Isolation of mitochondria from cells in suspension

Abstract

Methods for producing mitochondrial extracts are provided, including providing primary human hematopoietic cells in suspension, expanding the primary human hematopoietic cells, and isolating mitochondria. Mitochondrial extracts, compositions comprising the mitochondrial extracts, and methods for using the extracts and compositions are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 447,675, filed February 23, 2023, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present invention is in the field of mitochondrial isolation and transplantation. [Background technology]

[0003] Mitochondria play a key role in the homeostasis of most body cells. A decline in mitochondrial function can lead to a variety of diseases. In recent years, it has been reported that transplantation of mitochondria into autologous or non-autologous cells, tissues, model systems, and patients is not only possible but also therapeutically effective. Research on mitochondrial extraction methods that best preserve mitochondrial function and structural integrity is limited. Currently, there is an unmet need for an effective and reproducible method for producing functional mitochondria in therapeutic doses to treat diseases and disorders associated with non-functional or dysfunctional mitochondria. Summary of the Invention

[0004] The present invention provides a method for producing a mitochondrial extract, which includes providing primary human hematopoietic cells in suspension, expanding the primary human hematopoietic cells, and isolating mitochondria. Also provided are mitochondrial extracts, compositions comprising the mitochondrial extracts, and methods of using the extracts and compositions.

[0005] According to a first aspect, there is provided a method of producing a mitochondrial extract, the method comprising: a. Providing primary human hematopoietic cells in suspension; b. Growing the primary human hematopoietic cells for a time sufficient to produce an expanded population of human hematopoietic cells comprising at least 10 times the number of primary human hematopoietic cells provided; and c. isolating mitochondria from the expanded population; thereby producing a mitochondrial extract.

[0006] According to some embodiments, the primary hematopoietic cells are obtained from a blood sample from a human subject.

[0007] According to some embodiments, the primary hematopoietic cells are isolated from peripheral blood mononuclear cells (PBMCs).

[0008] According to some embodiments, the primary human hematopoietic cells are selected from T cells, B cells, NK cells and hematopoietic stem cells (HSCs).

[0009] According to some embodiments, the primary human hematopoietic cells are primary human immune cells.

[0010] According to some embodiments, the primary human immune cells are selected from T cells, B cells and NK cells.

[0011] According to some embodiments, the primary human hematopoietic cells are an isolated population of T cells.

[0012] According to some embodiments, the time period is 7 to 14 days.

[0013] According to some embodiments, the expanded population of human hematopoietic cells comprises at least 20 times the number of primary human hematopoietic cells provided.

[0014] According to some embodiments, the expanded population of human hematopoietic cells comprises at least 50 times the number of primary human hematopoietic cells provided.

[0015] According to some embodiments, the isolated mitochondria contain at least 20 micrograms of protein for every 1 million primary human hematopoietic cells prepared.

[0016] According to some embodiments, the isolated mitochondria contain at least 50 micrograms of protein for every 1 million primary human hematopoietic cells prepared.

[0017] According to some embodiments, the isolated mitochondria contain at least 80 micrograms of protein for every 1 million primary human hematopoietic cells prepared.

[0018] According to some embodiments, the protein is determined by a Bradford assay.

[0019] According to some embodiments, the expanding comprises contacting the prepared primary human hematopoietic cells with at least one of an antigen-presenting cell (APC), a component of an APC, a component that mimics APC activity, a factor secreted by activated T cells, and any combination thereof.

[0020] According to some embodiments, the provided primary human hematopoietic cells are primary T cells, and expanding comprises contacting the primary T cells with at least one of an anti-CD3 antibody or antigen-binding fragment thereof, an anti-CD28 antibody or antigen-binding fragment thereof, an anti-CD2 antibody or antigen-binding fragment thereof, interleukin 2 (IL-2), and any combination thereof.

[0021] According to some embodiments, the expanding comprises contacting the primary T cells with an anti-CD3 antibody or antigen-binding fragment thereof, an anti-CD28 antibody or antigen-binding fragment thereof, an anti-CD2 antibody or antigen-binding fragment thereof, and IL-2.

[0022] According to some embodiments, the expanding comprises culturing a suspension of primary human hematopoietic cells in a gas-permeable container.

[0023] According to some embodiments, the culturing is in a bioreactor.

[0024] According to some embodiments, the gas permeable container is a gas permeable rapid expansion (G-REX) well.

[0025] According to some embodiments, the isolating comprises lysing the cells to create a cell lysate, wherein the lysing comprises at least one of adding a lysis buffer, needle shearing, homogenization with a Dounce homogenizer, and nitrogen cavitation.

[0026] According to some embodiments, the isolating comprises disrupting the membranes of the immune cells by nitrogen cavitation or dounce homogenization.

[0027] According to some embodiments, the isolating comprises disrupting the membrane of the immune cells by nitrogen cavitation.

[0028] According to some embodiments, the method includes contacting the lysate with an artificial support comprising an anti-TOM22 binding agent, and isolating the artificial support and any mitochondria bound thereto.

[0029] According to some embodiments, the method further comprises eluting the mitochondria from the artificial scaffold.

[0030] According to some embodiments, the method includes centrifuging the lysate at about 3000 g to remove cellular debris and produce a supernatant, and centrifuging the supernatant at about 12,000 g to produce a mitochondrial precipitate.

[0031] According to another aspect, there is provided a mitochondrial extract produced by the method of the invention.

[0032] According to another embodiment, a mitochondrial extract is provided that is at least 90% T cell mitochondria, at least 90% B cell mitochondria, at least 90% NK cell mitochondria, or at least 90% HSC mitochondria.

[0033] According to some embodiments, the mitochondrial extract is at least 95% T cell mitochondria.

[0034] According to another aspect, there is provided a pharmaceutical composition comprising the mitochondrial extract of the present invention.

[0035] According to some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0036] According to some embodiments, the pharmaceutical composition is formulated for administration to a subject.

[0037] According to some embodiments, the pharmaceutical composition is formulated for in vitro transfer into target cells.

[0038] According to another aspect, there is provided a recombinant cell comprising the mitochondrial extract of the present invention.

[0039] According to some embodiments, the recombinant cells are depleted of endogenous mitochondria.

[0040] According to some embodiments, the cells are non-hematopoietic cells.

[0041] According to another aspect, there is provided a pharmaceutical composition comprising a recombinant cell of the present invention and a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0042] According to another aspect, there is provided a method of treating a subject suffering from a mitochondrial disease, the method comprising administering to the subject a pharmaceutical composition of the invention, thereby treating the mitochondrial disease.

[0043] According to some embodiments, the mitochondrial disease is selected from diabetes, Parkinson's disease, cancer, Alzheimer's disease, genetic mitochondrial disorders, aging, and dilated cardiomyopathy.

[0044] According to some embodiments, the mitochondrial extract is autologous to the subject.

[0045] According to some embodiments, the mitochondrial extract is allogeneic to the subject.

[0046] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0047] [Figure 1] Figure 1: Line graph of T cell number and viability over two weeks of culture in G-REX plates. [Figure 2] Figure 2: Line graph of CS activity of mitochondria isolated from T cells on days 7, 10, and 13 of culture. [Figure 3] Figure 3: Oxygen consumption rate (OCR) of isolated mitochondria measured using a Seahorse Bioanalyzer with glutamate / malate as substrates. [Figure 4A] Figures 4A-4B: (4A) Bar graphs of mitochondrial membrane potential measurements of freshly isolated mitochondria, after 4 days at 4°C or -80°C. [Figure 4B]Figures 4A-4B: (4B) Line graph of oxygen consumption rate of freshly isolated mitochondria, and mitochondria after 4 days at 4°C or -80°C. [Figure 5-1] Figures 5A-5E: Photomicrographs showing H&E staining (left) and staining with Oil Red O (right, lipids are shown in red) for (5A) healthy control, group A; (5B) healthy control treated with mitochondria, group D; (5C) untreated and high-fat diet, group B; (5D) high-fat diet treated with mitochondria; and (5E) high-fat diet treated with half the mitochondria regimen, group C. [Figure 5-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0048] In some embodiments, the present invention provides a method for producing a mitochondrial extract, comprising providing primary human hematopoietic cells in suspension, expanding the primary human hematopoietic cells, and isolating mitochondria. The present invention further relates to mitochondrial extracts, compositions comprising the mitochondrial extracts, and methods of using the extracts and compositions.

[0049] According to one aspect, the present invention discloses a method for providing functional mitochondria from non-transformed primary hematopoietic cells (e.g., immune cells, T cells) in suspension. Advantageously, the amount of functional mitochondria derived from hematopoietic cells obtained from a single blood unit according to the method of the present invention is sufficient for at least one therapeutic dose, and also sufficient for multiple therapeutic doses. Multiple therapeutic doses may be provided for repeated administration to the same patient or several different patients. In some embodiments, the method produces a sufficient number of functional mitochondria to produce multiple doses of a mitochondrial therapeutic composition. In some embodiments, the composition is a composition of the present invention. In some embodiments, the multiple doses are at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 therapeutic doses. Each possibility represents a separate embodiment. In some embodiments, the multiple doses are generated from a single blood unit. In some embodiments, the single blood unit is a single blood sample. In some embodiments, the single blood sample is a single blood draw. In some embodiments, the generation is by the method of the present invention. In some embodiments, the multiple doses are at least two doses. In some embodiments, the multiple doses are at least five doses. In some embodiments, the multiple doses are at least 10 doses. In some embodiments, the multiple doses are at least 100 doses. Those skilled in the art will understand that because the cells themselves are not intended for use in cell therapy, their functionality, therapeutic potential, and / or cytotoxic activity in culture are not the limiting factor, but rather, only the quality of the mitochondria. Therefore, cells from a single blood unit can be expanded as long as the quality and / or function of the mitochondria is maintained. In this way, the method of the present invention has the enormous advantage of producing very large quantities of mitochondria.

[0050] The present invention is based, at least in part, on the remarkable yield of mitochondria produced from primary T cells in culture. The method of the present invention enabled the production of over 85 μg of isolated mitochondrial protein from a starting population of only 1 million primary T cells. Furthermore, optimization of the method resulted in as much as 220 μg of protein per million primary T cells. When the optimized method was performed in a bioreactor rather than just a tissue culture plate, yields of up to 1,000 μg and beyond were achieved. The use of hematopoietic cells in suspension for mitochondrial production enabled cell expansion not possible with primary cultures of adherent cells. Furthermore, these mitochondria were of high quality, fully functional, and suitable for therapeutic use in human subjects. The resulting composition was able to effectively treat mice with diabetes and fatty liver.

