minutes, 10 minutes, 30 minutes or 60 minutes. The size of the droplets is 1 μm to 100 μm, more typically 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 be stored for a period of even 1 or 2 years.

[0337] After being spray frozen, the composition can be separated from the freezing medium, for example from liquid nitrogen, by evaporating the freezing medium, for example at a temperature above 0° C., for example at room temperature (e.g., between 20 and 25° C.). The evaporation of the freezing medium can be carried out at atmospheric pressure (e.g., 1 atm or 1.01325 bar) or at a pressure below atmospheric pressure.

[0338] After separation from the freezing medium, for example after removal of the freezing medium at a pressure below atmospheric pressure, the composition obtained may be a lyophilized composition.

[0339] Spray-frozen compositions may be thawed (eg, freeze-dried) prior to use.

[0340] In another aspect, the present invention provides a method for cryopreservation of a composition according to any of the above embodiments, the composition comprising isolated mitochondria, such as isolated viable mitochondria, the method comprising the following steps:

[0341] (i) spray freezing the composition into a freezing medium, such as liquid nitrogen at a temperature of about -196°C; and

[0342] (ii) storing the frozen composition obtained according to step (i) at a temperature below 0°C, such as the temperature of liquid nitrogen.

[0343] In another aspect, the present invention provides a method for cryopreservation of a composition according to any of the above embodiments, the composition comprising isolated mitochondria, such as isolated viable mitochondria, the method comprising the following steps:

[0344] (i) spray freezing the composition into a freezing medium, such as liquid nitrogen at a temperature of about -196°C;

[0345] (ii) drying / evaporating a freezing medium, such as liquid nitrogen; and

[0346] (iii) storing the freeze-dried composition obtained according to step (ii) at a temperature below 0°C

[0347] In one aspect, the invention provides a method for cryopreservation of a composition according to any of the above embodiments, wherein the composition comprises 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:

[0348] (a) freezing the composition in liquid nitrogen (e.g., at a temperature of about -196°C) or in dry ice (e.g., at a temperature of -78.5°C), preferably in dry ice, for at least 1 minute; and

[0349] (b) storing the frozen composition obtained according to step (a) at a temperature below 0°C.

[0350] In a specific embodiment, the freezing step (a) or (a.2) of the method of any of the above embodiments is carried out in dry ice for a period of up to 5 minutes; the storage step (b) is carried out at a temperature below -5°C, for example at a temperature of -4°C, -8°C, -15°C, -20°C, -25°C, -50°C or -78.5°C, and lasts for a period of at least 1 week, for example 4 weeks or 8 weeks, preferably at least 12 weeks or 24 weeks, more preferably at least 32 weeks, for example 36 weeks or 72 weeks.

[0351] In one aspect, the method of the present invention is a method for cryopreserving a composition according to any of the above embodiments, wherein the composition comprises 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 of isolated mitochondria, such as isolated viable mitochondria. However, in another embodiment, the present invention provides a method for cryopreserving a composition according to any of the above embodiments, wherein the composition comprises 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 of isolated mitochondria, such as isolated viable mitochondria.

[0352] Therapeutic treatment

[0353] Embodiment C: Cryopreserved Compositions Containing Mitochondria for Treatment of Disease

[0354] The present invention provides herein a cryopreservation composition comprising mitochondria, such as human mitochondria, or a cryopreservation composition prepared by any of the above methods, which is used as a medicine. Specifically, the present invention provides a composition according to any of the above embodiments, which is used in a therapeutically effective amount for treating a condition that benefits from the mitochondrial function of the mitochondria that is improved or repaired in a subject in need thereof, and a condition that benefits from the combined use of mitochondria with another agent (such as a molecule, antibody, oligonucleotide, peptide or extracellular vesicle). Combination use may include concomitant application or administration of a medicament and mitochondria and a solution, in which the medicament and mitochondria are prepared as a therapeutic entity, such as, for example, prepared as a single therapeutic agent, wherein the medicament is introduced into or connected to the mitochondria (before or after its cryopreservation).

[0355] The present invention provides herein a cryopreservation composition according to any of the above embodiments, comprising isolated mitochondria, such as isolated viable mitochondria, for use in a therapeutically effective amount in a subject in need thereof for treating a disease or disorder. In a specific embodiment, the mitochondria contained 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 specific embodiment, the mitochondria contained in the composition for treating a disease are combined mitochondrial reagents, such as previously isolated and subsequently connected to a pharmaceutical agent, a therapeutic agent, a diagnostic agent, or an imaging agent. The combined mitochondrial reagent comprises isolated mitochondria, which is artificially combined with a therapeutic agent, a pharmaceutical agent, a diagnostic agent, an imaging agent, or any other reagent. The reagent is combined with the mitochondria in any manner, for example, connected to the mitochondria (e.g., chemically or electrostatically connected), attached to the mitochondria, such as attached 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 reagent are in physical contact with each other.

[0356] Thus, the present specification provides a composition according to any of the above embodiments for use in treating a disease in a patient in need thereof by delivering the composition and the mitochondria contained therein to cells and / or tissues of the patient or cells derived from an allogeneic donor. A composition according to any of the above embodiments comprising isolated mitochondria (e.g., isolated viable mitochondria) for use in treating a disease or condition that benefits from improved mitochondrial function has been frozen, stored (i.e., cryopreserved), and thawed directly prior to use.

[0357] The mitochondria contained in the composition according to any of the above embodiments for treating a variety of diseases or conditions have been frozen, stored, and subsequently thawed according to the cryopreservation method of any of the above embodiments prior to administration to a subject in need thereof, and the diseases or conditions include but are not limited to various forms of cancer, tumors, autoimmune diseases, aging-related diseases, metabolic diseases, ischemia-related or ischemia-reperfusion injury and / or disease or trauma.

[0358] In one aspect, the invention provides a composition according to any of the above embodiments for use in a therapeutically effective amount for treating a disease in a subject in need thereof, such as by therapeutic mitochondrial transplantation (TMT).

[0359] In some embodiments, the mitochondria (e.g., isolated viable mitochondria) contained in the composition according to any of the above embodiments are autologous (i.e., autogeneic or autogenous). In some embodiments, the mitochondria are autologous or autologous mitochondria with genetic modifications. In other embodiments, the mitochondria are autologous and connected to imaging agents, diagnostic agents, or pharmaceutical agents. In other embodiments, the reagent is embedded or incorporated into autologous mitochondria. In other embodiments, the mitochondria are allogenic. In some embodiments, the mitochondria are allogenic mitochondria with genetic modifications. In other embodiments, the mitochondria are allogenic mitochondria connected to imaging agents, diagnostic agents, or pharmaceutical agents. In other embodiments, the reagent is embedded or incorporated into allogenic mitochondria. In other embodiments, the mitochondria are heterogeneous. In some embodiments, the mitochondria are heterogeneous mitochondria with genetic modifications. In other embodiments, the mitochondria are heterogeneous mitochondria connected to imaging agents, diagnostic agents, or pharmaceutical agents. In other embodiments, the reagent is embedded or incorporated into heterogeneous mitochondria. In other embodiments, the mitochondria are heterogeneous. In some embodiments, the mitochondria are heterogeneous mitochondria with genetic modifications. In other embodiments, the mitochondria are heterogeneous mitochondria connected to imaging agents, diagnostic agents, or pharmaceutical agents. In other embodiments, the reagent is embedded or incorporated into heterogeneous mitochondria.

[0360] In one aspect, the composition of any one of the above embodiments has been subjected to freeze-thaw cycles prior to use in treating a disease.

[0361] In another aspect, the composition of any of the above embodiments has been subjected to freeze-thaw cycles according to the method of any of the above embodiments prior to use in treating a disease.

[0362] In another aspect, the present invention provides a composition according to any of the above embodiments, comprising isolated mitochondria, such as isolated viable mitochondria, for treating a disease in a subject (e.g., a patient) in need thereof, wherein the composition is administered to a subject in need thereof in a therapeutically effective amount, by a variety of routes, such as by topical or parenteral administration, such as by skin or subcutaneous administration, by nebulization administration, by direct injection, by vascular infusion, such as intravenous injection (i.e., iv injection), or by injecting the composition into a subject's blood vessel (e.g., by intra-arterial injection or infusion into a blood vessel), wherein the blood vessel is part of the subject's vascular system and delivers blood to a target site. The blood flowing in the blood vessel carries isolated viable mitochondria (e.g., isolated viable mitochondria modified by gene editing) or a combined mitochondrial agent to a 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, auditory nerve, brain, or skeletal muscle. In certain embodiments, the blood vessel is a coronary artery of the subject. In some embodiments, the mitochondria or combined mitochondrial agents contained in the composition of any of the above embodiments are delivered to target cells in vitro, ex vivo, and in vivo. In a specific embodiment, a composition comprising isolated viable mitochondria (e.g., mitochondria modified by gene editing or mitochondria connected to a therapeutic agent, diagnostic agent, or imaging agent) according to any of the above embodiments is used to treat ischemia-reperfusion injury, such as pulmonary, renal, cardiac, or cerebral ischemia-reperfusion injury, and the composition is administered to a subject in need thereof in a therapeutically effective amount, such as by direct injection or intravenous or intra-arterial injection.

[0363] In one aspect, the composition according to any of the above embodiments is used to treat mitochondrial or mitochondrial-related diseases in a subject in need thereof. In one embodiment, the composition according to any of the above embodiments is used to treat mitochondrial diseases caused by mutations in mitochondrial DNA (mtDNA) or nuclear genes encoding mitochondrial components, such as acquired or inherited mutations. In another embodiment, the composition according to any of the above embodiments is used to treat hereditary mitochondrial disorders, such as but not limited to mitochondrial encephalopathy, mitochondrial myopathy, lactic acidosis and stroke-like attacks (MELAS) syndrome, myoclonic epilepsy with broken red fiber syndrome (MERRF), neuropathy ataxia and retinitis pigmentosa (NARP) syndrome, myoneurogenic gastrointestinal encephalopathy (MNGIE), Leber's hereditary optic neuropathy, myoclonic epilepsy with broken red fiber syndrome (MERRF) disorders, maternal inherited diabetes and deafness (MIDD), Leigh's syndrome and Huntington's disease (HD). In another embodiment, the composition according to any of the above embodiments is used to treat acquired mitochondrial-related diseases caused by toxicity, chemotherapy or iatrogenic diseases. In another embodiment, the composition according to any of the above embodiments is used to treat acquired mitochondrial-related diseases or dysfunctions, such as but not limited to central nervous system (CNS) diseases, such as Alzheimer's disease, Parkinson's disease. In some embodiments, mitochondria and mitochondrial-related diseases / disorders are chronic.

[0364] In another aspect, the composition according to any of the above embodiments is used to treat ischemic or ischemia-reperfusion-related injuries in a subject in need thereof, for example, to promote the repair process of damaged cells or tissues. In one embodiment, the composition according to any of the above embodiments is used to promote the repair process of damaged cells or tissues, wherein the injury is caused by stroke, traumatic brain injury or spinal cord injury. In one embodiment, the composition according to any of the above embodiments is used to treat non-ischemic injuries.

[0365] In another aspect, the composition according to any of the above embodiments is used to increase blood flow in the vasculature in a patient in need thereof, for example, for angiography. In some embodiments, the composition is used to dilate a blood vessel, such as an artery or vein, in a patient in need thereof. In other embodiments, the composition is used to reduce vascular resistance in an organ (e.g., heart, kidney, liver, or lung). In certain embodiments, the composition is used to locate, identify, and / or remove obstructions in a blood vessel, such as, for example, a blood clot.

[0366] In another aspect, the composition according to any of the above embodiments is used to treat wounds, such as wounds showing impaired wound healing, such as wounds in diabetic patients. In another aspect, the composition according to any of the above embodiments is used to promote the repair response of acute wounds. In another aspect, the composition according to any of the above embodiments is used to promote the repair response of 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. (October 30, 2020); 48 (5): 1995–2002, Horn A. et al.; Cell Regeneration volume 10, Article number: 5 (2021), Ma Y. et al.)

