Mitochondrial isolation from suspension cells

Abstract

A method of producing an extract of mitochondria is provided, the method comprising providing suspended primary artificial blood cells, amplifying the primary artificial blood cells, and isolating mitochondria. Also provided are mitochondrial extracts, compositions including mitochondrial extracts, and methods of using the extracts and compositions.

Description

[0001] Cross-references to related applications This application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 447,675, filed February 23, 2023 (the contents of which are incorporated herein by reference in their entirety). Technical Field

[0002] This invention belongs to the field of mitochondrial isolation and transplantation. Background Technology

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

[0004] This invention provides a method for producing a mitochondrial extract, the method comprising providing a suspension of primary artificial blood cells, expanding the primary artificial blood cells, and isolating mitochondria. It also provides a mitochondrial extract, a composition comprising the mitochondrial extract, and methods for using the extract and the composition.

[0005] According to a first aspect, a method for producing a mitochondrial extract is provided, the method comprising: a. Provides suspended primary artificial blood cells; b. The time required to expand the primary artificial blood cells sufficiently to produce an expanded population of artificial blood cells, said expanded population comprising at least 10 times the number of primary artificial blood cells provided; and c. Isolate mitochondria from the amplified population; This allows for the production of mitochondrial extracts.

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

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

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

[0009] According to some implementation methods, the primary artificial blood cells are primary human immune cells.

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

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

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

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

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

[0015] According to some embodiments, the isolated mitochondria comprise at least 20 micrograms of protein per 1 million of the provided primary human hematopoietic cells.

[0016] According to some embodiments, the isolated mitochondria comprise at least 50 micrograms of protein per 1 million of the provided primary human hematopoietic cells.

[0017] According to some embodiments, the isolated mitochondria comprise at least 80 micrograms of protein per 1 million of the provided primary human hematopoietic cells.

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

[0019] According to some embodiments, 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 the activity of an APC, a factor secreted by an activated T cell, and any combination thereof.

[0020] According to some embodiments, the provided primary human hematopoietic cells are primary T cells, and wherein the 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 the primary hematopoietic cells in a gas permeable container.

[0023] According to some embodiments, the culturing is performed 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 produce a cell lysate, wherein the lysing comprises at least one of the following: 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 membranes of the immune cells by nitrogen cavitation.

[0028] According to some embodiments, the method comprises contacting the lysate with an artificial carrier comprising an anti-TOM22 binding agent and isolating the artificial carrier 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 comprises centrifuging the lysate at about 3000 g to remove cell debris and produce a supernatant and centrifuging the supernatant at about 12,000 g to produce a mitochondria pellet.

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

[0032] According to another aspect, there is provided 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.

[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 application.

[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 a target cell.

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

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

[0040] According to some embodiments, the cell is a non-hematopoietic cell.

[0041] According to another aspect, there is provided a pharmaceutical composition comprising the recombinant cell of the application 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 the pharmaceutical composition of the application, thereby treating the mitochondrial disease.

[0043] According to some embodiments, the mitochondrial disease is selected from the group consisting of diabetes, Parkinson's disease, cancer, Alzheimer's disease, inherited 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] Other embodiments and full scope of the application will become apparent from the detailed description given hereinbelow. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 Line graph of the number and viability of T cells cultured for 2 weeks in G-REX plates.

[0048] Figure 2 Line graph of CS activity of mitochondria isolated from T cells at day 7, day 10 and day 13 of culture.

[0049] Figure 3 Oxygen consumption rate (OCR) of isolated mitochondria using glutamate / malate as substrate, measured using a Seahorse Bioanalyzer.

[0050] Figures 4A-4B: Bar graph of membrane potential measurements of freshly isolated mitochondria, mitochondria after 4 days at 4°C or -80°C. (4B) Line graph of oxygen consumption rate of freshly isolated mitochondria, mitochondria after 4 days at 4°C or -80°C.

[0051] Figures 5A-5E Micrographs showing H&E staining (left) and Oil Red O staining (right, lipids shown in red) of the following groups: (5A) healthy controls, i.e. Group A, (5B) healthy controls treated with mitochondria, i.e. Group D, (5C) high fat diet without treatment, i.e. Group B, (5D) high fat diet treated with mitochondria, i.e. Group E, (5E) high fat diet treated with mitochondria on a half regimen, i.e. Group C. DETAILED DESCRIPTION

[0052] The present invention provides, in some embodiments, methods of producing a mitochondrial extract, the method comprising providing primary human hematopoietic cells in suspension, expanding the primary human hematopoietic cells, and isolating mitochondria. The present invention also relates to mitochondrial extracts, compositions comprising mitochondrial extracts, and methods of using the extracts and compositions.