[0051] According to a first aspect, there is provided a method of producing a mitochondrial extract, the method comprising: a. Preparing cells in suspension; b. Growing the cells for a sufficient time to produce an expanded population of cells; and c. isolating mitochondria from the expanded population; thereby producing a mitochondrial extract.

[0052] In some embodiments, the method further comprises storing the produced mitochondrial extract. In some embodiments, the storage technique is selected from cryopreservation, thawing, freezing, vitrification, dry state storage, cryogenic storage, normothermic storage, encapsulation, entrapment in a matrix, and encapsulation and entrapment in a polymer. In some embodiments, the storing is freezing. In some embodiments, the storing is entrapment. In some embodiments, the storing is encapsulation. In some embodiments, the storing is drying. In some embodiments, the storing is lyophilization.

[0053] In some embodiments, the method further comprises providing the mitochondrial extract or the preserved mitochondrial extract to a subject in need thereof. In some embodiments, providing is administering.

[0054] In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is a culture method. In some embodiments, the method is a cell culture method. In some embodiments, the culture is in suspension. In some embodiments, the culture is in a tissue culture plate or well. In some embodiments, the culture is in a bioreactor.

[0055] In some embodiments, the mitochondrial extract comprises isolated mitochondria. In some embodiments, the mitochondrial extract is isolated mitochondria. In some embodiments, the isolated mitochondria are purified mitochondria. In some embodiments, the extract is at least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% mitochondria. Each possibility represents a separate embodiment of the present invention. In some embodiments, the extract is at least 90% mitochondria. In some embodiments, the extract comprises at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% purity. Each possibility represents a separate embodiment of the present invention. In some embodiments, the extract comprises at least 90% purity.

[0056] In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are primary cells. In some embodiments, the cells are obtained from a subject. In some embodiments, providing is obtaining. In some embodiments, obtaining is providing. In some embodiments, the cells are obtained directly from the subject. In some embodiments, the cells are differentiated from stem cells. In some embodiments, the stem cells are embryonic stem cells. In some embodiments, the embryonic stem cells are stem cells derived from umbilical cord blood. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the stem cells are hematopoietic stem cells (HSCs). In some embodiments, the method further comprises extracting primary cells from the subject. In some embodiments, the subject is human. In some embodiments, the cells are from a healthy subject. In some embodiments, the subject does not suffer from a mitochondrial disease. In some embodiments, the subject suffers from a condition that would benefit from increased mitochondrial function.

[0057] In some embodiments, the cells are primary cells. In some embodiments, the cells are not immortalized. In some embodiments, the cells are not cells of a cell line. In some embodiments, the cells are not transformed. In some embodiments, the cells are healthy cells. In some embodiments, the cells are not cancerous. The term "primary cells" is well known in the art and refers to cells taken directly from a living organism. In some embodiments, the cells are from a sample provided by a subject. In some embodiments, the method further comprises receiving a sample from the subject. In some embodiments, the method further comprises collecting a sample from the subject. In some embodiments, the sample is a sample comprising cells. In some embodiments, the sample comprises a bodily fluid. In some embodiments, the bodily fluid is selected from blood, serum, plasma, gastric fluid, intestinal fluid, saliva, bile, breast milk, urine, interstitial fluid, cerebrospinal fluid, and stool. In some embodiments, the fluid is blood. In some embodiments, the blood is peripheral blood. In some embodiments, the primary cells are obtained from a blood sample. In some embodiments, the method also comprises differentiating the obtained cells into hematopoietic cells in suspension. In some embodiments, the method also includes differentiating the iPSCs into hematopoietic cells.

[0058] In some embodiments, the cells are suspension cells. In some embodiments, the cells are non-adherent cells. In some embodiments, the cells are grown in suspension. In some embodiments, the cells are hematopoietic cells. In some embodiments, the hematopoietic cells are blood cells. In some embodiments, the hematopoietic cells are isolated from peripheral blood mononuclear cells (PBMCs). In some embodiments, the hematopoietic cells are differentiated from stem cells. In some embodiments, the hematopoietic cells are selected from T cells, B cells, natural killer (NK) cells, and hematopoietic stem cells (HSCs). In some embodiments, the cells are immune cells. In some embodiments, the hematopoietic cells are immune cells. In some embodiments, the immune cells are lymphoid cells. In some embodiments, the cells are lymphocytes. In some embodiments, the immune cells are selected from T cells, B cells, and NK cells. In some embodiments, the cells are T cells. In some embodiments, the cells are B cells. In some embodiments, the cells are NK cells. In some embodiments, the cells are HSCs. In some embodiments, the cells are human cells. In some embodiments, the cells are from a subject.

[0059] In some embodiments, the cells are a mixture of hematopoietic cells. In some embodiments, the cells are an isolated population of cells. In some embodiments, the cells are a homogeneous population of cells. In some embodiments, the cells are an enriched population of cells. In some embodiments, isolated is purified. In some embodiments, isolated is enriched. In some embodiments, enriched populations comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 99, or 100% homogeneous cell types. Each possibility represents a separate embodiment of the present invention. In some embodiments, purified populations comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 99, or 100% purity. Each possibility represents a separate embodiment of the present invention. In some embodiments, enriched or purified populations comprise at least 90% purity. In some embodiments, the cells are an isolated population of T cells. In some embodiments, the cells are an isolated population of B cells. In some embodiments, the cells are an isolated population of NK cells. In some embodiments, the cells are an isolated population of HSCs. In some embodiments, the cells are an enriched population of T cells. In some embodiments, the cells are an enriched population of B cells. In some embodiments, the cells are an enriched population of NK cells. In some embodiments, the cells are an enriched population of HSCs. In some embodiments, the cells are a mixture of hematopoietic cells comprising at least two, three, four, or five different types of cells. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cells are a mixture of B cells and additional hematopoietic cells. In some embodiments, the cells are a mixture of B cells and additional PBMCs. In some embodiments, the cells are a mixture of T cells and additional hematopoietic cells. In some embodiments, the cells are a mixture of T cells and additional PBMCs. In some embodiments, the cells are a mixture of NK cells and additional hematopoietic cells. In some embodiments, the cells are a mixture of NK cells and additional PBMCs.In some embodiments, the cells are a mixture of immune cells comprising at least 2, 3, 4, or 5 different types of immune cells, each possibility representing a separate embodiment of the present invention.

[0060] In some embodiments, the method comprises determining the cell type of the resulting population prior to expansion, hi some embodiments, the method comprises isolating a particular cell type from the resulting population prior to expansion.

[0061] In some embodiments, the cells are grown in solution. In some embodiments, the cells are grown in culture. In some embodiments, growing refers to culturing. In some embodiments, the cells are grown for at least a predetermined time. In some embodiments, the cells are grown for a time sufficient to generate an expanded population. In some embodiments, the time is at least 4, 5, 6, 7, 8, 9, or 10 days. Each possibility represents a separate embodiment of the present invention. In some embodiments, the time is at least 7 days. In some embodiments, the time is at most 10, 11, 12, 13, 14, 15, 16, 17, or 18 days. In some embodiments, the time is at most 10 days. In some embodiments, the time is at most 14 days. In some embodiments, the time is at most 13 days. In some embodiments, the time is between 7 and 14 days. In some embodiments, the time is between 7 and 13 days. In some embodiments, the time is between 7 and 10 days. In some embodiments, the time is between 10 and 14 days. In some embodiments, the time is between 10 and 13 days.

[0062] In some embodiments, the expanded population comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or 1000 times the number of provided cells. Each possibility represents a separate embodiment of the present invention. In some embodiments, the expanded population comprises at least 5 times the number of provided cells. In some embodiments, the expanded population comprises at least 10 times the number of provided cells. In some embodiments, the expanded population comprises at least 20 times the number of provided cells. In some embodiments, the expanded population comprises at least 30 times the number of provided cells. In some embodiments, the expanded population comprises at least 100 times the number of provided cells. In some embodiments, the expanded population comprises at least 200 times the number of provided cells. In some embodiments, the expanded population comprises at least 400 times the number of prepared cells. In some embodiments, the expanded population comprises at least 1000 times the number of prepared cells. In some embodiments, the time is sufficient to result in at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold expansion of the prepared cells. Each possibility represents a separate embodiment of the present invention. In some embodiments, the time is sufficient to result in at least a 2-fold expansion of the prepared cells. In some embodiments, the time is sufficient to result in at least a 3-fold expansion of the prepared cells. In some embodiments, the time is sufficient to result in at least a four-fold expansion of the prepared cells. In some embodiments, the time is sufficient to result in at least a five-fold expansion of the prepared cells. In some embodiments, the time is sufficient to result in at least a 100-fold expansion of the prepared cells. In some embodiments, the time is sufficient to result in at least a 200-fold expansion of the prepared cells.In some embodiments, the time is sufficient to result in at least a 300-fold expansion of the prepared cells. In some embodiments, the time is sufficient to result in at least a 400-fold expansion of the prepared cells. Methods for counting cells and determining cell number are well known in the art, and it is routine for one of ordinary skill in the art to count the number of cells initially plated (prepared cells) and the number of cells present after expansion (expanded population).

[0063] In some embodiments, the cells are cultured in a medium. In some embodiments, the medium is tissue culture medium. In some embodiments, the medium is a medium for suspension cells. In some embodiments, the medium is a medium for immune cells. In some embodiments, the medium is a chemically defined medium. In some embodiments, the medium is a medium for T cells. Media for suspension cells in general, and T cells in particular, are well known in the art, and any such medium can be used. Examples of commercially available media for use in the methods of the present invention include RMPI basal medium supplemented with 10% fetal bovine serum (FBS), CST™ OpTmizer™ T cell medium (ThermoFisher), TexMACS™ medium (Miltenyi Biotec), X-VIVO15 (Lonza), and 4Cell NutriT medium (Gibco). In some embodiments, the medium is 4Cell NutriT medium. In some embodiments, the medium is an animal component-free medium. In some embodiments, the medium is a chemically defined medium.