[0367] In another aspect, the composition according to any of the above embodiments is used to treat changes, disorders or diseases associated with aging. Some non-limiting examples of dysfunction / diseases associated with aging are neurodegenerative diseases (e.g., Parkinson's disease (PD), Alzheimer's disease (AD), dementia), skin aging, retinal dysfunction, hearing loss, metabolic diseases associated with aging (e.g., diabetes or cachexia) and muscle weakness, hypotonia, dystonia, malnutrition and / or atrophy (Biology (2019), 8, 48, RH Haas). Another example of a disease associated with aging is Hutchinson-Gilford progeria syndrome, a progressive genetic disorder that causes rapid aging in children.

[0368] In another aspect, the composition according to any of the above embodiments is used to treat a neurodegenerative disease, such as, for example, amyotrophic lateral sclerosis (ALS) or multiple sclerosis (MS).

[0369] In another aspect, the composition according to any of the above embodiments is used to treat sarcopenia, such as "primary" (or age-related) sarcopenia, which can be measured, for example, according to the diagnostic criteria of the European Working Group on Sarcopenia in Older People (EWGSOP), or for the treatment of "secondary" sarcopenia. Some non-limiting examples of secondary sarcopenia are: (a) sarcopenia caused by bed rest, sedentary lifestyle and inactivity, (b) disease-related sarcopenia, which is 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, caused by, for example, malabsorption, gastrointestinal disorders or the use of drugs that cause anorexia.

[0370] In another aspect, a composition according to any of the above embodiments is used for treating cancer in a human subject in need thereof. Some illustrative suitable cancers include, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, 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's lymphoma (HL), hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma (Kaposi 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 neck cancer with occult primary, midline tract cancer involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), 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, Sezary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumors, gastric cancer, T-cell lymphoma, teratoid tumor, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

[0371] In another aspect, a composition according to any of the above embodiments is used for treating an infectious disease in a human subject in need thereof.

[0372] In another aspect, the composition according to any of the above embodiments is used to treat or improve immune dysfunction or disease. In one aspect, the composition according to any of the above embodiments is used to mediate and regulate immune response. In a specific embodiment, the composition according to any of the above embodiments is used to treat or improve immune dysfunction or disease, wherein the mitochondria included in the composition described in any of the above embodiments are transplanted into naturally occurring immune cells to produce mitochondrial enhanced immune cells. It is not intended to be limited by any theory, in some embodiments, immune system damage (e.g., immune disorder or dysfunction) may be due to the reduction of immune diversity of initial T cells and the increase of aging effector cell number with age. In a specific aspect, the composition according to any of the above embodiments is used to treat autoimmune diseases. Some 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 (IBS), autoimmune Addison's disease, Grave's disease, Sjögren's syndrome, 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 a vital role not only in regulating the growth of immune cells, but also in regulating the function of immune cells. In addition to providing energy to support the synthesis of macromolecules necessary for immune cell proliferation, mitochondria also serve as signal transduction organelles, driving the activation of immune cells via metabolic intermediates, mitochondrial DNA (mtDNA) and reactive oxygen species (ROS). In addition, mitochondrial dynamics (fusion and fission), biogenesis (synthesis of new mitochondria) and mitochondrial autophagy (degradation of damaged mitochondria) also play an important role in regulating immune cell function. The connection between immune cell function and mitochondrial function is referred to as "immunometabolism" in the art. The relationship between immunity and metabolic pathways is crucial in many dysfunctions of immune response, such as in critical illness, particularly sepsis.

[0373] In another aspect, the composition according to any of the above embodiments is used in gene therapy. In a certain embodiment, gene therapy is used to treat cancer.

[0374] In another specific embodiment, gene therapy is used to treat infectious diseases. In another embodiment, gene therapy is used to treat heart, lung, kidney, bladder, prostate, uterus, pancreas or kidney disease. In another embodiment, gene therapy is used to treat hereditary gene defects or acquired genetic disorders. In a specific embodiment, gene therapy is used to treat autoimmune diseases. In another embodiment, gene therapy is used to treat mitochondrial-related diseases. Mitochondrial 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 broken red fiber syndrome, and subsequent stroke and traumatic brain or spinal cord injury (Mitochondrion (July 2017), 35: 70-79, Gollihue and Rabchevsky). Drugs can induce mitochondrial dysfunction by different mechanisms, including inhibition of fatty acid oxidation, damage to oxidative phosphorylation and respiratory chain activity, and changes in the integrity of mitochondrial membranes. Some drugs also damage mitochondrial function, via the generation of reactive oxygen species and the generation of reactive metabolites, which can be covalently bound to key mitochondrial proteins. Drug-induced mitochondrial dysfunction plays an important role in the pathogenesis of adverse effects such as liver damage, myopathy and cardiotoxicity. Some examples of drugs that can induce mitochondrial dysfunction are, for example, acetaminophen, amiodarone, doxorubicin, nucleoside reverse transcriptase inhibitors (e.g., stavudine, zidovudine, didanosine), statins (e.g., atorvastatin, cerivastatin, simvastatin) and valproic acid (Mitochondrial Biology and Experimental Therapeutics, (March 2018), pp 269–295, Massart).

[0375] In a specific aspect, the composition according to any of the above embodiments is used for gene therapy, wherein the mitochondria contained in the composition described in any of the above embodiments are transplanted into naturally occurring immune cells to produce mitochondrial enhanced immune cells. In another specific embodiment, the composition according to any of the above embodiments is used for gene therapy, wherein the mitochondria contained in the composition described in any of the above embodiments are transplanted into modified immune cells, such as but not limited to chimeric antigen receptor (CAR) T cells, CAR-NK cells, CAR- macrophages, neutrophils, tumor infiltrating lymphocytes (TIL), γ-δ T cells. In some aspects, immune cells are T lymphocytes, such as CD4 T cells, such as Treg cells. In some aspects, immune cells are natural killer (NK) cells. In some aspects, immune cells are mucosal-associated constant T cells. In some aspects, immune cells are γ-δ T cells. In some aspects, immune cells are monocytes or macrophages. In some aspects, immune cells are neutrophils. In some aspects, immune cells are B lymphocytes. In some aspects, the immune cells are generated from stem cells, mesenchymal stem cells or induced pluripotent stem cells (iPSC). In some aspects, the immune cells are allogeneic or autologous immune cells.

[0376] In a specific aspect, the composition according to the above embodiment is used for gene therapy, wherein the mitochondria contained in the composition in any of the above embodiments are first frozen and thawed according to any of the above methods, or frozen, stored and thawed, and then transplanted into immune cells to produce mitochondrial 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), γ-δ T cells. In another aspect, mitochondria are transplanted into immune cells before freezing to produce mitochondrial enhanced immune cells.

[0377] In another aspect, the composition according to any of the above embodiments is used for gene therapy, wherein the mitochondria contained in the composition described in any of the above embodiments are transplanted into stem cells, for example, the mitochondria in the composition are first frozen and thawed according to any 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 pluripotent stem cell-derived immune cells. In some aspects, the immune cells are T lymphocytes, such as helper T cells, cytotoxic T cells, regulatory T cells, memory T cells. In some aspects, the T lymphocytes are CD8 T cells. In some aspects, the T lymphocytes are CD4 T cells, such as Treg cells. In some aspects, the immune cells are natural killer (NK) cells. In some aspects, the immune cells are mucosal-associated constant T cells. In some aspects, the immune cells are γ-δ 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 cell is a B lymphocyte.In some aspects, the stem cell is an allogeneic or autologous stem cell.

[0378] In a specific aspect, the composition according to the above embodiments is used for gene therapy, wherein the mitochondria contained in the composition in any of the above embodiments are first frozen and thawed according to any of the above methods, or frozen, stored and thawed, and then transplanted into stem cells to produce mitochondrial enhanced stem cells. In another aspect, the mitochondria are transplanted into stem cells to produce mitochondrial enhanced stem cells before the freezing cycle.

[0379] In a specific aspect, the composition according to any one of the above embodiments is used to treat acute or chronic graft-versus-host disease (GVHD).

[0380] the term

[0381] Unless otherwise defined, all technical terms, symbols and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, for clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not be interpreted as representing differences that are commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are generally used using conventional methods. As appropriate, unless otherwise stated, procedures involving the use of commercially available kits and reagents are generally performed according to the protocols and conditions specified by the manufacturer.

[0382] As used herein, unmodified nouns mean one or more unless the context clearly indicates otherwise.

[0383] The terms "including", "such as", etc. are intended to express inclusion rather than limitation, unless specifically stated otherwise.

[0384] As used herein, the term "or" is generally used in its ordinary sense, including "and / or," unless the content clearly dictates otherwise. The term "and / or" means one or all of the listed elements or any combination of two or more of the listed elements.

[0385] Unless specifically stated otherwise, the term "comprising" as used herein also specifically includes the embodiments of "consisting of the recited elements" and "consisting essentially of the recited elements."

[0386] The term "about" as used herein refers to and encompasses a specified value and a range above and below that value. In certain embodiments, the term "about" refers to a specified value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term "about" refers to a specified value ± one standard deviation of that value.

[0387] As used herein, the term "isolated" means changed or removed from a natural state or environment. For example, a nucleic acid, peptide, or subcellular particle (e.g., mitochondria) naturally present in a living animal or cell is not "isolated," but the same nucleic acid, peptide, or subcellular particle (e.g., mitochondria) partially or completely separated from the coexisting materials of its natural state is "isolated."

[0388] "Mitochondria" used herein refers to viable mitochondria that are (substantially) free of eukaryotic cell material, such as foreign eukaryotic cell material, which is, for example, separated / purified from a cell or cell culture. Therefore, only a very small amount of cellular components (except mitochondria) are present in the mitochondria (composition) used herein. Preferably, in addition to mitochondria, no other cellular components are present in the mitochondria (composition) used herein. In this sense, "mitochondria" used herein are "isolated mitochondria", and the terms "mitochondria" and "isolated mitochondria" are used interchangeably. Any current technology known in the art can be used to isolate mitochondria, such as by repeated differential centrifugation (DC) or density gradient centrifugation (DGC) for subcellular fractionation (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). A preferred non-limiting example of how to obtain "isolated mitochondria" is provided in Preble et al., JOVE (2014): "Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration" and WO 2015 / 192020 A1 (McCully et al.), wherein mitochondria are separated by differential filtration, optionally followed by centrifugation. The term "isolated mitochondria" as used herein refers to mitochondria separated from other cellular components of donor cells. The term "donor cell" as used herein refers to a cell from which the mitochondria of the present invention are isolated. The terms "recipient cell", "accepting cell" and "host cell" are used interchangeably herein to describe cells that receive and encompass isolated mitochondria.

[0389] As used herein, the term "viable mitochondria" is used throughout the specification to describe viable mitochondria, which are complete, active, functional and respirable mitochondria. According to some embodiments, "viable mitochondria" refers to mitochondria that exhibit biological functions, such as, for example, respiration and ATP and / or protein synthesis.

[0390] The term "intact mitochondria" as used herein is used throughout the specification to describe mitochondria that include intact outer and inner membranes, intact intermembrane spaces, intact cristae (formed by the inner membrane), and intact matrix. Alternatively, intact mitochondria are mitochondria that retain their structure and ultrastructure. In another aspect, intact mitochondria include active respiratory chain complex IV embedded in the inner membrane, the ability to maintain membrane potential and synthesize ATP.