[0053] According to one aspect, the present application discloses methods for providing functional mitochondria from suspended, non-transformed primary hematopoietic cells (e.g., immune cells, T cells). Advantageously, the methods based on the present application yield a quantity of functional mitochondria from a single unit of blood sufficient for at least one therapeutic dose, and also sufficient for multiple therapeutic doses. Multiple therapeutic doses can be provided for repeated administration to the same patient or to several different patients. In some embodiments, the methods produce a sufficient quantity of functional mitochondria to produce multiple doses of a mitochondrial therapeutic composition. In some embodiments, the composition is a composition of the present application. 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 unit of blood. In some embodiments, the single unit of blood is a single blood sample. In some embodiments, the single blood sample is a single blood draw. In some embodiments, the generation is by a method of the present application. In some embodiments, the multiple doses are at least 2 doses. In some embodiments, the multiple doses are at least 5 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 appreciate that since the cells themselves are not intended for use in cell therapy, their functionality, therapeutic potential, and / or cytotoxic activity in culture are not limiting factors, but rather only the quality of the mitochondria. Thus, the cells of a single unit of blood are capable of being grown so long as the quality and / or function of the mitochondria is maintained. In this way, the methods of the present application have the great advantage of producing very large quantities of mitochondria.

[0054] The present application is based, at least in part, on the surprising yield of mitochondria produced from cultured primary T cells. The methods of the present application allow for the production of over 85 ug of isolated mitochondrial protein from a starting population of only 1 million primary T cells. Moreover, optimization of the methods produces yields as high as 220 ug of protein per 1 million primary T cells. When the optimized methods are performed in bioreactors, rather than just tissue culture plates, the yields reach up to and exceed 1000 ug. The use of suspended hematopoietic cells to generate mitochondria allows for cell expansion, which is not possible in primary cultures of adherent cells. Moreover, these mitochondria are of high quality, functionally intact, and suitable for therapeutic use in human subjects. The produced compositions are capable of effectively treating diabetic and fatty liver mice.

[0055] In a first aspect, there is provided a method for producing a mitochondrial extract, the method comprising: a. providing suspended cells; b. expanding the cells for a time sufficient to produce an expanded population of cells; and c. isolating mitochondria from the expanded population; thereby producing a mitochondrial extract.

[0056] In some embodiments, the method further comprises the step of preserving the produced mitochondrial extract. In some embodiments, the preservation technique is selected from the group consisting of: cryopreservation, thawing, freezing, vitrification, dry state preservation, cold storage, ambient storage, encapsulation, embedding in a matrix, and encapsulation and embedding in a polymer. In some embodiments, the preservation is freezing. In some embodiments, the preservation is embedding. In some embodiments, the preservation is encapsulation. In some embodiments, the preservation is drying. In some embodiments, the preservation is freeze-drying.

[0057] In some embodiments, the method further comprises the step of providing the mitochondrial extract or the preserved mitochondrial extract to a subject in need thereof. In some embodiments, the providing is administering.

[0058] 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 culturing is suspension culturing. In some embodiments, the culturing is culturing in a tissue culture plate or well. In some embodiments, the culturing is culturing in a bioreactor.

[0059] 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 application. In some embodiments, the extract is at least 90% mitochondria. In some embodiments, the extract comprises a purity of at least 70, 75, 80, 85, 90, 95, 97, 99, or 100%. Each possibility represents a separate embodiment of the present application. In some embodiments, the extract comprises a purity of at least 90%.

[0060] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is obtained from a subject. In some embodiments, providing is obtaining. In some embodiments, obtaining is providing. In some embodiments, the cell is directly obtained from a subject. In some embodiments, the cell is differentiated from a stem cell. In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the embryonic stem cell is an umbilical cord blood-derived stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the stem cell is a hematopoietic stem cell (HSC). In some embodiments, the method further comprises extracting a primary cell from a subject. In some embodiments, the subject is a human. In some embodiments, the cell is from a healthy subject. In some embodiments, the subject does not have a mitochondrial disease. In some embodiments, the subject has a condition that can benefit from an increase in mitochondrial function.