[0064] In some embodiments, the expanding comprises activating the cells. In some embodiments, the expanding comprises activating immune cells. Methods for activating immune cells are well known in the art, and any such known methods, reagents, or kits can be used for activation. In some embodiments, the expanding comprises contacting the prepared cells with an antigen-presenting cell (APC). In some embodiments, the expanding comprises contacting the prepared cells with a component of an antigen-presenting cell (APC). In some embodiments, the expanding comprises contacting the prepared cells with a component that mimics APC activity. In some embodiments, the component is an anti-CD2 antibody or an antigen-binding agent thereof. In some embodiments, the component is an anti-CD3 antibody or an antigen-binding agent thereof. In some embodiments, the component is an anti-CD28 antibody or an antigen-binding agent thereof. In some embodiments, the APC mimic is a bead or particle comprising a component of an APC. In some embodiments, the bead is an avidin bead. In some embodiments, the avidin is streptavidin. In some embodiments, the antibody or antigen-binding agent thereof is a biotinylated antibody or antigen-binding agent thereof. In some embodiments, the avidin beads have a biotinylated antibody or antigen-binding agent thereof conjugated to their surface, the antibody or antigen-binding agent being selected from anti-CD2, anti-CD3, anti-CD28, and combinations thereof. In some embodiments, the expanding comprises contacting the prepared cells with a factor secreted by activated T cells. In some embodiments, the factor is interleukin 2 (IL-2). In some embodiments, the expanding comprises contacting the prepared cells with IL-2. In some embodiments, IL-2 is present at a concentration of 10 to 600 IU / mL. In some embodiments, IL-2 is present at a concentration of about 200 IU / mL. In some embodiments, the factor is IL-7. In some embodiments, the factor is IL-15. In some embodiments, the factors are IL-7 and IL-15.In some embodiments, the expanding comprises contacting the prepared cells with IL-7. In some embodiments, the expanding comprises contacting the prepared cells with IL-15. In some embodiments, the expanding comprises contacting the prepared cells with IL-7 and IL-15. In some embodiments, the expanding comprises contacting the prepared cells with an anti-CD3 antibody or antigen-binding fragment thereof. In some embodiments, the antibody is an activating antibody. In some embodiments, the expanding comprises contacting the prepared cells with an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the expanding comprises contacting the prepared cells with an anti-CD2 antibody or antigen-binding fragment thereof. In some embodiments, the expanding comprises contacting the cells with a combination of an anti-CD3 antibody, an anti-CD2 antibody, an anti-CD28 antibody, and IL-2. In some embodiments, the expanding comprises contacting the cells with an anti-CD3 antibody, an anti-CD2 antibody, an anti-CD28 antibody, and IL-2. In some embodiments, the factors are replaced in culture every 1, 2, 3, 4, 5, 6, or 7 days. Each possibility represents a separate embodiment of the present invention. In some embodiments, factors are replaced in culture every three days.

[0065] In some embodiments, growing comprises culturing. In some embodiments, culturing is in suspension. In some embodiments, the method comprises culturing a suspension of cells in a gas-permeable container. In some embodiments, culturing is in a gas-permeable container. In some embodiments, culturing is in a culture bag. In some embodiments, culturing is in a gas-permeable bag. In some embodiments, the container is a well. In some embodiments, the container is a plate. In some embodiments, the container is a flask. In some embodiments, the container is sealed but gas-permeable. In some embodiments, at least one wall of the container is gas-permeable. In some embodiments, the gas-permeable container allows unlimited exchange of gas to the suspension medium. In some embodiments, the gas-permeable container allows unlimited oxygen to the cells. In some embodiments, the gas-permeable container is a gas-permeable rapid expansion (G-REX) container. In some embodiments, the G-REX container is a G-REX well. In some embodiments, the method comprises culturing a suspension of cells in a bioreactor. Examples of bioreactors include, but are not limited to, stirred tank bioreactors, wave mixer bioreactors, fixed bed bioreactors, microcarrier bioreactors, hollow fiber bioreactors, G-Rex bioreactors, and perfusion bioreactor systems.

[0066] In some embodiments, the expanded population is a mixture of hematopoietic cells. In some embodiments, the expanded population is a pure population. In some embodiments, the expanded population is an enriched population. In some embodiments, a pure population comprises at least 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 99, or 100% purity. Each possibility represents a separate embodiment of the present invention. In some embodiments, an enriched population comprises at least 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 99, or 100% homogeneity within the population. Each possibility represents a separate embodiment of the present invention. In some embodiments, a pure population comprises at least 90% purity. In some embodiments, an enriched population comprises at least 90% homogeneity. In some embodiments, purity and / or homogeneity relate to the cell types present in the population. In some embodiments, the expanded population is an essentially pure population of cells of a particular cell type. In some embodiments, the expanded population is an essentially homogeneous population of cells of a particular cell type. In some embodiments, the expanded population is an expanded population of T cells. In some embodiments, the expanded population is an expanded population of B cells. In some embodiments, the expanded population is an expanded population of NK cells. In some embodiments, the expanded population is an expanded population of HSCs. In some embodiments, the method further comprises measuring the purity of the population after expansion. In some embodiments, the method comprises isolating a particular cell type from the expanded population. In some embodiments, after measuring, only the pure population is used for mitochondria isolation.

[0067] In some embodiments, isolating refers to isolating a cell type. In some embodiments, isolating refers to isolating mitochondria. In some embodiments, isolating refers to purifying. In some embodiments, isolating refers to extracting. In some embodiments, isolating refers to disrupting a cell's membrane. In some embodiments, isolating refers to lysing the cells. In some embodiments, lysing the cells creates a lysate. In some embodiments, the lysate is a cell lysate. In some embodiments, disrupting a cell's membrane refers to lysing the cells. Methods for isolating mitochondria from cells are well known, and any method that removes intact, functional mitochondria from cells can be used. For example, a commercially available mitochondrial isolation kit is available from Miltenyi (130-094-532), although any commercially available kit may be used as part of the methods of the present invention. In some embodiments, lysing does not substantially lyse the mitochondria. In some embodiments, disrupting the membrane is disrupting the plasma membrane. In some embodiments, disrupting the plasma membrane does not substantially disrupt the mitochondrial membrane. In some embodiments, disrupting the membrane is by nitrogen cavitation. In some embodiments, disrupting the membranes is by homogenization. In some embodiments, homogenization uses a homogenizer. In some embodiments, the homogenizer is a Dounce homogenizer. In some embodiments, the homogenization is Dounce homogenization. In some embodiments, disrupting the membranes is by addition of a lysis buffer. In some embodiments, the lysis buffer includes a detergent. In some embodiments, the lysis buffer lyses by osmotic pressure. In some embodiments, disrupting the membranes is by needle shearing. In some embodiments, the needle is about a 30 gauge needle.

[0068] In some embodiments, the isolation comprises centrifuging the cells to remove the media. In some embodiments, the cells are washed. In some embodiments, the washing is in a wash buffer. In some embodiments, the wash buffer is PBS. In some embodiments, the centrifugation is at about 300 g. In some embodiments, the centrifugation is for a time sufficient to pellet the cells. In some embodiments, the sufficient time is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, or 20 minutes. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sufficient time is 5-15 minutes. In some embodiments, the sufficient time is 5-10 minutes. In some embodiments, the sufficient time is about 10 minutes. In some embodiments, the cells are suspended in a mitochondria isolation buffer prior to lysis / membrane disruption. Isolation buffer options include, but are not limited to, sugar, HEPES, Tris-HCl, EDTA, EGTA, magnesium chloride (MgCl), potassium chloride (KCl), phosphate, glutamic acid, malic acid, ATP, dithiothreitol (DTT), and reduced glutathione (GSH) buffers. In some embodiments, the isolation buffer is a sugar buffer. In some embodiments, the sugar is a monosaccharide. In some embodiments, the sugar is a disaccharide. In some embodiments, the sugar is a polysaccharide. In some embodiments, the isolation buffer is a sucrose buffer. In some embodiments, the isolation buffer comprises about 320 mM sucrose, about 5 mM Tris-HCl, and about 2 mM EGTA. In some embodiments, the isolation buffer is a mannitol buffer. In some embodiments, the isolation buffer is a trehalose buffer. In some embodiments, the isolation buffer comprises a neutral pH. In some embodiments, the isolation buffer comprises a very low basic pH. In some embodiments, the isolation buffer comprises a pH of about 7.4. In some embodiments, the isolation buffer comprises fatty acid-free bovine serum albumin (BSA). In some embodiments, the BSA concentration is about 0.5%. In some embodiments, isolating mitochondria comprises centrifuging the cell lysate.In some embodiments, the centrifuging is to remove cellular debris.

[0069] In some embodiments, the method comprises contacting the lysate with an anti-mitochondrial protein antibody or antigen-binding fragment thereof. In some embodiments, the contacting comprises incubating. In some embodiments, the incubating is for a time sufficient to allow the mitochondrial protein to bind to the antibody or antigen-binding fragment thereof. In some embodiments, the mitochondrial protein is TOM22. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a solid support. In some embodiments, the solid support is an artificial solid support. In some embodiments, the antibody or antigen-binding fragment thereof is immobilized to a solid support. In some embodiments, the solid support is a synthetic solid support. In some embodiments, the solid support is a non-natural solid support. In some embodiments, the solid support is an artificial solid support. In some embodiments, the solid support is a column. In some embodiments, the solid support is a bead. In some embodiments, the bead is a magnetic bead. In some embodiments, the bead is a paramagnetic bead. In some embodiments, the bead is configured for isolation. In some embodiments, the bead is isolatable in a column. In some embodiments, the bead is an avidin bead. In some embodiments, the avidin is streptavidin. In some embodiments, isolating comprises isolating the solid support and any mitochondria bound thereto. In some embodiments, isolating further comprises eluting the mitochondria from the solid support. In some embodiments, eluting uses an elution buffer. In some embodiments, the elution buffer is a salt buffer. In some embodiments, the salt buffer is a high-salt buffer. In some embodiments, the salt concentration is high enough to elute the mitochondria from the antibody or antigen-binding fragment thereof.

[0070] In some embodiments, the isolating comprises centrifuging the lysate. In some embodiments, the centrifugation is at a speed sufficient to precipitate cellular debris from the lysate. In some embodiments, the centrifugation is for a time sufficient to precipitate cellular debris from the lysate. In some embodiments, a sufficient speed is about 3000 g. In some embodiments, the sufficient time is about 5 minutes. In some embodiments, the centrifugation to remove cellular debris is repeated. In some embodiments, the centrifugation produces a supernatant. In some embodiments, the supernatant comprises mitochondria. In some embodiments, the lysate is substantially depleted of cellular debris. In some embodiments, the lysate is substantially depleted of organelles other than mitochondria. In some embodiments, the isolating further comprises centrifuging the supernatant. In some embodiments, the centrifugation is at a speed sufficient to precipitate mitochondria. In some embodiments, the centrifugation is for a time sufficient to precipitate mitochondria. In some embodiments, a sufficient speed is about 12,000 g. In some embodiments, the sufficient time is about 10 minutes. In some embodiments, precipitating mitochondria comprises creating a mitochondrial precipitate. In some embodiments, precipitating mitochondria comprises creating a mitochondrial pellet. In some embodiments, the precipitate is a pellet. In some embodiments, the isolating further comprises resuspending the mitochondrial pellet in an isolation buffer.