[0391] The term "modified mitochondria" used herein refers to mitochondria that have been modified by gene editing. Some 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 systems. Alternatively, mitochondria, such as isolated viable mitochondria containing exogenous mtDNA, such as mitochondria modified by transplanting exogenous mtDNA into autologous mitochondria. Alternatively, mitochondria have been modified by gene editor TALED technology, for example, the technology can rely on TALE proteins to target specific mitochondrial DNA sequences, and the desired adenine to guanine editing, and the enzymes for the reversal of cytosine to thymine are applied.

[0392] The term "mitochondrial DNA (mtDNA)" as used herein refers to the DNA of mitochondria, which is a double-stranded circular molecule containing 16,569 base pairs and 37 genes [external heavy chain (H chain) and internal light chain (L chain)] (Anderson et al., 1981). mtDNA has two non-coding regions: one is the light chain replication origin (OL), and the other is the displacement loop (D-loop), also known as the control region (CR), which contains the heavy chain replication origin (OH), the heavy chain promoter and the light chain promoter (Lotte et al., 2013). The mtDNA of mammalian cells is relatively small and genetically compact, containing two overlapping genes and very few non-coding sequences (Ojala et al., 1981).

[0393] As used herein, the phrases "cryoprotective composition," "cryoprotecting composition," "cryopreservation composition," "cryoprotectant," "cryoprotective agent," "cryoprotecting agent," or "cryopreservative agent" refer to chemical mixtures, chemical solutions, or chemical compounds that promote the cryoprotection process by reducing damage to mitochondria (e.g., isolated viable mitochondria) during freezing and thawing (e.g., during freeze-thaw cycles). Cryoprotectants protect mitochondria from damage associated with storage at subzero temperatures and / or freezing, such as mitochondrial membrane damage due to ice crystal formation.

[0394] The term "permeable cryoprotectant" as used herein refers to a cryoprotectant that is designed to pass through biological membranes and exert its effect particularly within biological structures or cells. "Permeable cryoprotectants" are also referred to as "intracellular cryoprotectants". Permeable cryoprotectants are small non-ionic molecules that have high water solubility at low temperatures and high diffusivity to lipid membranes. This group includes propylene glycol, ethylene glycol, glycerol, and dimethyl sulfoxide (DMSO). These substances diffuse efficiently through cell membranes and bind to water within cells, which results from imagination.

[0395] The term "cryopreserved mitochondria" or "frozen mitochondria" as used herein refers to mitochondria preserved by cooling to subzero temperatures, such as isolated viable mitochondria. "Cryopreserved mitochondria" include autologous (e.g., self- or autogenous), allogenic or xenogeneic mitochondria. The term "cryopreserved mitochondria" refers to, for example, mitochondria that have been isolated and frozen from any type of organ, tissue or cell (e.g., cultured cells). In some embodiments, frozen mitochondria are stored for a period of time. In some embodiments, mitochondria have undergone one or more freezing and thawing cycles. In some embodiments, mitochondria that have undergone freeze-thaw cycles are isolated mitochondria with viability. In some embodiments, cryopreserved isolated viable mitochondria are contained in a cryopreservation composition (e.g., contained therein). In some embodiments, mitochondria have undergone a spray freezing step.

[0396] As used herein, the term "cryopreservative amount" or "cryoprotective amount" refers to an amount of a cryopreservative or cryoprotective compound / agent that allows the mitochondria to maintain their physical, chemical, functional, and structural stability and integrity during and after a freezing cycle or a freeze-thaw cycle. The term "less than a cryopreservative amount" refers to an amount of a cryopreservative / cryoprotective compound / agent that is insufficient to maintain the physical, chemical, functional, or structural stability and integrity of the mitochondria during and after a freezing cycle of a freeze-thaw cycle.

[0397] As used herein, the terms "autologous," "self," or "autogenic" refer to any material derived from the same individual that is subsequently reintroduced into that individual.

[0398] As used herein, the term "allogeneic" refers to any material that originates from an animal of the same species as the individual into which the material is introduced or from a different patient than the individual into which the material is introduced. Two or more individuals are considered allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently different genetically to interact antigenically.

[0399] As used herein, the term "xenogeneic" refers to a transplant derived from an animal of a different species, ie, "xenogeneic mitochondria" refers to mitochondria obtained from a species different from that of the subject being treated.

[0400] The term "freeze-thaw cycle" as used herein 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 determined period of time, and thawing the mitochondria to room temperature or body temperature or any temperature above 0°C, which enables the mitochondria to be administered to a person in need of treatment, such as a person suffering from, for example, a mitochondrial or mitochondrial-related disease or disorder, inflammatory, infectious, autoimmune disease or disorder, cancer, ischemia or ischemia-related disorders and injuries, blood clots and blood flow dysfunction. Each possibility represents a separate embodiment of the present disclosure. The term "room temperature" as used herein refers to a temperature of 18°C ​​to 25°C. The term "body temperature" as used herein refers to a temperature of 35.5°C to 37.5°C, preferably 37°C.

[0401] As used herein, the term "flash-freezing" or "snap-freezing" refers to rapidly freezing mitochondria or compositions comprising mitochondria by subjecting them to cryogenic temperature.

[0402] As used herein, the terms "buffering agent," "buffer," "aqueous buffer," or "buffer system" are understood to mean a substance that maintains the pH of an aqueous medium within a narrow range even when small amounts of acid or base are added. The term "buffer" means those single substances or combinations of substances that prevent changes in hydrogen ion concentration when an acid or base is added.

[0403] As used herein, the term "chelating agent" or "chelator" refers to any organic or inorganic compound that will bind to a metal ion having a valence greater than 1. Exemplary chelating agents are compounds that bind calcium ions (i.e., calcium chelating agents or calcium chelators), such as EDTA and EGTA.

[0404] As used herein, the term "sugar" or "sugar component" refers to any sugar, such as general monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides. In some 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 (e.g., deoxyribose sugar or sucralose). In some 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.

[0405] The term "trehalose" as used herein refers to a sugar composed of two glucose molecules. 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) Alk-2-yl]oxy Alkane-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 (also called neotrehalose), and β,β-trehalose (also called isotrehalose).

[0406] The term "amino acid" as used herein refers to naturally occurring or non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. The term "amino acid" refers to proteinogenic or non-proteinogenic amino acids. Naturally encoded amino acids are 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 refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., α carbon is combined with hydrogen, carboxyl, amino group and R group, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. As used herein, the term "amino acid" may be referred to herein by either its commonly known three letter symbols or by the single letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may also be referred to by their commonly accepted single letter codes.

[0407] The term "protein" as used herein refers to large biomolecules and macromolecules comprising one or more long chains of amino acid residues. These biomolecules can be the product of biosynthesis or chemical (organic) synthesis technology. The term "post-translationally modified protein" is a protein modified according to "post-translational modification (post-translational modification, PTM)". PTM refers to covalent, and usually enzymatic post-translational modification, which refers to any change in the amino acid sequence of a protein after its synthesis. PTM may involve modification of amino acid side chains, terminal amino groups or carboxyl groups by covalent or enzymatic methods after protein biosynthesis. Generally speaking, these modifications affect the structure, stability, activity, cellular localization or substrate specificity of proteins. Post-translational modification makes the proteome complex because of its diverse functions and the limited number of genetic modifications of proteins after protein biosynthesis. Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which can then form mature protein products through PTM. PTM is an important component in cell signaling, such as when prohormones are converted into hormones. Post-translational modification can occur at the C- or N-terminus of amino acid side chains or proteins. They can expand the chemical repertoire of the 20 standard amino acids by modifying existing functional groups or introducing new ones, for example, by phosphorylation, glycosylation, sulfation, hydroxylation, methylation, SUMOylation (i.e., the functional group is SUMO (small ubiquitin-related modifier and protein is a protein of 100 amino acid residues that binds to the target protein in the same way as ubiquitin), disulfide bond formation, lipidation, acetylation, prenylation, etc.

[0408] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds comprising 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 a protein or peptide sequence can contain. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. The term as used herein refers to both short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, and long chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

[0409] The term "polymer" as used herein refers to a polymer suitable for clinical and medical applications. The polymer is biocompatible, for example, suitable for exposure to body and body fluids. The polymer can be synthetic or natural, hydrophilic or amphiphilic. The polymer can be biocompatible, and therefore suitable for medical and clinical applications. Some non-limiting examples of polymer are polyethylene glycol polymers (PEG), which can be, but are not limited to, PEG200, PEG400, PEG550, PEG600, PEG800, PEG1000, PEG3350, PEG4000, PEG8000 or PEG10000.

[0410] As used herein, the term "antibody" is used in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen binding domains that specifically bind to an antigen or epitope. The 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 antibody (e.g., camelid V HH ), other antibody fragments or forms known to those skilled in the art), and antibody mimetics (e.g., adnectins, affibodies, affilins, anticalins, avimers, DARPins, knottins, etc.). Antibodies can be monospecific, bispecific, and multispecific.

[0411] The term "antigen binding domain" as used herein refers to a portion of an antibody or T cell receptor that is capable of specifically binding to an antigen or epitope through its variable domain. An example of an antigen binding domain is the variable domain V and V of an antibody heavy chain and light chain, respectively. H and V L The antigen binding domain formed by the interface of the antibody mimics. Another example of an antigen binding domain is an antigen binding domain formed by the diversification of certain loops of an antibody mimic. Another example of an antigen binding domain is a variable domain of a TCR, such as a TCR domain containing CDRs, such as αCDR1, αCDR2, αCDR3, βCDR1, βCDR2, and βCDR3.

[0412] As used herein, "variable domain" refers to a variable nucleotide sequence resulting from a recombination event, for example, it may include the V, J and / or D regions of a T cell receptor (TCR) sequence from a T cell (e.g., an activated T cell), or it may include the V, J and / or D regions of an antibody. The term "antigen binding fragment" refers to at least a portion of an antibody or TCR or a recombinant variant thereof, which comprises an antigen binding domain, i.e., a variable domain and a hypervariable loop, the so-called complementarity determining region (CDR), which is sufficient to confer recognition and specific binding to the antigen binding fragment for a target (e.g., an antigen and its defined epitope). Some examples of antigen binding fragments include, but are not limited to, Fab, Fab', F(ab') and 2 and Fv fragments, single-chain (sc) Fv ("scFv") antibody fragments, linear antibodies, single domain antibodies (abbreviated as "sdAb") (V L or V H ), Camelidae V HH Domains (nanobodies), multispecific antibodies produced by antibody fragments, and TCR fragments. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), the entire contents of which are incorporated herein by reference.

[0413] The term "scFv" refers to an antibody heavy chain variable fragment (V H ) fusion protein, the antibody heavy chain variable fragment at its C-terminus through a flexible peptide linker with the antibody light chain variable fragment (V L ) and can be expressed as a single polypeptide chain, wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0414] The term "antigen" or "Ag" as used herein refers to a molecule that can be specifically bound by an antibody, or a molecule that triggers an immune response when the antigen is processed by an antigen presenting cell (APC). This immune response may involve the production of antibodies, or the activation of specific immunocompetent cells, or both.

[0415] Those skilled in the art will appreciate that any macromolecule, including almost all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will appreciate that any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response thus encodes the term "antigen" as used herein. In addition, those skilled in the art will appreciate that antigens do not need to be encoded only by the full-length nucleotide sequence of a gene. Obviously, the present disclosure includes but is not limited to the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in a variety of combinations to encode polypeptides that elicit a desired immune response. In addition, those skilled in the art will appreciate that antigens do not need to be encoded by a "gene" at all. Obviously, antigens can be produced by chemical synthesis; they can also be derived from biological samples, or can be macromolecules other than polypeptides, such as lipids or carbohydrates. Such biological samples can include but are not limited to tissue samples, tumor samples, cells, or fluids with other biological components.

[0416] The term "biocompatible" as used herein refers to materials that are non-toxic and conform to, for example, the United States Pharmacopoeia (USP) and the European Pharmacopoeia (Eur.Ph), such as polymers, ingredients / excipients in compositions / formulations. In biotechnology, a biocompatible material is a material that has no negative impact on the metabolism of living cells, tissues or organs when in direct contact with them.