[0061] In some embodiments, the cell is a primary cell. In some embodiments, the cell is not immortal. In some embodiments, the cell is not a cell of a cell line. In some embodiments, the cell is not transformed. In some embodiments, the cell is a healthy cell. In some embodiments, the cell is not cancerous. The term "primary cell" is well known in the art and refers to a cell taken directly from a living organism. In some embodiments, the cell is from a sample provided by a subject. In some embodiments, the method further comprises receiving a sample from a subject. In some embodiments, the method further comprises taking a sample from a 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 the group consisting of: blood, serum, plasma, gastric fluid, intestinal fluid, saliva, bile, breast milk, urine, interstitial fluid, cerebrospinal fluid, and fecal matter. In some embodiments, the fluid is blood. In some embodiments, the blood is peripheral blood. In some embodiments, the primary cell is obtained from a blood sample. In some embodiments, the method further comprises differentiating the obtained cell into a suspended hematopoietic cell. In some embodiments, the method further comprises differentiating an iPSC into a hematopoietic cell.

[0062] In some embodiments, the cell is a suspension cell. In some embodiments, the cell is a non-adherent cell. In some embodiments, the cell is grown in suspension. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the hematopoietic cell is a blood cell. In some embodiments, the hematopoietic cell is isolated from peripheral blood mononuclear cells (PBMCs). In some embodiments, the hematopoietic cell is differentiated from a stem cell. In some embodiments, the hematopoietic cell is selected from the group consisting of T cells, B cells, natural killer cells, and hematopoietic stem cells (HSCs). In some embodiments, the cell is an immune cell. In some embodiments, the hematopoietic cell is an immune cell. In some embodiments, the immune cell is a lymphoid cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the immune cell is selected from the group consisting of T cells, B cells, and NK cells. In some embodiments, the cell is a T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is an NK cell. In some embodiments, the cell is an HSC. In some embodiments, the cell is a human cell. In some embodiments, the cell is from a subject.

[0063] 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 homogenous 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, the enriched population comprises at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 99, or 100% homogenous cell type. Each possibility represents a separate embodiment of the application. In some embodiments, the purified population comprises 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 application. In some embodiments, the enriched or purified population comprises 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 2, 3, 4, or 5 different types of cells. Each possibility represents a separate embodiment of the application. In some embodiments, the cells are a mixture of B cells and another hematopoietic cell. In some embodiments, the cells are a mixture of B cells and another PBMC. In some embodiments, the cells are a mixture of T cells and another hematopoietic cell. In some embodiments, the cells are a mixture of T cells and another PBMC. In some embodiments, the cells are a mixture of NK cells and another hematopoietic cell. In some embodiments, the cells are a mixture of NK cells and another PBMC. In some embodiments, the cells are an immune cell mixture comprising at least 2, 3, 4, 5 different immune cells. Each possibility represents a separate embodiment of the application.

[0064] In some embodiments, the method comprises determining the cell type of the obtained population prior to expansion. In some embodiments, the method comprises isolating a specific cell type from the obtained population prior to expansion.

[0065] In some embodiments, the cells are expanded in solution. In some embodiments, the cells are expanded in culture. In some embodiments, expansion is culturing. In some embodiments, the cells are expanded for at least a predetermined time. In some embodiments, the cells are expanded for a time sufficient to produce 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 application. 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 7-14 days. In some embodiments, the time is between 7-13 days. In some embodiments, the time is between 7-10 days. In some embodiments, the time is between 10-14 days. In some embodiments, the time is between 10 and 13 days.

[0066] 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 cells provided. Each possibility represents a separate embodiment of the application. In some embodiments, the expanded population comprises at least 5 times the number of cells provided. In some embodiments, the expanded population comprises at least 10 times the number of cells provided. In some embodiments, the expanded population comprises at least 20 times the number of cells provided. In some embodiments, the expanded population comprises at least 30 times the number of cells provided. In some embodiments, the expanded population comprises at least 100 times the number of cells provided. In some embodiments, the expanded population comprises at least 200 times the number of cells provided. In some embodiments, the expanded population comprises at least 400 times the number of cells provided. In some embodiments, the expanded population comprises at least 1000 times the number of cells provided. In some embodiments, the time is sufficient to produce at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 fold expansion of the provided cells. Each possibility represents a separate embodiment of the application. In some embodiments, the time is sufficient to produce at least 2 fold expansion of the provided cells. In some embodiments, the time is sufficient to produce at least 3 fold expansion of the provided cells. In some embodiments, the time is sufficient to produce at least 4 fold expansion of the provided cells. In some embodiments, the time is sufficient to produce at least 5 fold expansion of the provided cells. In some embodiments, the time is sufficient to produce at least 100 fold expansion of the provided cells. In some embodiments, the time is sufficient to produce at least 200 fold expansion of the provided cells. In some embodiments, the time is sufficient to produce at least 300 fold expansion of the provided cells. In some embodiments, the time is sufficient to produce at least 400 fold expansion of the provided cells. Cell counting and methods of determining the number of cells are well known in the art, and it is routine technique for the skilled artisan to count the number of cells initially plated (the provided cells) and the number of cells present after expansion (the expanded population).