[0071] In some embodiments, the isolated mitochondria are mitochondrial extracts. In some embodiments, the isolated mitochondria are intact mitochondria. In some embodiments, the isolated mitochondria are functional mitochondria. In some embodiments, at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100% of the mitochondria are functional. Each possibility represents a separate embodiment of the present invention. In some embodiments, the function comprises ATP production. In some embodiments, the function comprises oxygen consumption. In some embodiments, the function comprises membrane potential. In some embodiments, the function comprises citrate synthase. Methods for testing mitochondrial functionality, including testing the functions listed herein above, are well known in the art and are also described below. Thus, one skilled in the art is well able to determine the functional capacity of extracted and / or isolated mitochondria. In some embodiments, the method further comprises testing the quality of the mitochondrial extract. In some embodiments, a cell lysate is tested. In some embodiments, isolated mitochondria are tested. In some embodiments, intact cells are tested. In some embodiments, the quality of the mitochondrial extract is determined by testing. In some embodiments, the testing comprises a citrate synthase assay. In some embodiments, the testing comprises a JC-1 assay. In some embodiments, the testing comprises determining the copy number of mitochondrial DNA (mtDNA). In some embodiments, the quantification of mitochondria in the extract is by the copy number of mtDNA. In some embodiments, the quantification of mitochondria in the extract is by the total amount of protein present after mitochondrial isolation / extraction.

[0072] In some embodiments, the isolated mitochondria contain at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 220, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 micrograms (ug) of protein per million cells prepared. Each possibility represents a separate embodiment of the present invention. In some embodiments, the isolated mitochondria contain at least 20 μg of protein per million cells prepared. In some embodiments, the isolated mitochondria contain at least 50 μg of protein per million cells prepared. In some embodiments, the isolated mitochondria contain at least 80 μg of protein per million cells prepared. In some embodiments, the isolated mitochondria contain at least 100 μg of protein per million cells prepared. In some embodiments, the isolated mitochondria contain at least 110 μg of protein per million cells prepared. In some embodiments, the isolated mitochondria contain at least 200 μg of protein per million cells prepared. In some embodiments, the isolated mitochondria contain at least 220 μg of protein per million cells prepared. It will be appreciated that the amount of protein present in an extract is proportional to the total number of mitochondria present. Thus, the greater the amount of protein, the greater the number of mitochondria. In some embodiments, the isolated mitochondria are at a concentration proportional to a protein concentration of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50, 75, 80, 90, 100, 150, 200, 220, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 ug / ml per million cells prepared.Each possibility represents a separate embodiment of the present invention. In some embodiments, the isolated mitochondria are at a concentration of at least 8 μg / ml. In some embodiments, the isolated mitochondria are at a concentration of at least 16 μg / ml.

[0073] The method for determining protein concentration and total protein amount is well known in the art, and any such method can be used.Examples of such methods include, but are not limited to, BCA assay, UV-Vis absorbance assay, Western blotting, immunostaining, Bradford assay and protein array.In some embodiments, the amount of existing protein is determined by Bradford assay.

[0074] According to another aspect, there is provided a mitochondrial extract produced by the method of the invention.

[0075] According to another aspect, a mitochondrial extract from a population of hematopoietic cells is provided.

[0076] According to another aspect, a mitochondrial extract from a pure population of hematopoietic cells is provided.

[0077] According to another aspect, a mitochondrial extract is provided that is pure mitochondria from a single cell type of hematopoietic cell.

[0078] In some embodiments, the mitochondrial extract is in suspension. In some embodiments, the mitochondrial extract is from cells in suspension. In some embodiments, the population of hematopoietic cells is in suspension. In some embodiments, the population is a pure population. In some embodiments, the population is an enriched population. In some embodiments, the population is a mixed population.

[0079] In some embodiments, the mitochondrial extract is a mitochondrial composition. In some embodiments, the extract comprises mitochondria. In some embodiments, the extract comprises isolated mitochondria. In some embodiments, the extract comprises purified mitochondria. In some embodiments, the extract consists essentially of mitochondria. In some embodiments, the extract comprises human mitochondria. In some embodiments, the extract consists of human mitochondria. In some embodiments, the extract comprises mitochondria from a subject. In some embodiments, the extract is mitochondria of hematopoietic cells. In some embodiments, the extract is at least 30, 40, 50, 60, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100% of the mitochondria of hematopoietic cells. Each possibility represents a separate embodiment of the present invention. In some embodiments, the extract is mitochondria of immune cells. In some embodiments, the extract is at least 30, 40, 50, 60, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100% of the mitochondria of immune cells. Each possibility represents a separate embodiment of the present invention. In some embodiments, the extract is pure T cell mitochondria. In some embodiments, the extract is at least 30, 40, 50, 60, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100% T cell mitochondria. Each possibility represents a separate embodiment of the present invention. In some embodiments, the extract is at least 90% T cell mitochondria. In some embodiments, the extract is at least 95% T cell mitochondria. In some embodiments, the extract is at least 97% T cell mitochondria. In some embodiments, the extract is at least 99% T cell mitochondria. In some embodiments, the extract is pure B cell mitochondria. In some embodiments, the extract is at least 30, 40, 50, 60, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100% B cell mitochondria. Each possibility represents a separate embodiment of the present invention.In some embodiments, the extract is at least 90% B cell mitochondria. In some embodiments, the extract is at least 95% B cell mitochondria. In some embodiments, the extract is pure NK cell mitochondria. In some embodiments, the extract is at least 30, 40, 50, 60, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100% NK cell mitochondria. Each possibility represents a separate embodiment of the present invention. In some embodiments, the extract is at least 90% NK cell mitochondria. In some embodiments, the extract is at least 95% NK cell mitochondria. In some embodiments, the extract is pure HSC mitochondria. In some embodiments, the extract is at least 30, 40, 50, 60, 70, 75, 80, 85, 90, 92, 95, 97, 99, or 100% HSC mitochondria. Each possibility represents a separate embodiment of the present invention. In some embodiments, the extract is at least 90% HSC mitochondria. In some embodiments, the extract is at least 95% mitochondria of HSCs.

[0080] In some embodiments, the mitochondrial extract comprises a protein concentration of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, or 1000 μg / ml. Each possibility represents a separate embodiment of the present invention. In some embodiments, the concentration is per million cells prepared. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 8 μg / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 16 μg / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 100 μg / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 110 μg / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 200 μg / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 220 μg / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, or 1000 μg / ml. Each possibility represents a separate embodiment of the present invention. In some embodiments, the mitochondrial extract comprises a protein concentration of about 16 μg / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of about 110 μg / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of about 220 μg / ml.

[0081] According to another aspect, there is provided a composition comprising the mitochondrial extract of the present invention.

[0082] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a therapeutic composition. In some embodiments, the composition consists essentially of the mitochondrial extract. In some embodiments, the composition lacks active ingredients or agents other than the mitochondrial extract. In some embodiments, the composition consists of the mitochondrial extract. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or adjuvant. In some embodiments, the composition consists of the mitochondrial extract and a pharmaceutically acceptable carrier, excipient, or adjuvant. In some embodiments, the composition comprises a therapeutically effective amount of the mitochondrial extract. In some embodiments, the effective amount is effective in treating a mitochondrial-associated disease. In some embodiments, the composition is for use in treating a subject. In some embodiments, the composition is for use in treating a condition that would benefit from increased mitochondrial function. In some embodiments, the composition is for use in treating a mitochondrial-associated disease. In some embodiments, the composition is for use in treating a disease or condition in a subject in need of treatment. In some embodiments, treating a mitochondrial-associated disease comprises producing a medicament for use in treating a mitochondrial-associated disease.

[0083] As used herein, the term "carrier," "excipient," or "adjuvant" refers to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation auxiliary, or simply a sterile aqueous medium, such as physiological saline. Some examples of materials which can function as pharmaceutically acceptable carriers are sugars such as mannose, lactose, glucose and sucrose, trehalose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, and other non-toxic, compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can function as carriers herein include sugar, starch, cellulose and its derivatives, enriched tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oil, polyol, pyrogen-free water, isotonic saline, phosphate buffer solution, cocoa butter (suppository base), emulsifier, and other non-toxic pharmaceutically compatible materials used in other pharmaceutical preparations.Wetting agents and lubricants such as sodium lauryl sulfate, as well as colorants, flavorings, excipients, stabilizers, antioxidants, and preservatives may also be present.Any non-toxic, inert, and effective carrier can be used to formulate the compositions contemplated herein.In this regard, suitable pharmaceutically acceptable carriers, excipients and diluents are well known to those skilled in the art, and are, for example, those listed in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the "Inactive Ingredient Guide," US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management (all of the contents of which are incorporated herein by reference in their entirety).The examples of pharmaceutically acceptable carriers, carriers and diluents useful in the compositions of the present invention include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution and DMSO. These additional inactive ingredients, as well as effective formulation and administration procedures, are well known in the art and are described in standard textbooks, e.g., Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated herein by reference in its entirety.The compositions described herein may also be contained in artificially created structures such as liposomes, ISCOMS, sustained-release particles, and other vehicles that extend the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the peptides described herein are generally formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and a sterol, such as cholesterol. The choice of lipid is generally determined by considerations such as liposome size and stability in the blood. Various methods for preparing liposomes are available, as reviewed, for example, in Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York; see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0084] The mitochondria of the present invention may be encapsulated or entrapped in a biocompatible natural polymer, such as a gel or hydrogel, e.g., hyaluronic acid, chitosan, heparin, alginate, fibrin, collagen, chondroitin sulfate, or silk. The hydrogel can be further formulated with an agent for reverse thermal gelation, such as Pluronic or methylcellulose. Synthetic polymers, such as poly(ethylene glycol) [PEG], poly(vinyl alcohol) [PVA], poly(N-isopropylacrylamide) [PNIPAAm], and polycaprolactone [PCL] or PCLA-PEG-PCLA, can also be used to encapsulate or formulate mitochondria for injection into tissues. In another embodiment, the polymer or gel may be a combination of natural and synthetic polymers, and in another embodiment, the gel or polymer may be crosslinked or chemically modified to include additional functional groups, such as thiols. In some embodiments, the mitochondria are encapsulated or entrapped in a biocompatible polymer. In some embodiments, the polymer is a natural polymer. In some embodiments, the polymer is an artificial polymer.

[0085] In another embodiment, the mitochondria of the present invention are encapsulated in a particle. In some embodiments, encapsulated in a particle means encapsulated in a particle. In some embodiments, the particle is a lipid particle. In some embodiments, the lipid particle is a liposome. In some embodiments, the lipid particle is a micelle. Any particle delivery method can be used to formulate mitochondria for delivery.

[0086] In another embodiment, the mitochondria of the present invention may be frozen or lyophilized.