[0417] The term "protease" as used herein refers to an enzyme that catalyzes (increases the reaction rate or "speeds up") proteolysis, breaking down proteins into smaller polypeptides or individual amino acids. Some examples of proteases are subtilisin, proteinase K, pepsin, trypsin, chymotrypsin, elastase, neutral protease, and other similar proteases.

[0418] The term "combined mitochondrial reagent" used herein refers to an isolated mitochondria artificially combined with a therapeutic agent, a pharmaceutical agent, a diagnostic agent, an imaging agent or any other reagent. The reagent is combined with the mitochondria in any way, for example, connected to the mitochondria (for example, chemically or electrostatically connected), attached to the mitochondria, embedded in the mitochondrial membrane, substantially wrapped in the mitochondria, or completely encapsulated by the mitochondria, as long as the mitochondria and the reagent are in physical contact with each other. The combined mitochondrial reagent is designed so that the mitochondria act as such a "carrier": it can be transported to the patient's cells / tissues, and the reagent (for example, a cargo payload) can be released into the cells / tissues, for example, after skin / subcutaneous administration, aerosol administration or direct injection. The combined mitochondrial reagent can be prepared by a method comprising the following steps: isolating mitochondria from cells or tissues, and mixing mitochondria with an effective amount of therapeutic agents, diagnostic agents or imaging agents under conditions sufficient to allow the therapeutic agent, diagnostic agent or imaging agent to be connected to the mitochondria. In some embodiments, mitochondria are mixed with imaging agents.

[0419] As used herein, the term "transplantation of mitochondria" or "mitochondria transplantation" or "mitochondrion transplantation" or "mitochondrial transfer" is used throughout the specification as a general term to describe the process of transferring at least one mitochondria, such as isolated viable mitochondria, such as autologous, allogeneic or xenogeneic isolated viable mitochondria, to a recipient. The term includes all types of mitochondrial transplantation known in the art, including but not limited to direct (micro) injection of mitochondria into recipient cells or tissues, and vascular infusion, such as intravenous injection (i.e., iv injection), or intra-arterial injection or infusion into a subject's blood vessel, wherein the blood vessel is part of the subject's vascular system and carries blood to the target site.

[0420] The term "transplantation" used herein is used as a general term throughout the specification to describe the process of implanting an organ, tissue, a large number of cells, a single cell or an organelle into a recipient. The term "cell transplantation" is used throughout the specification to describe the process of transferring at least one cell, such as the enhanced immune cell described herein, to a recipient. The term includes all classes of transplants known in the art, including blood transfusions. Transplantation is classified according to the position and genetic relationship between the donor and the recipient. The term includes, for example, autologous transplantation (cells or tissues are removed from a part of the patient and transferred to the same or another part of the same object), allograft (transplantation between members of the same species) and xenograft (transplantation between members of different species).

[0421] The term "stem cell" as used herein 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, a daughter cell will also become a stem cell. Stem cells can be obtained from embryonic, postnatal, juvenile or adult tissues. Stem cells can be multifunctional or multipotent. The term "progenitor cell" used herein refers to an undifferentiated cell derived from a stem cell, and it is not a stem cell itself. Some progenitor cells can produce offspring that can differentiate into more than one cell type. Stem cells include pluripotent stem cells, which can form cells of any body tissue lineage (mesoderm, endoderm and ectoderm). Therefore, for example, stem cells can be selected from human embryonic stem (ES) cells; human inner cell mass (inner cell mass, ICM) / epiblast cells; human primitive ectoderm cells, human primitive endoderm cells; human primitive mesoderm cells; and human primitive germ (EG) cells. Stem cells also include pluripotent stem cells, which can form a multicellular lineage that constitutes an entire tissue or multiple tissues, such as, but not limited to, hematopoietic stem cells or neural precursor cells. Stem cells also include totipotent stem cells, which can form a complete organism. In some embodiments, stem cell is mesenchymal stem cell.Term " mesenchymal stem cell " or " MSC " are interchangeably used to refer to non-terminally differentiated adult cells, which can split and produce cells as stem cells, or irreversibly differentiate and produce cells (for example, fat, bone, cartilage, elastic and fibrous connective tissue, myoblast) of mesenchymal cell lineage and tissue (for example, neural cell) except those derived from embryonic mesoderm, this depends on the multiple impacts of bioactive factors such as cytokines.In some embodiments, stem cell is partially differentiated or differentiated cell.In some embodiments, stem cell is induced pluripotent stem cell (iPSC), which has been reprogrammed or dedifferentiated.Stem cell can be obtained from embryo, fetus or adult tissue.

[0422] The term "treatment" (and variations thereof) as used herein refers to a clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment may be performed in both prevention and during clinical pathology. The desired effects of treatment include preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis.

[0423] As used herein, "therapeutically effective amount" is the amount of a composition or its active ingredient, which is sufficient to provide a beneficial effect to an individual to whom the composition is applied or to reduce harmful non-beneficial events in other cases. "Therapeutically effective dose" herein means a dose that produces one or more desired or desirable (e.g., beneficial) effects of applying the composition, such application occurring once or more times within a given time period. The exact dosage will depend on the purpose of the treatment, and will be determined by those skilled 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)). In addition, the term "therapeutically effective amount" means any amount of mitochondria or compositions comprising mitochondria that results in treatment, cure, prevention or amelioration of a disease, disorder or side effect, or a reduction in the rate of progression of a disease or disorder, compared to a corresponding subject not receiving such an amount. The term also includes within its scope amounts effective to enhance normal physiological function.

[0424] As used herein, the term "therapeutic effect" refers to the effect of a therapeutic drug, substance, biological particle or composition (e.g., such as a composition comprising isolated viable mitochondria or isolated mitochondria) that is achieved by alleviating, inhibiting, alleviating or eradicating a disease state.

[0425] As used herein, the term "prevention" means prophylactic or protective treatment against a disease or disease state.

[0426] The term "subject" as used herein means a mammalian subject. Exemplary subjects include humans or mammals, such as monkeys, dogs, cats, mice, rats, cattle, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. The terms "subject in need," "subject in need thereof," "subject in need of treatment," or "patient" are intended to include living organisms that suffer from a disease, disorder, or condition or are at risk of developing a disease, disorder, or condition, or that otherwise require the compositions and methods provided herein, i.e., subjects.

[0427] As used herein, "prevention" refers to preventing a disease or condition, such as tumor formation, in a patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the invention, and does not subsequently develop a tumor or other form of cancer, the disease is prevented in the individual, at least for a period of time.

[0428] The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredients and / or maintains or enhances the viability of the biological entities (e.g., cells) contained therein to effectively treat a subject, and does not contain additional components that are unacceptably toxic to a subject in the amounts provided in the pharmaceutical composition.

[0429] The term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are compatible with drug administration. In some embodiments, the pharmaceutically acceptable carrier is phosphate buffered saline, physiological saline, Krebs buffer, Tyrode's solution, contrast media, or omnipaque, or a mixture thereof. The term "pharmaceutically acceptable carrier" also includes sterile mitochondrial buffer (300 mM sucrose; 10 mM K + -HEPES (potassium-buffered (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.2); 1 mM K + -EGTA (potassium-buffered ethylene glycol tetraacetic acid, pH 8.0)). The term also includes respiratory buffer (250 mM sucrose, 2 mM KH 2 PO 4 、10mM MgCl 2 , 20 mM K-15 HEPES buffer (pH 7.2) and 0.5 mM K + -EGTA (pH 8.0)). The term also includes T cell culture media, such as RPMI 1640 medium GlutaMAXTM supplement 500 ml (ThermoFisher, 61870010).

[0430] The term "ischemia-related disease" as used herein refers to a disease involving ischemia. Ischemia as used herein is a reduction in blood flow to an organ and / or tissue. The reduction in blood flow may be caused by any suitable mechanism, including partial or complete blockage (occlusion), narrowing (constriction) and / or leakage / rupture of one or more blood vessels supplying blood to an organ and / or tissue, etc.

[0431] The term "tumor" refers to the growth and proliferation of all tumor cells, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorders," "proliferative disorders," and "tumor" as used herein are not mutually exclusive. The terms "cell proliferative disorders" and "proliferative disorders" 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 aspects, the tumor is a hematological malignancy (hematological tumor).

[0432] The phrase "disease associated with [target] expression" includes, but is not limited to, diseases associated with [target] expression or conditions associated with cells expressing [target], including, for example, proliferative diseases such as cancer or malignancies or precancerous conditions. 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. Non-cancer related indications associated with [target] expression include, but are not limited to, for example, autoimmune diseases (e.g., lupus, rheumatoid arthritis, colitis), inflammatory diseases (allergies and asthma), and transplantation.

[0433] The term "endogenous" refers to any substance that originates or originates from within an organism, cell, tissue or system.

[0434] The term "exogenous" refers to any substance introduced or produced from outside an organism, cell, tissue or system. In the case of a patient, the term "exogenous" can refer to a substance of patient, donor or cell culture origin. For example, mitochondria isolated from a patient's muscle tissue and subsequently introduced into an immune cell population, which can be autologous or self-generated by the patient, are considered exogenous. The term "exogenous mitochondria" refers to any mitochondria isolated from autologous sources, allogeneic sources and / or xenogeneic sources, where the nature of the source can be tissue, blood or cultured cells.

[0435] In the context of the present invention, the following abbreviations for cryoprotectants are used. "CRYO" refers to a commercially available cryoprotectant identified by a code number. For example, CRYO33 represents cryoprotectant 70% w / v D-(+)-glucose monohydrate. Examples of commercially available cryoprotectants or cryoprotecting agents are provided in Table 1b of the specification.

[0436] As used herein, the term "parenteral" administration of a composition (e.g., a composition comprising mitochondria) refers to administration by various routes, such as, for example, by subcutaneous (sc), transdermal (with systemic effect), intradermal, intraocular, intravitreal, intranasal, transmucosal, intravenous (iv), intramuscular (im), perivascular, intraarticular, intraosseous, epidural, intrathecal, intratechal, intracerebral, intraventricular, extra-amniotic, intrauterine, intravaginal, intracavernous, intracardiac, intravesical, intraperitoneal, intrasternal, intratumoral, or intralesional (into skin lesions) injection / infusion.

[0437] As used herein, the term "topical" administration of a composition (eg, a composition comprising mitochondria) refers to administration of a composition or drug to a localized area of ​​the body or to a composition or drug that is applied to a surface and has a local effect.

[0438] The term "cancer" as used herein refers to various types of malignant tumors, most of which can invade surrounding cells, tissues or organs and can metastasize to different sites, as defined in Stedman's Medical Dictionary 25' edition (Hen syl ed. 1990). Some 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 cancer are mesothelioma, papillary serous ovarian cancer, clear cell ovarian cancer, mixed Mullerian ovarian carcinoma, endometrioid mucinous ovarian carcinoma, malignant pleural disease, pancreatic cancer, ductal pancreatic cancer, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct cancer, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, or breast adenocarcinoma.

[0439] Scope: Throughout the disclosure, aspects of the disclosure may be presented in the form of a range. It should be understood that the description in the form of a range is only for convenience and brevity, and should not be interpreted as an unchangeable limitation on the scope of the disclosure. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, a description such as a range from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6. As another example, a range such as 95 to 99% homogeneity includes things with 95%, 96%, 97%, 98% or 99% homogeneity, and includes, for example, sub-ranges of 96 to 99%, 96 to 98%, 96 to 97%, 97 to 99%, 97 to 98% and 98 to 99% homogeneity. This applies regardless of the breadth of the scope.

[0440] Example

[0441] The following are examples of methods and compositions of the present invention.It is understood that numerous other embodiments may be practiced, given the general description provided herein.