[0067] In some embodiments, the cells are cultured in a culture medium. In some embodiments, the culture medium is a tissue culture medium. In some embodiments, the culture medium is a suspension cell culture medium. In some embodiments, the culture medium is an immune cell culture medium. In some embodiments, the culture medium is a chemically defined medium. In some embodiments, the culture medium is a T cell culture medium. General suspension cell culture media and specific T cell culture media 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 application include RPMI basal medium supplemented with 10% fetal bovine serum (FBS), CST™ OpTmizer™ T cell medium (ThermoFisher), TexMACS™ medium (Miltenyi Biotec), X-VIVO 15 (Lonza), and 4Cell NutriT medium (Gibco). In some embodiments, the culture medium is 4Cell NutriT medium. In some embodiments, the culture medium is an animal component-free medium. In some embodiments, the culture medium is a chemically defined medium.

[0068] In some embodiments, expanding comprises activating the cells. In some embodiments, expanding comprises activating the immune cells. Methods of activating immune cells are well known in the art, and any such method, reagent, or kit known for use in activation purposes can be used. In some embodiments, expanding comprises contacting the provided cells with an antigen presenting cell (APC). In some embodiments, expanding comprises contacting the provided cells with a component of an APC. In some embodiments, expanding comprises contacting the provided cells with a component that mimics the activity of an APC. In some embodiments, the component is an anti-CD2 antibody or antigen binding moiety thereof. In some embodiments, the component is an anti-CD3 antibody or antigen binding moiety thereof. In some embodiments, the component is an anti-CD28 antibody or antigen binding moiety 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 moiety thereof is a biotinylated antibody or antigen binding moiety thereof. In some embodiments, the avidin bead has a surface conjugated to a biotinylated antibody or antigen binding moiety thereof selected from the group consisting of anti-CD2, anti-CD3, anti-CD28, and combinations thereof. In some embodiments, expanding comprises contacting the provided cells with a factor secreted by an activated T cell. In some embodiments, the factor is interleukin 2 (IL-2). In some embodiments, expanding comprises contacting the provided cells with IL-2. In some embodiments, the IL-2 is present at a concentration of 10-600 IU / mL. In some embodiments, the 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 factor is IL-7 and IL-15. In some embodiments, expanding comprises contacting the provided cells with IL-7. In some embodiments, expanding comprises contacting the provided cells with IL-15. In some embodiments, expanding comprises contacting the provided cells with IL-7 and IL-15. In some embodiments, expanding comprises contacting the provided cells with an anti-CD3 antibody or antigen binding fragment thereof. In some embodiments, the antibody is an activating antibody. In some embodiments, expanding comprises contacting the provided cells with an anti-CD28 antibody or antigen binding fragment thereof. In some embodiments, expanding comprises contacting the provided cells with an anti-CD2 antibody or antigen binding fragment thereof. In some embodiments, 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, 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 factor in the culture is replaced every 1, 2, 3, 4, 5, 6, or 7 days.Each possibility represents a separate embodiment of the present application. In some embodiments, the factors in the culture are replaced every 3 days.

[0069] In some embodiments, the expanding comprises culturing. In some embodiments, the culturing is suspension culture. In some embodiments, the method comprises culturing the cell suspension in a gas permeable container. In some embodiments, the culturing is in a gas permeable container. In some embodiments, the culturing is in a culture bag. In some embodiments, the 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 for unrestricted exchange of gases to the suspension culture. In some embodiments, the gas permeable container allows for unrestricted access of 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 the cell suspension in a bioreactor. Examples of bioreactors include, but are not limited to, a Stirred Tank Bioreactor, a Wave-Mixed Bioreactor, a Fixed-Bed Bioreactor, a Microcarrier-Based Bioreactor, a Hollow Fiber Bioreactor, a G-Rex Bioreactor, and a Perfusion Bioreactor System.