[0087] Carriers may comprise, in total, from about 0.1% to about 99.99999% (by weight) of the pharmaceutical compositions presented herein.

[0088] In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is a subject in need thereof. In some embodiments, the subject is a subject suffering from a mitochondrial-associated disease. In some embodiments, the subject is a subject suffering from a disease treatable by a mitochondrial extract. In some embodiments, the composition is formulated for systemic administration. In some embodiments, the composition is formulated for systemic administration to a subject. In some embodiments, the composition is formulated for topical administration.

[0089] As used herein, the terms "administer," "administration," and the like refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in sound medical practice. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of the subject composition to a patient in need thereof. Other suitable routes of administration may include intravascular, intrathecal, intracranial, parenteral, subcutaneous, oral, topical, inhalation, intramuscular, intraocular, or intraperitoneal.

[0090] The dosage administered will depend upon the age, health, and weight of the recipient, type of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0091] In some embodiments, the composition is formulated for in vitro administration to a cell. In some embodiments, in vitro administration is in vitro transfer to a cell. In some embodiments, to is into. In some embodiments, into a cell is into the cytoplasm of a cell.

[0092] According to another aspect, there is provided a cell comprising the mitochondrial extract of the invention.

[0093] In some embodiments, the cells are recombinant cells. In some embodiments, the cells are not genetically modified. In some embodiments, the cells are of the same species as the mitochondria. In some embodiments, the cells are of the same cell type from which the mitochondrial extract was made. In some embodiments, the cells are of a different cell type from which the mitochondrial extract was made. In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are not hematopoietic cells. In some embodiments, the cells are for adoptive cell transfer (ACT). In some embodiments, the cells are cells from a subject. In some embodiments, the cells are allogeneic to the subject. In some embodiments, the cells are syngeneic to the subject. In some embodiments, the cells are autologous to the subject.

[0094] In some embodiments, the cells are depleted of endogenous mitochondria. In some embodiments, the cells are substantially depleted of endogenous mitochondria. In some embodiments, the cells lack endogenous mitochondria. In some embodiments, the substantially depleted comprises less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1% of the endogenous mitochondria that were present in the cell. Each possibility represents a separate embodiment of the present invention. In some embodiments, the substantially depleted comprises less than 20% of the endogenous mitochondria that were present in the cell.

[0095] According to another aspect, there is provided a composition comprising the cells of the invention.

[0096] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or adjuvant. In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the composition is formulated for administration to an organ containing cells of the same cell type as the cells of the present invention. In some embodiments, the composition is for use in ACT.

[0097] According to another aspect, there is provided a method of treating a subject in need thereof, the method comprising administering to the subject a composition of the present invention, thereby treating the subject.

[0098] In some embodiments, the subject is suffering from a disease or condition. In some embodiments, the disease or condition is an age-related disease or condition. In some embodiments, the disease or condition is a degenerative disease or condition. In some embodiments, the degenerative disease or condition is a neurodegenerative disease or condition. In some embodiments, the disease or condition is a mitochondrial-associated disease or condition. In some embodiments, the mitochondrial-associated disease or condition is a mitochondrial disease or condition. In some embodiments, the disease or condition is treatable with the compositions of the present invention. In some embodiments, the disease is a metabolic disease or condition. In some embodiments, the disease is an energy homeostasis disease or condition. In some embodiments, the disease is a mitochondrial disease. In some embodiments, the disease is a cardiac disease or condition. In some embodiments, the disease is a cognitive disease. In some embodiments, the disease is a neurodegenerative disease or condition. In some embodiments, the disease is a developmental disease or condition. In some embodiments, the disease is a genetic disease or condition. In some embodiments, the disease is a cardiovascular disease or condition. In some embodiments, the disease is a neuro-ophthalmological disease or condition. In some embodiments, the disease is an ophthalmological disease or condition. In some embodiments, the disease is an ophthalmic disease or condition. Non-limiting examples of ophthalmic diseases and conditions include manifest optic atrophy (DOA), Leber's hereditary optic neuropathy (LHON), chronic progressive external ophthalmoplegia (CPEO), neurogenic weakness, ataxia, retinitis pigmentosa (NARP), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS); myoclonic epilepsy and ragged-red fibers (MERRF), and Kearns-Sayre syndrome (KSS). In some embodiments, the ophthalmic disease is LHON. In some embodiments, the ophthalmic disease is MELAS. In some embodiments, the disease is a muscular disease or condition. In some embodiments, the disease is a disease treatable by mitochondrial concentration therapy. In some embodiments, the disease is a disease treatable by mitochondrial transplantation therapy.In some embodiments, the disease is a disease treatable by mitochondrial replacement therapy. In some embodiments, the disease is a disease treatable by mitochondrial replacement and / or concentration therapy. In some embodiments, the mitochondrial replacement and / or mitochondrial concentration therapy is mitochondrial donation. In some embodiments, the disease is a systemic disease. In some embodiments, the disease is a multi-organ disease. In some embodiments, the disease is a multifocal disease. In some embodiments, the disease is an organ-specific disease.

[0099] In some embodiments, the disease or condition is diabetes. In some embodiments, the diabetes is diabetes mellitus. In some embodiments, the diabetes is type II diabetes. In some embodiments, the diabetes is type I diabetes. In some embodiments, the disease or condition is prediabetes. In some embodiments, the disease or condition is elevated blood glucose. In some embodiments, the disease is obesity. In some embodiments, the disease is fatty liver disease. In some embodiments, the fatty liver disease is non-alcoholic fatty liver disease (NAFLD). In some embodiments, the fatty liver disease is steatotic liver disease. In some embodiments, the NAFLD is metabolic dysfunction-associated fatty liver disease (MASLD). In some embodiments, the disease or condition is stroke. In some embodiments, the stroke is metabolic stroke. In some embodiments, the disease or condition is stroke. In some embodiments, the disease or condition is cardiomyopathy. In some embodiments, the cardiomyopathy is dilated cardiomyopathy. In some embodiments, the disease or condition is arrhythmia. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is a precancerous malignancy. In some embodiments, the disease or condition is Parkinson's disease. In some embodiments, the disease or condition is Alzheimer's disease. In some embodiments, the disease or condition is amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or condition is multiple sclerosis (MS). In some embodiments, the disease or condition is a genetic mitochondrial disorder. In some embodiments, the genetic disorder is primary mitochondrial myopathy. In some embodiments, the genetic disorder is mitochondrial encephalopathy. In some embodiments, the genetic disorder is mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. In some embodiments, the genetic disorder is myoclonic epilepsy with ragged-red fibers (MERRF).In some embodiments, the genetic disorder is Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome. In some embodiments, the genetic disorder is Leber's Hereditary Optic Neuropathy. In some embodiments, the disease or condition is optic neuropathy. In some embodiments, the disease or condition is aging. In some embodiments, the method is a method of treating aging. In some embodiments, the method is a method of ameliorating at least one age-related symptom. In some embodiments, the aging comprises muscle aging. In some embodiments, the muscle aging comprises muscle atrophy. In some embodiments, the disease or condition is sarcopenia. In some embodiments, the aging is skin aging. In some embodiments, the aging is ocular aging. In some embodiments, the ocular aging comprises macular degeneration. In some embodiments, the aging is cognitive aging. In some embodiments, cognitive aging comprises dementia.

[0100] In some embodiments, the disease is not a localized disease. In some embodiments, the disease is a regional disease. In some embodiments, the disease is not ischemia. In some embodiments, the disease is not cardiac ischemia. In some embodiments, the disease is not a cardiac disease. In some embodiments, the disease is not myocardial infarction. In some embodiments, the heart is myocardium. In some embodiments, the disease is not a disease treatable with a total amount of mitochondria of less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg. Each possibility represents a separate embodiment of the present invention. In some embodiments, the disease is not a disease treatable with a total amount of mitochondria of less than 12 mg. In some embodiments, the disease is not a disease treatable with a total amount of mitochondria of less than 17 mg. In some embodiments, the disease is not a disease treatable by a total amount of mitochondria of less than 50 mg. In some embodiments, the disease is a disease treatable only by a total amount of mitochondria of more than 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg. Each possibility represents a separate embodiment of the present invention. In some embodiments, the disease is a disease treatable only by a total amount of mitochondria of more than 16 mg. In some embodiments, the disease is a disease treatable only by a total amount of mitochondria of more than 100 mg. In some embodiments, the disease is one that can only be treated with a total amount of mitochondria exceeding 200 mg. In some embodiments, only what is treatable is effectively treated. In some embodiments, only what is treatable is a clinical benefit that can only be achieved with the recited amount of mitochondria. In some embodiments, the total is the total in a single administration.In some embodiments, the total is the sum of all administrations.

[0101] In some embodiments, the extract is autologous to the subject. In some embodiments, the composition is autologous to the subject. In some embodiments, the mitochondria are autologous to the subject. In some embodiments, the extract is allogeneic to the subject. In some embodiments, the composition is allogeneic to the subject. In some embodiments, the mitochondria are allogeneic to the subject. In some embodiments, the extract is syngeneic to the subject. In some embodiments, the composition is syngeneic to the subject. In some embodiments, the mitochondria are syngeneic to the subject.

[0102] In some embodiments, the method comprises extracting hematopoietic cells from the subject. In some embodiments, the method comprises taking a blood sample from the subject. In some embodiments, the method comprises receiving a blood sample from the subject. In some embodiments, the method comprises receiving hematopoietic cells from the subject. In some embodiments, the method comprises obtaining a blood sample from the subject. In some embodiments, the method comprises obtaining hematopoietic cells from the subject. In some embodiments, the blood sample is a peripheral blood sample. In some embodiments, the blood sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the hematopoietic cells are PBMCs.

[0103] In some embodiments, T cells, B cells, NK cells, or HSCs are isolated from a blood sample. In some embodiments, T cells, B cells, NK cells, or HSCs are isolated from hematopoietic cells. In some embodiments, T cells are isolated from a blood sample. In some embodiments, T cells are isolated from hematopoietic cells. Methods for isolating T cells are well known in the art, and any such method or known kit can be used for isolation. For example, isolation can be performed using anti-CD3 antibodies, anti-CD3 beads, or T cell isolation kits such as those sold by Miltenyi Biotec, Thermo Fisher, and many others.

[0104] In some embodiments, the mitochondrial extract is made from a blood sample. In some embodiments, the mitochondrial extract is made from hematopoietic cells. In some embodiments, the mitochondrial extract is made by a method of the present invention. In some embodiments, the mitochondrial extract is an autologous mitochondrial extract. In some embodiments, the mitochondrial extract is an allogeneic mitochondrial extract. In some embodiments, the mitochondrial extract is a syngeneic mitochondrial extract.