[0442] abbreviation

[0443] Table 1b

[0444]

[0445]

[0446] In Table 1b above, examples of commercially available cryoprotectants are provided.

[0447] The cryoprotectants described in Table 1b have been used to prepare compositions by diluting the solutions to 5%, 10% and 20% (v / v) in the following mitochondrial "trehalose-based" isolation buffer (1) (see Figure 3A and Figure 3B and CRYO1 to CRYO47 of Table 1b), 5%, 10%, 15%, 20%, 25% and 30% (v / v) (see Figure 3C For CRYO6, CRYO12, CRYO15, CRYO22, CRYO25 and CRYO33) and 10%, 20% and 30% (v / v) ( Figure 4A to D for CRYO6+CRYO12, CRYO6+CRYO25, CRYO6+CRYO33 and CRYO33+CRYO25).

[0448] 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 CRYO6, CRYO12, CRYO25 and / or CRYO33.

[0449] Separation buffer

[0450] (1) "Trehalose-based" aqueous buffer: 10 mM HEPES, 1 mM EGTA, 300 mM trehalose; or

[0451] (2) "Sucrose-based" aqueous buffer: 10 mM HEPES, 1 mM EGTA, 300 mM sucrose

[0452] Example 1a: Isolation of mitochondria from tissue samples or cultured cells

[0453] Experiments were performed to isolate mitochondria from tissue samples.

[0454] preparation

[0455] Prepare the following solutions to isolate intact, viable, respiring mitochondria. To successfully isolate mitochondria using the methods of the present invention, solutions and tissue samples should be kept on ice to maintain mitochondrial viability. Even when kept on ice, isolated mitochondria will show a decrease in functional activity over time (Olson et al., J Biol Chem 242:325-332, 1967). If possible, prepare solutions in advance:

[0456] - 1 M K-HEPES stock solution (pH adjusted to 7.2 with KOH).

[0457] - 0.5 M K-EGTA stock solution (pH adjusted to 8.0 with KOH).

[0458] -1M MgCl 2 Stock solution (if the ionic component should be in the composition, this solution is used).

[0459] -1×PBS (ThermoFisher, 10010031)

[0460] - Prepare a stock solution of Subtilisin A by weighing out 2 mg of Subtilisin A into a 1.5 mL microcentrifuge tube. Store at -20°C until use. Prepare to 2 mg / ml in separation buffer.

[0461] Using the above stock solution, prepare a separation buffer consisting of 300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA at pH 7.2 and store it at 4 °C.

[0462] Isolation of mitochondria from tissues

[0463] Two 6 mm biopsy fresh punches from skeletal muscle were transferred to 5 mL of isolation buffer in a gentleMACS C tube (Miltenyi Biotec, Somerville, MA) and isolated using the gentleMACS TM The sample was homogenized using a 1-minute homogenization program of a separator (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 × g for 4 minutes (as an optional step). The homogenate was then filtered through a pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge tube on ice. The filtrate was filtered again through a new pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge on ice. In a 50 mL conical centrifuge tube on ice, the filtrate was re-filtered through a new pre-wetted 10 μm mesh filter. The filtrate was filtered again 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 × g for 10 minutes at 4 ° C. Remove the supernatant and resuspend the pellet containing mitochondria and mix in 1 mL of isolation buffer.

[0464] Example 1b: Isolation of mitochondria from cultured cells

[0465] Experiments were performed to isolate mitochondria from cultured cells.

[0466] preparation

[0467] Prepare the following solutions to isolate intact, viable, respirable mitochondria. In order to successfully isolate mitochondria using the methods of the present invention, solutions and tissue samples should be kept on ice to maintain mitochondrial viability. Even when kept on ice, isolated mitochondria will show a decrease in functional activity over time (Olson et al., J Biol Chem 242:325-332, 1967). If possible, prepare the following solutions in advance:

[0468] - 1 M K-HEPES stock solution (pH adjusted to 7.2 with KOH).

[0469] - 0.5 M K-EGTA stock solution (pH adjusted to 8.0 with KOH).

[0470] -1×PBS (ThermoFisher, 10010031)

[0471] - Prepare a stock solution of Subtilisin A by weighing out 2 mg of Subtilisin A into a 1.5 mL microcentrifuge tube. Store at -20°C until use. Prepare to 2 mg / ml in separation buffer.

[0472] The above stock solution was used to prepare the separation buffer (pH 7.2): 300 mM sucrose, 10 mM K-HEPES and 1 mM K-EGTA. It was stored at 4°C.

[0473] -Trypsin (Catalog No. 0103; ScienCell Research Laboratories, Carlsbad, CA).

[0474] Using the above stock solution, prepare a separation buffer consisting of 300 mM sucrose, 10 mM K-HEPES and 1 mM K-EGTA at pH 7.2 and store it at 4°C.

[0475] Isolation of mitochondria from cultured cells

[0476] Mitochondria are also isolated from cultured cells, for example, from the human cardiac fibroblast (HCF) cell line (obtained from ScienCell Research Laboratories, Carlsbad, CA).

[0477] Culture of human cardiac fibroblast (HCF) cells

[0478] Human cardiac fibroblasts (HCF) were maintained in Fibroblast Medium-2 containing fetal bovine serum, Fibroblast Growth Supplement-2, and antibiotic (penicillin / streptomycin) solution according to the supplier's instructions (ScienCell). Cells were incubated at 37°C in 5% CO. 2 Cells were maintained as monolayers in a humidified atmosphere of 5% CO and passaged when 80% confluence was reached.

[0479] Preparation of human cardiac fibroblasts (HCF)

[0480] 80% confluent HCF cells from two bottles (T150) were washed once with PBS. The cells were then isolated with trypsin according to the supplier's instructions (ScienCell Research Laboratories, Carlsbad, CA). The reaction was terminated by adding trypsin neutralization solution according to the supplier's instructions (ScienCell Research Laboratories, Carlsbad, CA). The cells were collected in a 50 mL centrifuge tube and centrifuged at 1000 rpm (190 × g) for 5 minutes. The supernatant was discarded and washed three times in total with 1 × PBS.

[0481] Preparation of cultured cells other than HCF should be performed according to the manufacturer's instructions. Of note, cells used as a source of mitochondria can be adherent, semi-adherent, or in suspension.

[0482] Isolation of mitochondria from HCF cultured cells

[0483] HCF cells from each flask were transferred to 5 mL of separation buffer (ie, "sucrose-based" separation buffer) in a gentleMACS C tube (Miltenyi Biotec, Somerville, MA) and isolated using a gentleMACS TM The sample was homogenized by a 1-minute homogenization procedure of a separator (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 filtered again through a new pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge on ice. The filtrate was re-filtered again in a 50 mL conical centrifuge tube on ice through a new pre-wetted 10 μm mesh filter. Optionally, the filtrate was re-filtered again in a 50 mL conical centrifuge tube on ice through a new pre-wetted 5 μm mesh filter. The resulting filtrate was concentrated by centrifugation. After centrifugation, the filtrate was transferred to a 1.5 mL microcentrifuge tube and centrifuged at 9500 × g for 5 minutes at 4 ° C. Three washes were performed at the same centrifugal speed.

[0484] Quantification of isolated mitochondria

[0485] The isolated mitochondria were suspended in the isolation buffer of Example 1b and kept on ice until use. TM Fluorometer (ThermoFisher Scientific / Invitrogen) using Qubit TMThe amount of mitochondria in the preparations administered at different doses was measured using a protein assay kit according to the manufacturer's instructions. For protein concentration measurement, mitochondria were resuspended in PBS (ThermoFisher, 10010031). Mitochondrial doses were estimated based on protein content in μg.

[0486] Example 2: Viability of frozen-thawed mitochondria

[0487] Experiments were performed with isolated mitochondria to assess their functional status after freezing and thawing under different conditions.

[0488] 2.1 Mitochondrial Isolation

[0489] Mitochondria were isolated as in Example 1b with minor modifications as described below. 2 HCF cells were cultured in FM-2 medium (ScienCell) in T150 flasks (Sarstedt) at 4 °C. Cells at 90% confluence from two T150 flasks were collected by trypsinization, washed twice with Gibco PBS pH 7.4 (Fisher Scientific, Cat. No. 10010023), and stained using gentleMACS TM The cells were homogenized using a 1-minute homogenization program of a dissociator (Miltenyi Biotec) and incubated on ice for 10 minutes with 0.1 mg / ml subtilisin A (protease from Bacillus licheniformis, Sigma-Aldrich) in a dissociation buffer containing 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 passed through two 40 μm ( 40μm Cell Strainer, CorningCat.Nr.352340) and a 10μm filter ( The mitochondria were filtered through a 10 μm (Cell Strainer, PluriSelect Cat. No. 43-50010-03) and the mitochondria were pelleted at 9500 g for 5 minutes. An additional spin was performed at 500 g for 5 minutes at 4°C to remove unopened cells. 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 separation buffer.

[0490] 2.2 Freezing of isolated mitochondria

[0491] Isolated mitochondria were frozen under different conditions (ie, liquid nitrogen or dry ice) and then thawed for subsequent functional analysis.

[0492] Procedure: The isolated mitochondria in pellet form were resuspended in two different isolation buffers containing 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 to ice. Subsequently, the thawed isolated mitochondria were compared to unfrozen controls using membrane potential assays, ATP assays or by assessing cytochrome c release (Figure 1).

[0493] 2.3 Mitochondrial membrane potential measurement

[0494] In order to evaluate the mitochondrial membrane potential, the isolated mitochondria were resuspended in a separation buffer containing sucrose or a separation buffer containing trehalose at 1 mg / ml, and 15 μg of mitochondrial suspension was used for each sample. Unfrozen mitochondria and mitochondria frozen and thawed according to the procedure described in Section 2.2 were stored on ice before use, for example, for a period of 5 minutes to a maximum of 60 minutes, preferably for a period of a few minutes (e.g., a maximum of 15 minutes, such as a period of 5 minutes or 10 minutes). For Mitotracker Red CMXRos / Mitotracker Green FM staining, a labeling solution was prepared by adding 200nM MitoTracker Red CMXRos and 200nM MitoTracker Green FM (Thermo Fisher) in the separation buffer. The isolated mitochondria were then suspended in 1ml of the labeling solution, vortexed and incubated in a 37°C incubator for 15 minutes. Mitochondria were precipitated by centrifugation at 9500g for 5 minutes at 4°C, washed once with respective separation buffers, and resuspended in separation buffers at a concentration of 1mg / ml. Subsequently, 15 μl of mitochondrial suspension was mixed with 15 μl of 40% PEG 8000 and the mitochondria labeled with MitoTracker Red (579 / 599 nm) and MitoTracker Green (490 / 516 nm) were imaged using an automated microscope Keyence BZ-8000 by taking 3 images / sample at different positions. MitoTracker Red or MitoTracker Green signals were quantified using the Keyence BZ-800 analyzer software, calculating the integrated brightness of each image and averaging images taken from one sample.

[0495] Results: This experiment showed that mitochondria frozen in both sucrose-containing and trehalose-containing isolation buffers could not maintain the inner membrane potential ( Figure 1A , Figure 1B , Figure 1C and Figure 1D ).

[0496] 2.4 ATP determination

[0497] ATP measurements were performed using the ATPlite luminescent assay system (Catalog Number: 6016941, Perkin Elmer) according to the manufacturer's instructions. Each sample used 10 μg of mitochondria suspended in separation buffer (i.e., in a "sucrose-based" or "trehalose-based" buffer). Unfrozen mitochondria (control samples) were stored on ice. Mitochondria were frozen by placing them on dry ice, thawed in a 37°C water bath, precipitated at 9500 g for 5 minutes at 4°C, resuspended in 50 μl of separation buffer, and transferred to OptiPlate-96, i.e., white opaque 96-well microplates (Catalog Number: 6005290; Perkin Elmer). 25 μl of mammalian cell lysis solution (i.e., a solution as provided in the ATPlite luminescent assay, Perkin Elmer) was added, and the plate was shaken at 700 rpm for 5 minutes. Afterwards, 25 μl of lyophilized substrate solution (as provided in the ATPlite luminescence assay, Perkin Elmer) dissolved in ATPlite buffer was added, followed by further shaking at 700 rpm for 5 minutes. The plate was kept in the dark and the luminescence was measured 10 minutes after mixing. The values ​​were normalized to the control and the results are expressed as ATP content relative to the control.