[0070] 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, the 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 application. In some embodiments, the enriched population comprises at least 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 99, or 100% homogeneity in the population. Each possibility represents a separate embodiment of the application. In some embodiments, the pure population comprises at least 90% purity. In some embodiments, the enriched population comprises at least 90% homogeneity. In some embodiments, the purity and / or homogeneity is with respect to the type of cells present in the population. In some embodiments, the expanded population is a substantially pure population of a particular cell type. In some embodiments, the expanded population is a substantially homogeneous population 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, only the pure population is used for mitochondrial isolation after measurement.

[0071] In some embodiments, the isolating is isolating a cell type. In some embodiments, the isolating is isolating mitochondria. In some embodiments, the isolating is purifying. In some embodiments, the isolating is extracting. In some embodiments, the isolating comprises disrupting the membranes of the cells. In some embodiments, the isolating comprises lysing the cells. In some embodiments, lysing the cells produces a lysate. In some embodiments, the lysate is a cell lysate. In some embodiments, disrupting the membranes of the cells is lysing the cells. Methods of isolating mitochondria from cells are well known and any method of removing intact and functional mitochondria from cells can be used. For example, a commercially available mitochondria isolation kit is available from Miltenyi (130-094-532), but any commercially available kit can be used as part of the methods of the present application. In some embodiments, the lysing is substantially non-lysing of the mitochondria. In some embodiments, the membrane disruption is plasma membrane disruption. In some embodiments, the plasma membrane disruption is substantially non-disrupting of the mitochondrial membranes. In some embodiments, the disrupting the membranes is by nitrogen cavitation disruption. In some embodiments, the disrupting the membranes is by homogenization disruption. In some embodiments, the homogenization is homogenization with a homogenizer. In some embodiments, the homogenizer is a Dounce homogenizer. In some embodiments, the homogenization is Dounce homogenization. In some embodiments, the disrupting the membranes is by addition of a lysis buffer. In some embodiments, the lysis buffer comprises a detergent. In some embodiments, the lysis buffer lysis is by osmotic lysis. In some embodiments, the disrupting the membranes is by needle shearing. In some embodiments, the needle is a needle of about 30 gauge.

[0072] In some embodiments, isolating comprises centrifuging the cells to remove the culture medium. In some embodiments, the cells are washed. In some embodiments, washing is washing in a wash buffer. In some embodiments, the wash buffer is PBS. In some embodiments, centrifuging is at 300 g. In some embodiments, centrifuging 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 application. In some embodiments, the sufficient time is between 5-15 minutes. In some embodiments, the sufficient time is between 5-10 minutes. In some embodiments, the sufficient time is about 10 minutes. In some embodiments, the cells are suspended in a mitochondrial isolation buffer prior to lysis / membrane disruption. Options for isolation buffers include, but are not limited to, sugar, HEPES, Tris-HCl, EDTA, EGTA, magnesium chloride (MgCl2), 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 alkaline pH. In some embodiments, the isolation buffer comprises a pH of about 7.4. In some embodiments, the isolation buffer comprises bovine serum albumin (BSA) that is free of fatty acids. In some embodiments, the BSA concentration is about 0.5%. In some embodiments, isolating mitochondria comprises centrifuging the cell lysate. In some embodiments, centrifuging is to remove cellular debris.

[0073] 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 incubation. In some embodiments, the incubation is for a time sufficient for 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 the 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 separation. In some embodiments, the bead is separable on a column. In some embodiments, the bead is a streptavidin bead. In some embodiments, the streptavidin is streptavidin. In some embodiments, the separating comprises separating the solid support and any mitochondria bound thereto. In some embodiments, the separating further comprises eluting the mitochondria from the solid support. In some embodiments, the eluting is with 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 sufficiently high to elute the mitochondria from the antibody or antigen-binding fragment thereof.