[0105] In some embodiments, the prepared mitochondrial extract is administered to a subject. In some embodiments, administering is reconstitution. In some embodiments, administering is systemic administration. In some embodiments, administering is intravenous administration. In some embodiments, administering is topical administration. In some embodiments, administering is intravenous administration. In some embodiments, administering is intraocular administration. In some embodiments, administering is to the site of disease. It will be appreciated by those skilled in the art that by using the subject's own blood cells as the source of expanded mitochondria, an inexpensive, non-immunogenic, and inexhaustible source of high-quality mitochondria has been identified. If the subject is suffering from a genetic mitochondrial disease or a condition in which all of the subject's mitochondria are affected, it will be necessary to prepare an extract from donor blood cells. However, many diseases characterized by mitochondrial dysfunction are characterized by dysfunction only in a target set of diseased cells (e.g., liver cells, pancreatic cells), and the mitochondria in the subject's blood cells will be healthy and available for treatment.

[0106] As used herein, the term "treatment" or "treating" of a disease, disorder, or condition includes alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, a composition or method useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide an improvement in the quality of life of the patient or subject.

[0107] As used herein, the term "about," when used in conjunction with a value, refers to ±10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ±100 nm.

[0108] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a polynucleotide" includes a plurality of such polynucleotides; a reference to "the polypeptide" includes a reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements or the use of "negative" limitations.

[0109] Where a convention similar to "at least one of A, B, and C, etc." is used, generally, such configuration is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those of ordinary skill in the art that virtually any disjunctive word and / or phrase presenting two or more terms to choose from, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" would be understood to include the possibilities of "A" or "B" or "A and B."

[0110] It is understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0111] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0112] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. [Example]

[0113] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are fully explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual" by Sambrook et al. (1989); "Current Protocols in Molecular Biology" Volumes I-III, Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA," Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series," Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); methodologies described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "Cell Biology: A Laboratory Handbook," Volumes I-III, Cellis, JE, ed. (1994); "Culture of Animal Cells—A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology," Volumes I-III, Coligan, JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated by reference. Other general references are provided throughout this document.

[0114] material and method Isolation of PBMCs from blood samples: The blood sample bag was washed with ethanol, and 13 mL of blood was transferred to 17 mL of RPMI medium and mixed. 13 mL of Ficoll was added to a new tube, and the blood mixture was maintained at 45°C. The Ficoll-blood tube was centrifuged at 800 g for 30 minutes at room temperature (RT, 25°C) with the slowest acceleration and the brake off. The PBMC layer (a whitish layer formed at the interface between the plasma and Ficoll) was transferred to a new 50 mL tube, which was then filled to the 45 mL mark with 37°C RPMI. After centrifugation at 330 g for 10 minutes at room temperature, the supernatant was discarded, and the cells were resuspended by tapping the tube until no clumps were visible. 5 mL of RPMI was added, and the cells were counted and resuspended at a concentration of 50 × 10^6 cells / mL in freezing medium (for freezing) or AutoMacs running buffer (Miltenyi) for T cell isolation.

[0115] Isolation of T cells from PBMC: Isolation of CD3-positive T cells from PBMC was performed according to the protocol of the Pan T Cell Isolation Kit Human (Miltenyi) with several adaptations.

[0116] For MACSiBead particle preparation, 200 μL of CD2-biotin, 200 μL of CD3-biotin, and 200 μL of CD28-biotin were added to a tube and mixed, followed by the addition of 1 mL of anti-biotin MACSiBead particles and 400 μL of AutoMacs running buffer (Miltenyi) and incubation for 2 hours at 4°C with gentle rotation.

[0117] For T cell isolation, PBMCs were suspended in AutoMacs running buffer (40 μL buffer per 10 x 106 total cells), and pan T cell biotin-antibody cocktail (10 μL per 10 x 106 total cells) was added, followed by a 5-minute refrigerated incubation (2-8°C). Next, pan T cell microbead cocktail (20 μL per 10 x 106 total cells) was added, followed by a 10-minute refrigerated incubation (2-8°C).

[0118] The mesh-loaded LS column was placed over a tube in the magnetic field of an appropriate MACS separator and rinsed with 3 mL of buffer, followed by a new tube. The cell suspension was applied to the mesh, and the flow-through, containing unlabeled cells representing enriched T cells, was collected. Following this, the column was washed with 9 mL of buffer, and the flow-through was collected in the same tube. The column was removed from the separator and placed in a new tube. The magnetically labeled non-T cells were then flushed out by pipetting 5 mL of buffer and firmly depressing the plunger onto the column.

[0119] T cell activation and proliferation: Cells were seeded in G-REX 24-well plates or G-REX 6-well plates in suspension cell medium (4Cell NutriT medium (Gibco)) with or without beads coated with antibodies against human CD2, CD3, and CD28 to mimic antigen-presenting cells, and containing IL-2 as an activator. Cells were incubated for 14 days in a 37°C incubator supplemented with 5% CO2. The IL-2-containing medium was refreshed on day 4 (6 mL of 8) and on days 7, 9, and 11 to 8 mL. In G-REX 6-well plates, 1–5 × 10^6 cells were seeded in each well and 100 mL of medium was added. Cells were incubated in a 37°C incubator supplemented with 5% CO2. The medium was not changed, and IL-2 was added to the wells every 3 days. Cell counts were performed on days 7, 9, 11, and 14.

[0120] MACSiBead particles (beads coated with antibodies against human CD2, CD3, and CD28 to mimic antigen-presenting cells) were prepared using a T Cell Activation / Proliferation Kit, Human (Miltenyi Biotec, catalog number 130-091-441) and MACS buffer (Miltenyi, catalog number 130-091-221) according to the manufacturer's instructions.

[0121] Mitochondrial quantity: Relative mitochondrial quantity was determined by testing the protein concentration of the mitochondrial purified fraction with BCA reagent, normalized to the total number of cells used to generate mitochondria (initial cell plating).

[0122] Western Blot: Total protein extraction was performed by lysing cells with RIPA lysis buffer supplemented with protease inhibitors, incubating at 4°C for 30 minutes, followed by centrifugation at 14,000 g and 4°C for 10 minutes, and collecting the supernatant. Isolated mitochondria were used as the mitochondrial subfraction. The supernatant above the mitochondrial pellet was used as the cytosolic subfraction. Protein concentration was determined by Bradford assay. Samples were separated on SDS-PAGE gels, and proteins were then electrotransferred to an Inmobilon-P transfer membrane. The membrane was then blotted with anti-pyruvate dehydrogenase E1α and anti-α-tubulin.

[0123] ATP assay: ATP from expanded T cells was assayed using the ATPlite 1-step kit (PerkinElmer) according to the manufacturer's instructions. Briefly, cells were transferred to a 96-well plate in a total volume of 100 μL, and 100 μL of kit reagent was added. Luminescence from the whole cell lysate was measured using a plate reader.

[0124] Oxygen consumption: Expanded T cells were tested for oxygen consumption using the MitoXpress Xtra Oxygen Consumption Assay Kit (Aligent) according to the manufacturer's instructions. Briefly, cells were transferred to a 96-well plate, followed by addition of kit reagents, sealing the wells with oil, and recording fluorescence using a plate reader (Ex / Em 380 / 650). Assays were performed on isolated mitochondria or intact cells.

[0125] Membrane potential: Membrane potential was tested using JC-1 reagent (Sigma) according to the manufacturer's instructions. Briefly, JC-1 was added to cells (5 μM) for 30 min, followed by three washes, and the fluorescence of the cells was recorded using a plate reader at Ex / Em 550 / 600 and 485 / 535. Assays were also performed using isolated mitochondria.

[0126] Citrate synthase activity: Mitochondrial citrate synthase (CS) activity in samples (either isolated mitochondria or whole cells / cell lysates) was determined by an immunocapture-based assay. The assay principle involves capturing the enzyme in a microplate well and then determining activity by recording the color development of TNB, which is produced from DTNB in ​​the citrate synthesis reaction. The total reaction product, TNB, absorbs at 412 nm. The reaction proceeds as follows: Oxaloacetate + acetyl CoA + H2O → citrate + CoA-SH + H+ CoA-SH+DTNB→TNB+CoA-SS-TNB (↑Absorbance at 412 nm)

[0127] Antibody ab119692 specifically immunocaptures only native citrate synthase from the applied test sample. In general, this immunocapture-based activity assay allows for the measurement of citrate synthase activity with simple sample preparation and without the need for mitochondrial isolation.

[0128] Mitochondrial isolation: Cells were centrifuged at 300 × g for 10 min, washed with PBS, recentrifuged, and resuspended in mitochondrial isolation buffer (320 mM sucrose, 5 mM Tris-HCl, pH 7.4, 2 mM EGTA, with or without fatty acid-free BSA (0.5%), protease inhibitor cocktail, and PMSF) and homogenized using a Dounce homogenizer, needle shearing (passing the cells 10 times with a 30G needle), or nitrogen cavitation. Nitrogen cavitation was performed using a disruption vessel (Parr Instruments) at 800 psi for 20 min. The cell homogenate was centrifuged at 3,000 × g for 5 min, the supernatant was collected, and centrifuged again at 3,000 × g for 5 min. The supernatant was then centrifuged at 12,000 × g for 10 min. The mitochondrial pellet was resuspended in mitochondrial isolation buffer. Mitochondrial concentration was determined by Bradford assay.

[0129] Alternatively, mitochondria were isolated using a mitochondrial isolation kit (Miltenyi, 130-094-532) according to the manufacturer's instructions. Briefly, the cell homogenate was suspended in the kit's isolation buffer, anti-TOM22 MicroBeads were added, and the suspension was incubated for 1 h in a refrigerator (2-8°C) with gentle shaking. The suspension was then applied to the kit's column, washed with the isolation buffer, and the column was removed from the separator. Mitochondria were obtained by adding mitochondrial isolation buffer and pushing the plunger into the column. Mitochondrial concentration was determined by Bradford assay.

[0130] Liver analysis: Liver samples were collected from 36 mice, fixed in 4% formaldehyde, and maintained for 48 hours for further fixation. The tissues were then trimmed, placed in embedding cassettes, and routinely processed for paraffin embedding. Two cassettes were prepared per animal. Paraffin sections (4 microns thick) were cut, placed on glass slides, and stained with hematoxylin and eosin (H&E) for general histology. Additionally, frozen sections (8 microns thick) of liver samples (n=36) were prepared using a cryostat and stained with Oil Red-O for triglyceride detection. Slides were subjected to histopathological evaluation by pathologists at PATHO-LOGICA Ltd.

[0131] Photographs were taken at objective magnifications of X1.25, X4 and X10 using an Olympus microscope (BX60, serial number 7D04032) and a microscope camera (Olympus DP73, serial number OH05504).