[0498] Results: This experiment showed that mitochondria frozen in both sucrose-containing isolation buffer and trehalose-containing isolation buffer did not maintain their ATP content when compared to freshly isolated mitochondria ( Figure 1E and Figure 1F ).

[0499] 2.5 Cytochrome c release assay

[0500] The release of cytochrome c was assessed by separating mitochondrial-bound cytochrome c from released cytochrome c and analyzed by western blotting. Mitochondria isolated in sucrose-based buffer or trehalose-based buffer were frozen / thawed on a dry ice / 37°C water bath, incubated at 37°C for 15 minutes, and precipitated at 20,000 g for 20 minutes. The supernatant was separated from the precipitate and transferred to a clean test tube. Both the precipitate and supernatant samples were lysed in a final volume of 20 μl of 1× NuPage loading buffer (Thermo Fisher, Cat. Nr NP0008) and the NuPAGE system was used with Bolt TM 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 15 wells (Thermo Fisher, NW00125BOX) and NuPAGE TM The analysis was performed using MES SDS running buffer (20X) (Thermo Fisher, NP0002). Proteins were transferred to Power Blotter Pre-cut membranes and filters, PVDF, regular size (Thermo Fisher PB9320) using the Tetra system (BioRad Cat. No. 1703930), blocked with 5% skim milk dissolved in phosphate-buffered saline with 0.1% Tween 20 (Thermo Fisher Cat. No. 14190144), and stained with SuperSignal using anti-cytochrome c primary antibody (Proteintech Cat. No. 10993-1-AP) and secondary antibody (SA00001-2). TM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Cat.Nr.34577) and CL-XPosure TM Membrane, 5×7 inches (13×18 cm) (Thermo Fisher Cat. Nr. 34090) membrane detects cytochrome c.

[0501] Results: Mitochondria frozen and thawed in isolation buffer containing sucrose did not maintain outer membrane integrity, whereas mitochondria frozen and thawed in isolation buffer containing trehalose did maintain outer membrane integrity ( Figure 1G ).

[0502] Example 3: Effect of freezing and thawing rates on mitochondrial viability

[0503] Mitochondria were isolated as described in Example 2.1 and processed 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 assessed by MitoTracker staining, and ATP content was determined as described in Examples 2.3 and 2.4.

[0504] 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., by placing them on dry ice, in liquid nitrogen, or first on dry ice and then transferred to liquid nitrogen). Mitochondrial viability 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 thawing them on ice, and ATP content was determined using ATP assay.

[0505] Results: Freezing and thawing of mitochondria in trehalose-containing isolation buffer resulted in different degrees of functional and structural damage, depending on the freezing rate and thawing temperature ( Figure 3A to 3G).

[0506] Example 4: Viability of frozen-thawed mitochondria contained in a composition containing one or more cryoprotectants

[0507] 4.1 Integrity of the mitochondrial inner membrane of frozen-thawed mitochondria.

[0508] Mitochondria isolated according to Example 2.1 were resuspended in a "trehalose-based" isolation buffer. The effect of adding cryoprotectants on maintaining the integrity of the mitochondrial inner membrane was investigated using the MitoTracker staining assay, as described in Example 2.3. Mitochondria were frozen on dry ice after adding a single cryoprotectant selected from the list of cryoprotectants provided in Table 1b to the mitochondria, wherein the cryoprotectants had three different concentrations of 5% (v / v), 10% (v / v) and 20% (v / v). Mitochondria of the control samples were frozen on dry ice without any 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 unfrozen control, and the results are shown in the form of a heat map. Figure 3AMito Tracker Red / Green staining and MitoTracker Red / Green signal assays of four selected cryoprotectants (i.e., CRYO12, CRYO06, CRYO33, and CRYO25) are shown in Figure 3B middle.

[0509] Results: Mito Tracker Red staining assay showed that cryoprotectants CRYO12, CRYO06, CRYO33, and CRYO25 maintained the integrity of the mitochondrial inner membrane.

[0510] 4.2 ATP content of frozen-thawed mitochondria.

[0511] 4.2.1 Use of a single cryoprotectant. Different cryopreservation compositions with increasing concentrations of a single cryoprotectant selected from proline, sucrose, glucose, PEG400, PEG1000 and pentaerythritol propoxylate were prepared by adding CRYO6, CRYO12, CRYO15, CRYO22, CRYO25, CRYO33 to mitochondria resuspended in a trehalose-based isolation buffer. The obtained samples were then frozen on dry ice (about -78°C) and thawed in a 37°C water bath. The ATP content was determined relative to an unfrozen control (stored on ice at about 4°C).

[0512] Results: It was shown that the mitochondrial inner membrane potential and ATP content depended on the type and concentration of cryoprotectant ( Figure 3C ).

[0513] 4.2.2 Using two cryoprotectants. Different cryopreservation compositions were prepared, wherein each composition contained an increasing concentration of one of the two cryoprotectants. The cryoprotectant was selected from proline, sucrose, glucose or PEG400.

[0514] 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 specific concentrations of cryoprotectants, as described in Table 2.

[0515] Table 2

[0516]

[0517]

[0518] These compositions were then added to isolated mitochondria (10 μg per sample in triplicate as described in Example 2.4) that had been previously suspended in an isolation buffer containing trehalose as described in Example 2. The obtained samples were then frozen on dry ice (approximately -78°C) and thawed in a water bath at 37°C. The ATP content was determined relative to an unfrozen control (stored on ice (approximately 4°C)). The ATP assay was performed according to the procedure provided in Example 2.

[0519] Results: The mitochondrial inner membrane potential and ATP content were dependent on the type and concentration of cryoprotectant ( Figures 4A to 4D ).

[0520] Example 5: Effect of controlled freezing rate on mitochondrial viability

[0521] Mitochondria were isolated from HCF cells as described in Example 2 and resuspended in a trehalose-based separation 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). 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 by placing it in a -80°C freezer. These samples were later used for normalization of mitochondrial content between different mitochondrial isolates. The remaining mitochondria were diluted to a concentration of 0.166 μg / μl with a trehalose-based separation 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). Each 50 μl of this mitochondrial suspension is filled into 12 wells of a Greiner 96-well μFT flat-bottom microplate (i.e., 100ug mitochondrial suspension in the plate). The microplate is mounted to an ILK multiwellRACK heat exchanger and frozen in a SY-LAB IceCube 15M controlled rate freezer using a sample-specific temperature program. After precooling to -5°C in 17 minutes, ice nucleation is mechanically triggered and frozen 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). A sample-specific temperature program was developed by repeatedly adjusting the temperature envelope of the IceCube to the desired sample freezing rate, which was monitored at different positions of the microplate using a 20ωTJC2-NNIN-IM050U-800 0.50mm N-type thermocouple operated by a Delphin ExpertKey 200 measurement system. The frozen plates were stored at -80°C for one month and subsequently analyzed using ATP assays and MitoTracker staining as described in Example 2. The ATP content in samples frozen in Eppendorf tubes at -80°C was also determined to normalize for differences in mitochondrial mass caused by differences between mitochondrial isolations. For MitoTracker Red / MitoTracker Green staining, fresh mitochondrial isolation was performed and mitochondrial staining intensity was compared between fresh mitochondria and mitochondria frozen at a controlled rate and stored for one month.

[0522] Results: The results show that mitochondria frozen at a rate of 10K / min have higher ATP content and higher membrane potential than mitochondria frozen at slower freezing rates (2K / min and 6K / min). In addition, this experiment shows that compositions containing isolated viable mitochondria can be frozen using the method described in Example 5 and stored for at least one month.

[0523] Example 6: In vivo coronary blood flow experiment

[0524] Mitochondria were isolated as described in Example 2, resuspended in cryopreservation buffer (7 mM HEPES, pH 7.2, 0.7 mM EGTA, 210 mM trehalose, 1.2 M proline, and 353 mM D-glucose) at a concentration of 10 mg / ml, and frozen in 500 ug aliquots in 1.5 ml Eppendorf tubes by placing the tubes on dry ice and then storing them at -80° for up to one week. For each in vivo experiment, the desired number of aliquots (as indicated in the examples) were thawed in a 37°C water bath and immediately diluted in 6.5 ml of sucrose-based isolation buffer. Within 5 minutes of thawing, mitochondria were injected intracoronarily into the hearts of healthy anesthetized pigs (Sus scrofa). In some experiments (as indicated in the Examples), mitochondria were freshly isolated from HCF cells (as described in Example 2) or from animal skeletal muscle tissue (as described in Example 1a), stored on ice, and subsequently injected exactly as described above for frozen mitochondria.

[0525] Results: In preclinical trials in pigs, compositions comprising isolated mitochondria frozen under the conditions described in Example 6 had the same effect on coronary blood flow as freshly isolated mitochondria.

[0526] Example 7: Mitochondrial transfer improves the killing ability of CAR-T cells

[0527] CAR-T cells were implanted with mitochondria suspended in a solution containing 1.2 M proline, 353 mM glucose, 210 mM trehalose, 0.7 mM K-EGTA, 7 mM K-HEPES (pH 7.2) and previously subjected to freeze-thaw cycles.

[0528] In order to evaluate the effect of mitochondrial transplantation on the killing ability of CAR-T cells, a FACS-based killing assay was performed 24 hours after transplantation. CAR-T cells (CD19-41BB-CD3z, PMC746) were purchased from ProMab Biotechnologies (Richemond, CA 94806). In order to measure the percentage of target cells expressing early markers of apoptosis in the transition to apoptosis or apoptosis, the percentage of annexin V+, annexin V+ / PI+ or PI+ was evaluated respectively. Transplanted or untransplanted CAR-T cells were plated with an effector-to-target ratio of 5 to 1. CAR-T cells were co-incubated with target cells Daudi (ATCC CCL-213) (B lymphoblastoid cell line expressing CD19) for 4 hours. After co-incubation, cells were collected and stained with anti-human CD8 PerCP-Cy5.5 (Stemcell, 60022PS) and anti-human CD3 APC (Stemcell, 60011AZ) at 4 ° C for 20 minutes. After washing with FACS buffer, cells were stained with Annexin V FITC (Biolegend, 640914) and propidium iodide (PI, Biolegend, 640914) according to the supplier's instructions for 15 minutes at room temperature and acquired on FACSLyric (BD Biosciences).

[0529] program

[0530] (i) CAR-T cell culture was performed in the presence of 100U / ml recombinant human IL-2 (Peprotech, 200-02). CAR-T cells were cultured in RPMI 1640 medium supplemented with GlutaMAXTM supplement 500ml (ThermoFisher, 61870010): 1% L-glutamine (ThermoFisher, 25030024), 1% penicillin-streptomycin (10'000U / 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).

[0531] (ii) Mitochondrial Isolation: Mitochondria were isolated from human cardiac fibroblasts (HCF) according to the procedure described in Example 1b.

[0532] (iii) Quantification of isolated mitochondria: Mitochondrial dose was estimated based on protein content in μg according to the procedure described in Example 1b.

[0533] Mitochondrial transplantation enhanced the killing ability of CAR-T cells. When co-incubated with transplanted CAR-T cells, the percentage of target cells that turned to apoptosis and expressed early markers of apoptosis or expressed late markers of apoptosis increased.