[0074] In some embodiments, the isolating comprises centrifuging the lysate. In some embodiments, the centrifuging is at a speed sufficient to precipitate cellular debris from the lysate. In some embodiments, the centrifuging is for a time sufficient to precipitate cellular debris from the lysate. In some embodiments, the sufficient speed is about 3000 g. In some embodiments, the sufficient time is about 5 minutes. In some embodiments, the centrifuging is repeated to remove cellular debris. In some embodiments, the centrifuging 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 centrifuging is at a speed sufficient to precipitate mitochondria. In some embodiments, the centrifuging is for a time sufficient to precipitate mitochondria. In some embodiments, the sufficient speed is about 12,000 g. In some embodiments, the sufficient time is about 10 minutes. In some embodiments, the precipitating mitochondria comprises producing a mitochondrial pellet. In some embodiments, the precipitating mitochondria comprises producing a mitochondrial pellet. In some embodiments, the pellet is a pellet. In some embodiments, the isolating further comprises resuspending the mitochondrial pellet in an isolation buffer.

[0075] 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 application. In some embodiments, the function includes ATP production. In some embodiments, the function includes oxygen consumption. In some embodiments, the function includes membrane potential. In some embodiments, the function includes citrate synthase. Methods of testing the functionality of mitochondria, including testing the functions listed above, are well known in the art and are also described below. Thus, the skilled artisan is fully capable of determining 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, the cell lysate is tested. In some embodiments, the isolated mitochondria are tested. In some embodiments, the intact cell is tested. In some embodiments, the test determines the quality of the mitochondrial extract. In some embodiments, the test comprises a citrate synthase assay. In some embodiments, the test comprises a JC-1 assay. In some embodiments, the test comprises determining the copy number of mitochondrial DNA (mtDNA). In some embodiments, quantification of mitochondria in the extract is quantified by mtDNA copy number. In some embodiments, quantification of mitochondria in the extract is quantified by the total amount of protein present after mitochondrial isolation / extraction.

[0076] In some embodiments, the isolated mitochondria comprise 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 100 million cells provided. Each possibility represents a separate embodiment of the application. In some embodiments, the isolated mitochondria comprise at least 20 ug of protein per 100 million cells provided. In some embodiments, the isolated mitochondria comprise at least 50 ug of protein per 100 million cells provided. In some embodiments, the isolated mitochondria comprise at least 80 ug of protein per 100 million cells provided. In some embodiments, the isolated mitochondria comprise at least 100 ug of protein per 100 million cells provided. In some embodiments, the isolated mitochondria comprise at least 110 ug of protein per 100 million cells provided. In some embodiments, the isolated mitochondria comprise at least 200 ug of protein per 100 million cells provided. In some embodiments, the isolated mitochondria comprise at least 220 ug of protein per 100 million cells provided. It will be appreciated that the amount of protein present in the extract is directly 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 present at a concentration that is directly 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 100 million cells provided. Each possibility represents a separate embodiment of the application. In some embodiments, the concentration of the isolated mitochondria is at least 8 ug / ml. In some embodiments, the concentration of the isolated mitochondria is at least 16 ug / ml.

[0077] Methods of determining protein concentration and total protein mass are 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 blot, immunostaining, Bradford assay, and protein array. In some embodiments, the amount of protein present is determined by a Bradford assay.

[0078] In another aspect, a mitochondrial extract is provided that is produced by the methods of the application.

[0079] In another aspect, a mitochondrial extract is provided that is from a population of hematopoietic cells.

[0080] In another aspect, a mitochondrial extract is provided that is from a pure population of hematopoietic cells.

[0081] In another aspect, a mitochondrial extract is provided that is pure mitochondria from hematopoietic cells of a single cell type.

[0082] In some embodiments, the mitochondrial extract is a suspended mitochondrial extract. In some embodiments, the mitochondrial extract is from a suspended cell. In some embodiments, the population of hematopoietic cells is a suspended population of hematopoietic cells. 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.

[0083] 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 hematopoietic 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% hematopoietic cell mitochondria. Each possibility represents a separate embodiment of the application. In some embodiments, the extract is immune 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% immune cell mitochondria. Each possibility represents a separate embodiment of the application. 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 application. 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 application. 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 application. 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 application. In some embodiments, the extract is at least 90% HSC mitochondria. In some embodiments, the extract is at least 95% HSC mitochondria.

[0084] 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 ug / ml. Each possibility represents a separate embodiment of the application. In some embodiments, the concentration is per 100 million cells provided. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 8 ug / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 16 ug / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 100 ug / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 110 ug / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 200 ug / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of at least 220 ug / 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 ug / ml. Each possibility represents a separate embodiment of the application. In some embodiments, the mitochondrial extract comprises a protein concentration of about 16 ug / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of about 110 ug / ml. In some embodiments, the mitochondrial extract comprises a protein concentration of about 220 ug / ml.