[0132] H&E stained sections were examined, described, and scored by a research pathologist for the severity of histopathological changes using a semiquantitative grading scale (5-point scale) (the scale is disclosed in Schafer et al., "Use of severity grades to characterize histopathologic changes," Toxicologic Pathology 2018, 46:256-265, the contents of which are incorporated herein by reference in their entirety). The scale is as follows: Grade 0 - tissue appears normal, no changes at all; Grade 1 - minimal pathological findings; Grade 2 - mild pathological findings; Grade 3 - moderate pathological findings; Grade 4 - Severe pathological findings.

[0133] Oil Red-O staining for lipid droplets in hepatocytes was scored as follows: Grade 0 = normal liver without lipid accumulation; Grade 1 = very mild accumulation of lipid droplets; Grade 2 = mild accumulation of lipids; Grade 3 = moderate accumulation of lipids; Grade 4 = significant lipid accumulation.

[0134] Statistics: Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey HSD. Significance was considered at p<0.05.

[0135] Membrane potential measurement with JC-1: Mitochondrial membrane potential was determined using the cationic dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide). JC-1 exhibits potential-dependent accumulation in mitochondria, indicated by a shift from green fluorescence (monomers) to red fluorescence (aggregates). Mitochondria were isolated from T cells and resuspended in isolation buffer at a protein concentration of 1 mg / ml, as determined by the BCA assay.

[0136] Isolated mitochondria were incubated with JC-1 dye at a final concentration of 0.2 μg / ml in assay buffer for 30 min at 37 °C to allow for dye uptake and equilibration. After incubation, the fluorescence intensity of JC-1 monomers and aggregates was measured using a fluorescence plate reader with appropriate settings (green fluorescence: Ex / Em = 485 / 535 nm, red fluorescence: Ex / Em = 550 / 600 nm). All assays were performed in triplicate to ensure reproducibility.

[0137] Example 1: Expansion of primary T cells for mitochondrial isolation In an attempt to generate large quantities of functional therapeutic mitochondria from cells grown in culture, we decided to optimize mitochondrial production / extraction from primary T cells. Because mitochondrial viability and integrity are compromised in immortalized cells and mitochondrial damage is a hallmark of cancerous cells, primary cells were used, even though these cells are generally much more difficult to culture and grow. Hematopoietic cells, such as T cells, natural killer (NK) cells, B cells, and hematopoietic stem cells (HSCs), have the advantage that they grow in suspension and do not adhere to plates. This allows for the cultivation of much larger numbers of cells in the same amount of culture medium as is possible with adherent cells.

[0138] T cells were isolated from human PBMCs as described hereinabove (Materials and Methods). Flow cytometry was performed to confirm the homogeneity of the T cell population, and cultures were seeded with more than 90% T cells. The isolated T cells were then grown for two weeks in gas-permeable rapid expansion (G-REX) plates. These plates are specifically designed for suspension cells. The G-REX configuration provides unlimited access to nutrients without mixing (reducing contact with cells, which can result in mitochondrial damage). Notably, there is essentially unlimited access to oxygen, as the gas-permeable membrane increases oxygen diffusion throughout the medium. Flow cytometry was also performed after expansion, and the population was found to have a purity of more than 97%.

[0139] Cell number and viability were monitored over a two-week period (Figure 1). Cell numbers were found to peak at day 10, with 400 x 10^6 cells per 6 wells. This was an approximately 80-fold increase since the initial plating concentration was 5 x 10^6 cells / 6 wells. Cell viability, assessed by trypan blue staining, was also still very high at day 10 but declined sharply by day 14.

[0140] Example 2: Mitochondrial characterization and isolation Next, mitochondria were isolated from the expanded T cells. It is essential that the isolation process is not overly harsh, as there is a risk of damaging or rupturing the mitochondria. Two methods for cell disruption were tested: Dounce homogenization and nitrogen cavitation. Both methods are known in the art and are generally considered equivalent for adherent cells. However, adherent cells must first be trypsinized from the plate and are therefore already damaged or ruptured to some extent. Therefore, it was unknown whether the procedure for adherent cells was equivalent to that for suspension cells. The results are summarized in Table 1.

[0141] [Table 1]

[0142] Both methods result in robust yields of intact mitochondria. These yields were far greater than those produced from adherent cells. A rate of 220 μg of mitochondria from 1 million starting primary suspension cells is completely unknown in the art, and certainly 90 or 110 μg is well in excess of what can currently be produced from such small starting cells. Certainly, these quantities of mitochondria cannot be produced when seeding a similar initial amount of primary adherent cells. Culturing is also performed in a bioreactor rather than tissue culture cells. Yields from the bioreactor reach and exceed 1000 μg of protein from as few as 106 starting T cells.

[0143] Mitochondria from expanded T cells were assessed for quality. This included assays of oxygen consumption, membrane potential, and citrate synthesis (CS) activity. Mitochondria isolated from T cells were found to be functional and within the expected range for all of these assays. CS activity was assayed at three time points during T cell expansion and was found to be comparable at all time points, indicating that long-term expansion does not adversely affect mitochondrial quality (Figure 2 and Table 2).

[0144] [Table 2]

[0145] Isolating such a large number of mitochondria enabled the generation of therapeutic compositions with extremely high mitochondrial concentrations. Mitochondria were isolated from proliferating T cells on day 8 after seeding. The oxygen consumption rate (OCR) of isolated mitochondria was tested at four concentrations in the same total volume: 16 μg / mL, 8 μg / mL, 4 μg / mL, and 2 μg / mL. The results are shown in Figure 3. Substrate glutamate / malate levels were kept constant throughout the experiment, allowing for direct comparison of mitochondrial activity based on dose alone. Basal respiration levels were recorded first. Addition of ADP induced oxidative phosphorylation, evidenced by an increase in OCR, indicating ATP production by mitochondria. This response was concentration-dependent, with the 16 μg / mL preparation showing the most significant increase, indicating greater metabolic activity at higher mitochondrial densities.

[0146] The introduction of oligomycin resulted in a decrease in OCR at all concentrations, consistent with its role as an ATP synthase inhibitor. This demonstrates that the prior increase in OCR was due to ATP synthesis. The uncoupler FCCP caused a spike in OCR at all mitochondrial concentrations, as expected. This uncoupler collapses the proton gradient across the mitochondrial inner membrane, resulting in maximal electron transport chain activity without ATP generation. The particularly pronounced peak OCR at the highest mitochondrial concentration validates electron transport capacity and suggests robust inner membrane integrity and function. Finally, the addition of rotenone, an inhibitor of complex I, resulted in a decrease in OCR in all samples, confirming the role of complex I in the measured respiratory activity.

[0147] Example 3: Mitochondria retain their functionality after freezing The membrane potential of isolated mitochondria was tested with JC-1 dye on freshly isolated mitochondria kept at 4°C immediately after isolation, on isolated mitochondria kept at 4°C for 4 days, and on isolated mitochondria kept at -80°C for 4 days. Fresh mitochondria intentionally damaged by several freeze / thaw cycles were used as a negative control.

[0148] The ratio of red to green fluorescence intensity was calculated for each sample and used as an index of mitochondrial membrane potential. An increase in the red / green fluorescence ratio indicates a higher membrane potential due to intramitochondrial JC-1 aggregation, whereas a decrease in this ratio suggests mitochondrial depolarization, which correlates with loss of membrane potential. As can be seen in Figure 4A, when mitochondria were stored at 4°C for 4 days, the membrane potential decreased significantly, but freezing for 4 days had no apparent effect on the mitochondrial membrane potential upon thawing. Mitochondria that had undergone intentional damage exhibited essentially no membrane potential.

[0149] As a control, mitochondria were treated with the potassium ionophore valinomycin (0.5 μM) to dissipate the membrane potential before adding JC-1 dye. This served as a positive control for depolarization, and as expected, all valinomycin-treated mitochondria showed little to no membrane potential. The ATP synthase inhibitor oligomycin (1 μg / ml) was used to inhibit proton backflow into mitochondria and serve as a control for assessing the coupling efficiency of the electron transport chain. Blocking ATP synthase results in an increased proton gradient (i.e., an increase in membrane potential). While fresh mitochondria treated with oligomycin indeed showed the expected increase in membrane potential, mitochondria stored at 4°C for 4 days essentially failed to respond to oligomycin, indicating that these mitochondria were not functional. Importantly, mitochondria stored at -80°C and then thawed also showed an increase in membrane potential after oligomycin treatment, indicating that storage at -80°C does not significantly affect membrane potential maintenance.

[0150] Next, to confirm the functionality of isolated mitochondria stored at -80°C for 4 days, we measured the oxygen consumption rate using the Seahorse system. When mitochondria were stored at 4°C, the oxygen consumption rate was significantly reduced (Figure 4B). However, freezing at -80°C and then thawing only slightly reduced the oxygen consumption rate, confirming that the mitochondria were functional and active.

[0151] The respiratory control ratio (RCR) is a quantitative measure of mitochondrial efficiency calculated by dividing the oxygen consumption rate during ADP-stimulated respiration (state 3) by the OCR in the basal, nonphosphorylated state (state 4). High RCR values ​​indicate efficient coupling of electron transport to ATP synthesis, reflecting healthy mitochondrial function, whereas low RCR values ​​suggest mitochondrial dysfunction or uncoupling. The RCR of fresh mitochondria was calculated to be 5, while mitochondria kept at 4°C had an RCR of only 1.6. In contrast, frozen mitochondria had an RCR of 4.2, indicating they were still highly functional and active.

[0152] Example 4: Treatment of diabetes and NAFLD in mouse models Type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) are associated with mitochondrial dysfunction and oxidative stress. In diabetic patients, hyperglycemia enhances mitochondrial reactive oxygen species (ROS) generation, inducing oxidative damage and impaired insulin signaling. Mitochondrial biogenesis also regulates energy balance, and excessive ROS generation under high glucose conditions may exacerbate vascular complications. In nonalcoholic fatty liver disease (NAFLD), mitochondrial dysfunction leads to abnormal hepatic lipid metabolism and oxidative stress. Because mitochondrial DNA and proteins are irreversibly damaged, lifestyle interventions such as diet, exercise, and antioxidants offer limited protection. Replenishing and / or strengthening dysfunctional mitochondria with healthy ones may be a promising therapeutic approach for these diseases. Therefore, a mitochondrial composition prepared from T cells was tested in a mouse model for its ability to treat both diabetes and NAFLD.

[0153] Healthy male C57BL / 6J mice weighing 18-22 g were used in this study. Animals were maintained under standard housing conditions with access to standard laboratory mouse chow and water. All animal experiments were performed in accordance with guidelines.