[0534] Example 8: Therapeutic Mitochondrial Transplantation (TMT) - Preclinical Studies in Pigs

[0535] Cryopreserved mitochondria were tested in an animal model of cardiac ischemia-reperfusion injury. The experiment was conducted in an established porcine model of local ischemia-reperfusion injury. Mitochondria were isolated from human cardiac fibroblasts as described in Example 1b. The isolated mitochondria were 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 were added to the mitochondrial suspension, incubated for 2 minutes, and the test tubes were frozen on dry ice. After 10 minutes, the test tubes were transferred to a storage box at -80°C and stored frozen until use. For animal experiments, cardiac ischemia was induced in healthy female pigs (Susscrofa) by occluding the coronary artery (LAD) for 90 minutes. Cryopreserved mitochondria (stored at -80°C for no more than 2 weeks) were rapidly thawed by placing them in a metal block heated to 37°C, 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 coronary arteries of pigs within 15 minutes after reperfusion. Blood samples were collected on days 0, 3, 30, and 90, and infarct biomarkers (e.g., creatine kinase-myocardial band (CKMB) or troponin I) were measured in serum. Cardiac function was assessed 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 were sacrificed and histological samples were obtained from the heart and other organs for analysis. The therapeutic effect was evaluated by the reduction of serum infarct biomarkers, reduction of infarct size, and improvement of ejection fraction compared with the control group.

[0536] Results: Interim analysis at Day 3 and Day 28 showed the following:

[0537] Ejection fraction was measured using magnetic resonance imaging (MRI) in animals treated with the composition stored at -80°C for up to 2 weeks and thawed prior to use (n=2) and animals receiving an equal volume of vehicle. MRI was performed on days 3 and 28 after ischemia. Preliminary results showed that two of the three animals (67%) receiving vehicle had a decrease in ejection fraction between days 3 and 28, indicating impaired cardiac function, whereas in the two animals treated with mitochondria, ejection fraction increased, indicating improvement.

[0538] Example 9: Spray Freezing of Cryopreservation Compositions Comprising Isolated Mitochondria

[0539] Mitochondria were isolated from a HEPG2 cell line stably expressing mitochondrial-targeted green fluorescent protein (GFP) by the same method as in Example 1. The isolated mitochondria were resuspended in 350 μl of trehalose-based mitochondrial isolation buffer (300 mM trehalose, 1 mM EGTA, 10 mM HEPES, pH 7.2). 100 μl of 3.53 M D-glucose and 50 μl of 6 M L-proline were then added to the mitochondrial suspension. The composition formed after the addition of glucose and L-proline was then spray-frozen, i.e., the composition was directly sprayed into liquid nitrogen in the form of droplets using a spray device. The liquid nitrogen was then evaporated at room temperature, and the remaining frozen composition was collected in an Eppendorf tube that had been pre-cooled in dry ice. The frozen composition was thawed by placing the tube in a metal rack pre-heated to 37°C. The thawed composition was diluted to 1.5 ml with trehalose-based mitochondrial isolation buffer (300 mM trehalose, 1 mM EGTA, 10 mM HEPES, pH 7.2), pelleted by centrifugation at 9500 g for 5 minutes, and resuspended in 20 μl of trehalose-based mitochondrial isolation buffer.

[0540] After thawing, the suspension containing mitochondria was added to the wells of a 96-well plate with cultured human cardiac fibroblasts and incubated for 24 hours. After washing the cultured cells four times with warm cell culture medium, the internalization of frozen-thawed GFP-labeled mitochondria was observed using a fluorescence microscope. This experiment shows that the mitochondria contained in the composition (i.e., the cryopreservation composition containing trehalose, glucose, and proline) spray-frozen in liquid nitrogen retains the ability to be internalized by living cells.

[0541] Other Implementations

[0542] The disclosure set forth above may encompass a plurality of different inventions with independent uses. Although each of these inventions is disclosed in its preferred form, the specific embodiments disclosed and illustrated herein should not be considered to be restrictive, because many variations are possible. The subject matter of the present invention includes all new and non-obvious combinations and sub-combinations of the various elements, features, functions and / or characteristics disclosed herein. The attached claims specifically point out certain combinations and sub-combinations that are considered to be new 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 to this application, or a related application. Such claims, whether for different inventions or the same invention, whether wider, narrower, the same or different in scope than the original claims, are considered to be included within the subject matter of the invention of the present disclosure.

Claims

1. A composition comprising: (i) an aqueous buffer having a pH of 5.5 to 8.5, for example pH 7.2; (ii) trehalose at a concentration of at least 150 mM; (iii) a first cryoprotectant selected from one or more of (a) amino acids at a total concentration of at least 150 mM; and optionally (iv) a second cryoprotectant selected from one or more of (b) sugars, (c) polymers, or combinations thereof, wherein: (b) the total concentration of said sugars is at least 150 mM; and (c) The total concentration of the polymer is 2.5% (w / v) to 30% (w / v).

2. A composition comprising: (i) an aqueous buffer having a pH of 5.5 to 8.5, for example pH 7.2; (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) an amino acid, (c) a polymer, or a combination thereof, wherein: (a) the total concentration of the amino acids is at least 150 mM; and (c) The total concentration of the polymer is 2.5% (w / v) to 30% (w / v).

3. A composition comprising: (i) an aqueous buffer having a pH of 5.5 to 8.5, for example pH 7.2; (ii) trehalose: at a concentration of at least 150 mM; and (iii) Cryoprotectant: selected from one or more (c) polymers, wherein the total concentration of the polymers is 16% (w / v) to 30% (w / v).

4. The composition of any one of claims 1 to 3, wherein (i) the aqueous buffer comprises a buffering agent, wherein the buffering agent is selected from 2-[4-(2-hydroxyethyl)-piperazin-1-yl]-ethane-sulfonic acid (HEPES), piperazine-N,N'-bis(2-ethane-sulfonic 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 dimethylcacoate, tris-(hydroxymethyl)aminomethane hydrochloride) (Tris), 4-morpholinepropanesulfonic acid (MOPS), 1,3-bis-[tris-(hydroxymethyl)-methylamino]-propane (Bis-Tris propane), sodium acetate, or a combination thereof, preferably HEPES.

5. The composition of any one of claims 1 to 4, wherein the concentration of the buffer in the aqueous buffer is 0.5 mM to 50 mM, such as 2 mM to 35 mM, more particularly such as 5 mM to 30 mM, such as 10 mM.

6. The composition of any one of claims 1 to 5, further comprising a calcium chelator, wherein the calcium chelator: - selected from ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2,2',2",2"'-(ethane-1,2-diacid dinitrile)-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, preferably selected from EGTA or EDTA; and / or - The calcium chelator has a concentration of 0.1 mM to 10 mM, such as 0.2 mM to 5 mM, such as 1 mM.

7. The composition of any one of claims 1 to 6, further comprising an ionic component, wherein: - the concentration of the ionic component is from 0.1 mM to 100 mM, for example from 1 mM to 30 mM, more particularly from 5 mM to 15 mM; and / or - The ionic component is selected from the group consisting of Mg 2+ 、Na + , K + , Cl - 、HCO3 - a salt, acid or base, such as MgCl2.MgSO4, KCl, KH2PO4, NaHCO3, Na2HPO4, a formate anion, such as C2H2MgO4 (magnesium formate), a pyruvate anion, such as C3H3NaO3 (sodium pyruvate), an acetate anion, such as C2H3NaO2 (sodium acetate), a malate anion, an oxaloacetate anion, a glutamate anion, an α-ketoglutarate anion, a succinate anion, or a combination thereof.

8. The composition of any one of claims 1 to 7, 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), wherein the albumin is preferably bovine serum albumin (BSA), human serum albumin (HSA), or a combination thereof.

9. The composition of any one of claims 1 to 3, wherein (i) the buffer has: - pH 7.4 and containing 20 mM Tris, 2 mM EDTA and 10 mM MgCl2; - pH 7.25 and comprising 5 mM MOPS, 10 mM BAPTA and 5 mM sodium pyruvate; or - pH 7.2 and containing 10 mM HEPES and 1 mM EGTA.

10. The composition of any one of claims 1 to 9, wherein the composition comprises less than a cryopreservation amount of propylene glycol, ethylene glycol, glycerol, and dimethyl sulfoxide (DMSO), or does not comprise propylene glycol, ethylene glycol, glycerol, and dimethyl sulfoxide (DMSO).

11. The composition of any one of claims 1 to 10, wherein (ii) the concentration of trehalose does not exceed 1500 mM, such as does not exceed 1000 mM, in particular does not exceed 1300 mM, such as preferably does not exceed 450 mM, such as 300 mM.

12. The composition of any one of claims 1, 2 or 4 to 11, wherein: (a) the amino acid is selected from the group consisting of leucine, isoleucine (e.g., L-isoleucine), proline (e.g., L-proline), methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiproline, 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 acid (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.

13. The composition of claim 12, wherein: (a) 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.

14. The composition of claim 12, wherein: (a) The amino acid is selected from proline derivatives, such as methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof.

15. The composition of claim 12, wherein the amino acid of (a) is proline, such as L-proline.

16. The composition of any one of claims 1, 2 or 4 to 15, wherein the concentration of the amino acid (a) is at least 160 mM, such as at least 180 mM or at least 200 mM.

17. The composition of any one of claims 1, 2, or 4 to 16, wherein the sugar (b) is selected from: a monosaccharide, a disaccharide, or a trisaccharide, or a combination thereof.

18. The composition of any one of claims 1, 2, or 4 to 17, wherein the sugar (b) is selected from maltose, lactose, fructose, sucrose, glucose, dextran, melezitose, raffinose, nigerose, maltotriose, maltotrione, kestose, cellobiose, chitobiose, lactulose, or a combination thereof.

19. The composition of any one of claims 1, 2, or 4 to 18, wherein the sugar (b) is selected from sucrose, glucose, or a combination thereof.

20. The composition of claim 19, wherein the sugar (b) is a combination of glucose and sucrose.

21. The composition of claim 19, wherein the sugar (b) is glucose.

22. The composition of claim 19, wherein the sugar (b) is sucrose.

23. The composition of any one of claims 1, 2 or 4 to 22, wherein the concentration of the sugar (b) is at least 160 mM, such as at least 180 mM.

24. The composition of any one of claims 1 to 23, wherein (c) the polymer: - is a biocompatible polymer, a hydrophilic polymer, an amphiphilic polymer, or a combination thereof; and -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, polyvinyl pyrrolidone, or a combination thereof, preferably, wherein the polymer (c) is polyethylene glycol (PEG).

25. The composition of any one of claims 1, 2 or 4 to 24, wherein the concentration of (c) the polymer is 5% (w / v) to 25% (w / v), such as 10% (w / v) to 20% (w / v).

26. The composition of any one of claims 1, 4 to 11, or 15 to 25, wherein: (iii) the first cryoprotectant is (a) an amino acid, wherein the amino acid is proline, such as L-proline, 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 a combination thereof, wherein: (b) the sugar is glucose, sucrose, or a combination thereof, and the total concentration is at least 150 mM, such as at least 200 mM, more particularly at least 300 mM, such as at least 500 mM; and (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 particularly 10% (w / v) to 20% (w / v), such as 15% (w / v).

27. The composition of any one of claims 1, 4 to 11, 14, or 16 to 25, wherein: (iii) the first cryoprotectant is one or more (a) amino acids, wherein the amino acid is a proline derivative selected from the group consisting of methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof, 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 a combination thereof, wherein: (b) the sugar is glucose, sucrose, or a combination thereof, and the total concentration is at least 150 mM, such as at least 200 mM, more particularly at least 300 mM, such as at least 500 mM; and (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 particularly 10% (w / v) to 20% (w / v), such as 15% (w / v).

28. The composition of any one of claims 2 or 4 to 25, wherein: (iii) the first cryoprotectant is one or more (b) sugars, wherein the sugar is glucose, sucrose, or a combination thereof, in a total concentration of at least 160 mM, such as at least 200 mM, more particularly at least 300 mM, such as at least 500 mM; and (iv) the second cryoprotectant is selected from one or more of (a) amino acids, (c) polymers, or a combination thereof, wherein: (a) the amino acid is selected from the amino acids according to any one of claims 12 to 16, and the concentration is 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 particularly 10% (w / v) to 20% (w / v), such as 15% (w / v).

29. The composition of claim 28, wherein the amino acid in (a) is proline, such as L-proline.

30. The composition of any one of claims 3 to 11, wherein: (iii) the cryoprotectant is (c) a polymer, wherein the polymer is polyethylene glycol (PEG) and has a concentration of 16% (w / v) to 30% (w / v), such as 20% (w / v) to 30% (w / v), such as 25% (w / v).

31. The composition of any one of claims 26, 28 or 29, wherein: (a) The amino acid is proline, such as L-proline, at a concentration of at least 500 mM, such as at least 600 mM, in particular at least 800 mM.

32. The composition of any one of claims 26, 28 or 29, wherein: The amino acid is proline, such as L-proline, at a concentration of at least 1000 mM, such as 1200 mM.

33. The composition of any one of claims 26, 28 or 29, wherein: The amino acid is proline, such as L-proline, at a concentration of at least 1300 mM, such as at least 1500 mM.

34. The composition of any one of claims 26, 28 or 29, wherein: The amino acid is proline, such as L-proline, at a concentration of at least 1600 mM, such as at least 1800 mM, in particular at least 2000 mM.

35. The composition of any one of claims 26, 28, 29 or 31 to 34, wherein: The amino acid is proline, such as L-proline; and The sugar is glucose at a concentration of 200 mM to 1300 mM, such as 300 mM to 1200 mM, in particular 350 mM to 1100 mM.

36. The composition of any one of claims 26, 28, 29, or 31 to 34, wherein: The amino acid is proline, such as L-proline; and The sugar is sucrose at a concentration of 160 mM to 900 mM, such as 200 mM to 800 mM, in particular 250 mM to 650 mM.

37. The composition of any one of claims 26, 28, 29, or 31 to 34, wherein: The amino acid is proline, such as L-proline; and The polymer is polyethylene glycol (PEG) at a concentration of 5% (w / v) to 30% (w / v), 10% (w / v) to 20% (w / v), such as 15% (w / v).

38. The composition of any one of claims 1, 4 to 11, 15, 16 or 26, wherein the cryoprotectant consists of proline, such as L-proline, at a concentration according to any one of claims 31 to 34.

39. The composition of claim 38, wherein the concentration of proline, such as L-proline, is 600 mM or 1200 mM.

40. The composition of any one of claims 1, 4 to 11, 14, 16 or 27, wherein the cryoprotectant consists of one or more (a) amino acids selected from the group consisting of: Methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof.

41. The composition of any one of claims 2, 4 to 11, 19 or 28, 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 particularly at least 500 mM, and not more than 2000 mM, such as not more than 1700 mM.

42. The composition of any one of claims 2, 4 to 11, 19 or 28, 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 particularly at least 800 mM, and not more than 2000 mM, such as not more than 1650 mM.

43. The composition of any one of claims 2, 4 to 11, 19 or 28, 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, in particular at least 1350 mM, and not more than 2000 mM, such as not more than 1650 mM.

44. The composition of any one of claims 2, 4 to 11, 21 or 28, wherein the cryoprotectant consists of glucose at a concentration of: At least 300 mM, such as at least 500 mM, in particular at least 800 mM, but not more than 1200 mM, such as not more than 1000 mM.

45. The composition of any one of claims 2, 4 to 11, 22 or 28, wherein the cryoprotectant consists of sucrose at a concentration of: At least 200 mM, such as at least 250 mM, in particular at least 300 mM, but not more than 800 mM, such as not more than 650 mM.

46. ​​The composition of any one of claims 1, 4 to 11, 27 or 40, wherein the cryoprotectant consists of one or more (a) amino acids, wherein: The amino acid is a proline derivative selected from the following: methylproline, benzylproline, hydroxyproline, aminoproline, dehydroproline, aziridinecarboxylic acid, azetidinecarboxylic acid, pipecolic acid, oxaproline, thiaproline, or a combination thereof, and the concentration is at least 300mM, preferably at least 500mM, for example at least 600mM or at least 800mM.

47. The composition of any one of claims 1, 4 to 11, 27 or 40, wherein the cryoprotectant consists of one or more (a) amino acids, wherein: The amino acid is a proline derivative selected from the group consisting of 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, such as at least 1200 mM, in particular at least 1500 mM, such as 2000 mM.

48. The composition of any one of claims 1 to 47, further comprising isolated mitochondria, such as isolated viable mitochondria.

49. The composition of claim 48, wherein the isolated mitochondria are mammalian isolated mitochondria, such as mammalian isolated viable mitochondria.

50. The composition of claim 49, wherein the isolated mitochondria are human isolated mitochondria, such as human isolated viable mitochondria.

51. The composition of any one of claims 48 to 50, wherein the mitochondria have been isolated from a cell, tissue or organ.

52. The composition of claim 51, wherein the tissue is liver tissue, skeletal muscle, heart, brain, kidney, placenta, lung tissue, or adipose tissue.

53. The composition of claim 51, wherein the mitochondria have been isolated from cultured cells, such as cultured human cells.

54. The composition of any one of claims 51 or 53, wherein mitochondria have been 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.

55. The composition of any one of claims 48 to 53, wherein the concentration of the isolated mitochondria is at least 0.02 μg / μL, such as at least 0.05 μg / μL, in particular at least 0.1 μg / μL.

56. The composition of claim 55, wherein the concentration of the isolated mitochondria is at least 0.2 μg / μL, such as at least 0.5 μg / μL, in particular at least 0.75 μg / μL.

57. The composition of any one of claims 55 or 56, wherein the concentration of the isolated mitochondria is at least 1.0 μg / μL, such as at least 1.5 μg / μL, in particular at least 2 μg / μL, such as 2.5 μg / μL.

58. The composition of any one of claims 55 to 57, wherein the concentration of the isolated mitochondria is at least 5 μg / μL, such as at least 10 μg / μL, such as 20 μg / μL or 30 μg / μL.

59. The composition of any one of claims 48 to 58, wherein the concentration of the isolated mitochondria does not exceed 100 μg / μL, such as does not exceed 80 μg / μL, in particular does not exceed 50 μg / μL.

60. The composition of claim 59, wherein the concentration of the isolated mitochondria is no more than 25 μg / μL, such as no more than 15 μg / μL, in particular no more than 10 μg / μL, such as no more than 5 μg / μL.

61. The composition of any one of claims 48 to 60, wherein the isolated mitochondria are linked to a pharmaceutical agent, a diagnostic agent, an imaging agent, a therapeutic agent, or any other biocompatible substance.

62. The composition of any one of claims 48 to 60, wherein the mitochondria are linked to an antibody.

63. The composition of any one of claims 48 to 60, wherein the mitochondria are linked to an antigen binding fragment, wherein the antigen binding fragment is at least a portion of an antibody or TCR, or a recombinant variant thereof.

64. The composition of any one of claims 61 to 63, wherein the mitochondria is linked to the agent, antibody or antigen-binding fragment by a covalent bond.

65. The composition of any one of claims 61 to 63, wherein the mitochondria are linked to the agent, antibody or antigen via a non-covalent bond, such as an electrostatic bond.

66. The composition of any one of claims 61 to 63, wherein the agent, antibody or antigen is embedded in mitochondria, embedded in mitochondrial membranes, substantially enclosed within mitochondria or completely encapsulated by mitochondria.

67. The composition of any one of claims 61 to 65, wherein the agent, antibody or antigen is attached to the outer membrane of the mitochondria by a covalent bond or a non-covalent bond, such as an electrostatic bond.

68. The composition of any one of claims 48 to 67, wherein the isolated mitochondria are mitochondria modified by gene editing.

69. The composition of any one of claims 48 to 67, wherein the isolated mitochondria comprise exogenous mtDNA.

70. A method for cryopreservation of a composition comprising isolated mitochondria, such as isolated viable mitochondria, according to any one of the preceding claims, the method comprising the steps of: (a) freezing the composition at a temperature below 0°C; as well as (b) storing the frozen composition obtained according to step (a) at a temperature below 0°C, such as -4°C, preferably -20°C; and optionally comprising the steps of: (c) thawing the frozen composition at a temperature above 0°C.

71. The method of claim 70, wherein: (a) Freezing at a temperature below -20°C, such as below -50°C, preferably below 70°C.

72. The method of any one of claims 70 or 71, wherein: (a) Freezing at a temperature below -100°C, such as below -150°C, in particular below -180°C.

73. The method of any one of claims 70 to 72, wherein: (a) Freezing 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.

74. The method of any one of claims 70 to 73, wherein: (a) Freezing is rapid freezing.

75. The method of any one of claims 70 to 73, wherein: (a) Freezing is gradual freezing at a rate of at least 5°C / min, such as at least 10°C / min, such as 20°C / min or 30°C / min.

76. The method of any one of claims 70 to 75, wherein: (b) The duration of storage is at least 24 hours before thawing, such as 120 hours, preferably, wherein: (b) The duration of storage is at least 1 week, such as 4 weeks.

77. The method of any one of claims 70 to 75, wherein: (c) Thawing is performed at a temperature above 4°C and below 40°C, for example, at a temperature between 20°C and 38°C.

78. A composition for treating a disease in a subject in need thereof, e.g., by therapeutic mitochondrial transplantation (TMT), comprising a therapeutically effective amount of isolated mitochondria according to any one of the preceding claims, e.g., isolated viable mitochondria, or a composition prepared by the method of any one of claims 70 to 78.

79. The composition of claim 79 for use in treating a mitochondrial disease or a mitochondrial-related disease in a subject in need thereof.

80. The composition of claim 79 for use in treating cancer or a tumor in a subject in need thereof.

81. The composition of claim 79 for use in treating an autoimmune disease.

82. The composition of claim 79 for use in treating ischemia-related injury, such as lung, kidney, heart or brain ischemia-reperfusion injury.

83. A composition according to claim 79 for use in removing blockages in blood vessels, such as blood clots.

84. The composition according to claim 79, for use in gene therapy, in particular for use in gene therapy for the treatment of cancer, infectious diseases or autoimmune diseases.

85. A composition according to claim 79, which is used to transplant mitochondria, such as isolated viable mitochondria, into immune cells to produce mitochondria-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), γ-δ T cells.

86. The composition of claim 79 for use in treating a disease in a subject in need thereof, wherein the composition is to be administered to a subject in need thereof by topical or parenteral administration.

87. The composition of claim 79 for use in treating a disease in a subject in need thereof, wherein the composition is administered to a subject in need thereof in the form of an aerosol.

88. The composition of claim 79 for use in treating a disease in a subject in need thereof, wherein the composition is to be administered to a subject in need thereof by direct injection into a blood vessel, tissue or organ.

89. A composition according to any of the preceding claims for use in treating a disease in a subject in need thereof, wherein the composition is delivered in vitro to a target cell of a subject or donor in need thereof.

90. The composition of any of the preceding claims, wherein the mitochondria, e.g., viable mitochondria, contained in the composition are autologous, allogeneic, or xenogeneic.

91. The composition of any one of claims 79 to 91, wherein prior to use in treating a disease, the composition has been subjected to at least a freeze-thaw cycle, such as a freeze-thaw cycle according to the method of any one of claims 70 to 78.

92. Use of a composition as claimed in any one of the preceding claims for cryopreservation of viable mitochondria, such as isolated viable mitochondria.

93. Use of a composition prepared by the method of any one of claims 70 to 78 for cryopreservation of viable mitochondria, such as isolated viable mitochondria.

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