[0154] C57BL / 6J mice were randomly assigned to six groups (n=6 for each group). The experimental groups are listed in Table 3. Mice in groups A and D were fed a standard chow diet (fat content ∼6%) and served as normal controls, while mice in the other three groups (B, C, E) were intragastrically administered a high-lard fat and high-cholesterol diet in which 60% of total calories came from fat.

[0155] [Table 3]

[0156] After 8 weeks, mice in groups D and E were intravenously injected with a preparation of healthy mitochondria isolated from human T cells (0.5 mg / kg body weight, e.g., >9 mg of mitochondria per injection) once every three days for a total of six doses. Mice in group C received only half the dose, administered every three days, for a total of three doses. These mice had a more limited source of mitochondria and represented the best possible incomplete dosing. For groups A and B, mice were intravenously administered an equal volume of saline. Following the final mitochondrial treatment, all mice were fasted for 12 hours and then euthanized with an overdose of sodium pentobarbital. Mouse serum and liver tissue were collected.

[0157] Groups A and D (healthy control animals) showed normal morphology without any pathological changes or lipid metabolism abnormalities (score of 0 for all animals for both pathological severity score and lipid metabolism abnormalities) (Figure 5A-B).

[0158] Group B (high-fat diet, control) showed a score of 2.16 for both pathological severity and lipid metabolism abnormalities (Figure 5C). In addition to macrodroplet and microdroplet changes in hepatocytes, hepatocyte necrosis was also observed. H&E staining of the heart was also performed as a control, and all animals showed a pathology score of 0. Group E (high-fat diet, mitochondrial treatment) showed a significant decrease in the number and size of vesicles within hepatocytes, as well as a marked improvement in the area indicating hepatocyte regeneration (Figure 5D). Oil Red O staining strongly supported these findings. The grades of pathological severity (H&E) and Oil Red O staining were 1.5 and 1.33, respectively. No improvement was observed when only the half-dosing regimen was used (Group C) (Figure 5E). Statistical testing showed significant changes in pathological livers between Group B and Group E, but not between Group B and Group C.

[0159] When administered intravenously, a total large amount of mitochondria was required to produce a systemic effect. However, lipid levels and histology did not completely return to normal, so even higher doses of mitochondria may be required. It has not been possible to isolate such a large amount of therapeutic mitochondria until now. Mitochondria from mice or other test animals are not suitable for therapeutic administration to humans. Mitochondria from cancerous or immortalized cell lines have damaged mitochondria and are similarly not suitable for therapeutic administration. Prior to the mitochondrial production method provided herein, the only suitable method for producing therapeutic mitochondria was to grow adherent primary cells in culture or to produce mitochondria using direct tissue biopsy. As outlined herein above, primary adherent cell cultures only yield a small number of cells, and such high concentrations and total amounts of mitochondria could not be feasibly produced. Bharadwaj et al. taught the use of percutaneous needle biopsy to prepare skeletal muscle tissue for isolation of therapeutic mitochondria (Bharadwaj et al., "Preparation and respirometric assessment of mitochondria isolated from skeletal muscle tissue obtained by percutaneous needle biopsy," J Vis Exp. 2015;(96):52350, the contents of which are incorporated herein by reference in their entirety). As can be seen in Figure 5 of Bharadwaj, the maximum total mitochondrial yield from a single biopsy was approximately 2 mg. Most yielded even smaller amounts (<1.5 mg).

[0160] In the above experiment, 0.5 mg / kg of mitochondria was administered six times. For an average person weighing 70 kg, this amounts to 35 mg of mitochondria. People with diabetes or NAFLD may be even heavier. A single muscle needle biopsy cannot produce enough mitochondria for even a single therapeutic dose, let alone six (210 mg required). Furthermore, if the subject's diet is not changed, such treatment may need to be repeated. Therefore, it is clear that muscle biopsies are not a viable source of therapeutic mitochondria. In fact, with the exception of the T cell isolation method of the present invention, no source is sufficiently robust to produce the therapeutic effects demonstrated herein.

[0161] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. 1. A method for producing a mitochondrial extract, the method comprising: a. Providing primary human hematopoietic cells in suspension; b. growing the primary human hematopoietic cells for a sufficient time to produce an expanded population of human hematopoietic cells comprising at least 10 times the number of the primary human hematopoietic cells provided; and c. isolating mitochondria from the expanded population; thereby producing a mitochondrial extract.

2. The method of claim 1 , wherein the primary hematopoietic cells are obtained from a blood sample from a human subject.

3. 3. The method of claim 1 or 2, wherein the primary hematopoietic cells are isolated from peripheral blood mononuclear cells (PBMCs).

4. The method of any one of claims 1 to 3, wherein the primary human hematopoietic cells are selected from T cells, B cells, NK cells and hematopoietic stem cells (HSCs).

5. The method of any one of claims 1 to 4, wherein the primary human hematopoietic cells are primary human immune cells.

6. 6. The method of claim 5, wherein the primary human immune cells are selected from T cells, B cells, and NK cells.

7. The method of claim 6, wherein the primary human hematopoietic cells are an isolated population of T cells.

8. The method of any one of claims 1 to 7, wherein the period of time is between 7 and 14 days.

9. The method of any one of claims 1 to 8, wherein the expanded population of human hematopoietic cells comprises at least 20 times the number of the primary human hematopoietic cells provided.

10. 10. The method of claim 9, wherein the expanded population of human hematopoietic cells comprises at least 50 times the number of primary human hematopoietic cells provided.

11. 11. The method of any one of claims 1 to 10, wherein the isolated mitochondria contain at least 20 micrograms of protein per million primary human hematopoietic cells prepared.

12. 12. The method of claim 11, wherein the isolated mitochondria contain at least 50 micrograms of protein for every 1 million primary human hematopoietic cells prepared.

13. 13. The method of claim 12, wherein the isolated mitochondria contain at least 80 micrograms of protein for every 1 million primary human hematopoietic cells prepared.

14. The method of any one of claims 11 to 13, wherein the protein is determined by Bradford assay.

15. 15. The method of any one of claims 1 to 14, wherein the expanding comprises contacting the provided primary human hematopoietic cells with at least one of an antigen-presenting cell (APC), a component of an APC, a component that mimics APC activity, a factor secreted by activated T cells, and any combination thereof.

16. 16. The method of claim 15, wherein the provided primary human hematopoietic cells are primary T cells, and said expanding comprises contacting the primary T cells with at least one of an anti-CD3 antibody or antigen-binding fragment thereof, an anti-CD28 antibody or antigen-binding fragment thereof, an anti-CD2 antibody or antigen-binding fragment thereof, interleukin-2 (IL-2), and any combination thereof.

17. 17. The method of claim 16, wherein said expanding comprises contacting the primary T cells with an anti-CD3 antibody or antigen-binding fragment thereof, an anti-CD28 antibody or antigen-binding fragment thereof, an anti-CD2 antibody or antigen-binding fragment thereof, and IL-2.

18. The method of any one of claims 1 to 17, wherein said expanding comprises culturing a suspension of said primary human hematopoietic cells in a gas-permeable container.

19. 20. The method of claim 18, wherein the culturing is in a bioreactor.

20. 19. The method of claim 18, wherein the gas permeable container is a gas permeable rapid expansion (G-REX) well.

21. 21. The method of any one of claims 1 to 20, wherein the isolating comprises lysing the cells to produce a cell lysate, and the lysis comprises at least one of adding a lysis buffer, needle shearing, homogenization with a Dounce homogenizer, and nitrogen cavitation.

22. 22. The method of claim 21, wherein said isolating comprises disrupting the membranes of said immune cells by nitrogen cavitation or Dounce homogenization.

23. 23. The method of claim 22, wherein said isolating comprises disrupting the membranes of said immune cells by nitrogen cavitation.

24. 24. The method of any one of claims 21 to 23, comprising contacting the lysate with an artificial support comprising an anti-TOM22 binding agent, and isolating the artificial support and any mitochondria bound thereto.

25. 25. The method of claim 24, further comprising eluting the mitochondria from the artificial scaffold.

26. 24. The method of any one of claims 21 to 23, comprising centrifuging the lysate at about 3000 g to remove cellular debris and produce a supernatant, and centrifuging the supernatant at about 12,000 g to produce a mitochondrial precipitate.

27. A mitochondrial extract produced by the method of any one of claims 1 to 26.

28. A mitochondrial extract that is at least 90% T cell mitochondria, at least 90% B cell mitochondria, at least 90% NK cell mitochondria, or at least 90% HSC mitochondria.

29. 29. The mitochondrial extract of claim 28, which is at least 95% T cell mitochondria.

30. 30. The mitochondrial extract of claim 28 or 29, wherein the mitochondria are human mitochondria.

31. 31. The mitochondrial extract of any one of claims 28 to 30, comprising a mitochondrial concentration of at least 16 ug / ml.

32. A pharmaceutical composition comprising the mitochondrial extract of any one of claims 27 to 31.

33. 33. The pharmaceutical composition of claim 32, further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant.

34. 34. The pharmaceutical composition of claim 32 or 33, formulated for systemic administration to a subject.

35. 34. The pharmaceutical composition of claim 32 or 33, formulated for in vitro transfer into target cells.

36. A recombinant cell comprising the mitochondrial extract of any one of claims 27 to 31.

37. 37. The recombinant cell of claim 36, wherein the cell is depleted of endogenous mitochondria.

38. 38. The recombinant cell of claim 36 or 37, which is a non-hematopoietic cell.

39. A pharmaceutical composition comprising the recombinant cell of any one of claims 36 to 38 and a pharmaceutically acceptable carrier, excipient or adjuvant.

40. A method for treating a subject suffering from a mitochondrial disease, comprising administering to the subject a pharmaceutical composition according to any one of claims 32 to 35 and 39, thereby treating the mitochondrial disease.

41. 41. The method of claim 40, wherein the administration is systemic.

42. 42. The method of claim 40 or 41, wherein the mitochondrial disease is selected from diabetes, fatty liver disease, Parkinson's disease, cancer, Alzheimer's disease, genetic mitochondrial disorders, aging, and dilated cardiomyopathy.

43. 43. The method of claim 42, wherein the mitochondrial disease is selected from diabetes and non-alcoholic fatty liver disease (NAFLD).

44. 44. The method of any one of claims 40 to 43, wherein the mitochondrial extract is autologous to the subject.

45. 44. The method of any one of claims 40 to 43, wherein the mitochondrial extract is allogeneic to the subject.

46. 45. The method of any one of claims 40 to 44, comprising extracting a blood sample from the subject, producing a mitochondrial extract from the blood sample by the method of any one of claims 1 to 26, and administering the produced mitochondrial extract to the subject.

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