[0085] In another aspect, a composition is provided comprising a mitochondrial extract of the application.

[0086] 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 is free of 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, effective is effective to treat a mitochondrial-related disease. In some embodiments, the composition is used to treat a subject. In some embodiments, the composition is used to treat a condition that benefits from increased mitochondrial function. In some embodiments, the composition is used to treat a mitochondrial-related disease. In some embodiments, the composition is used to treat a disease or condition in a subject in need thereof. In some embodiments, treating a mitochondrial-related disease comprises manufacturing a medicament for treating a mitochondrial-related disease.

[0087] As used herein, the term "carrier," "excipient," or "adjuvant" refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid, liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or just a sterile aqueous medium, such as saline. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars, such as mannose, lactose, glucose and sucrose, starches, such as corn starch and potato starch, cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients, such as cocoa butter and suppository waxes; 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; buffering agents, such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can be used as carriers herein include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifying agents, and other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives can 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 of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the entire contents of each of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents for use in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulation and administration procedures, are well known in the art and are described in standard textbooks, such as 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 hereby incorporated by reference in its entirety. The compositions described herein can also be contained in artificially created structures such as liposomes, ISCOMS, slow release particles, and other vehicles that increase the half-life of a peptide or polypeptide 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 formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and a steroi, such as cholesterol. The choice of lipid is typically dependent on considerations such as the size and stability of the liposome in the blood. For example, Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, reviews various methods that can be used to prepare liposomes, and see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0088] The mitochondria of the present application can be embedded or encapsulated in a biocompatible natural polymer (such as a gel or hydrogel, for example, hyaluronic acid, chitosan, heparin, alginate, fibrin, collagen, chondroitin sulfate, or silk). The hydrogel can be further formulated with a reverse thermal gelation agent 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 embed or formulate the mitochondria for injection into a tissue. In another embodiment, the polymer or gel can be a combination of a natural polymer and a synthetic polymer, and in another embodiment, the gel or polymer can be cross-linked, and can also be chemically modified to include additional functional groups such as thiols. In some embodiments, the mitochondria are encapsulated or embedded in a biocompatible polymer. In some embodiments, the polymer is a natural polymer. In some embodiments, the polymer is a man-made polymer.

[0089] In another embodiment, the mitochondria of the present application are encapsulated with a particle. In some embodiments, what is encapsulated with the particle is the mitochondria. 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 the mitochondria for delivery.

[0090] In another embodiment, the mitochondria of the present application can be frozen or lyophilized.

[0091] The carrier can comprise from about 0.1% to about 99.99999% by weight of the pharmaceutical composition presented herein.

[0092] In some embodiments, the composition is configured 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 having a mitochondria-related disease. In some embodiments, the subject is a subject having a disease that can be treated 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 local administration.

[0093] As used herein, the terms "administering," "administration," and like terms mean any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in the context of reasonable medical practice. One aspect of the present subject matter provides intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration can include intravascular, intrathecal, intracranial, parenteral, subcutaneous, oral, topical, inhalation, intramuscular, intraocular, or intraperitoneal.

[0094] The dose administered will depend on the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0095] In some embodiments, the composition is formulated for in vitro administration to a cell. In some embodiments, the in vitro administration is in vitro transfection to a cell. In certain embodiments, the in vitro administration is into a cell. In some embodiments, the into a cell is into the cytoplasm of a cell.

[0096] In another aspect, a cell is provided, comprising a mitochondrial extract of the present invention.

[0097] In some embodiments, the cell is a recombinant cell. In some embodiments, the cell is not genetically modified. In some embodiments, the cell is of the same species as the mitochondria. In some embodiments, the cell is of the same cell type as the cell type from which the mitochondrial extract was produced. In some embodiments, the cell is of a different cell type than the cell type from which the mitochondrial extract was produced. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is not a hematopoietic cell. In some embodiments, the cell is a cell for adoptive cell transfer (ACT). In some embodiments, the cell is a cell from a subject. In some embodiments, the cell is allogeneic to a subject. In some embodiments, the cell is syngeneic to a subject. In some embodiments, the cell is autologous to a subject.

[0098] 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, being substantially depleted comprises less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1% of the endogenous mitochondria present in the cells. Each possibility represents a separate embodiment of the application. In some embodiments, being substantially depleted comprises less than 20% of the endogenous mitochondria present in the cells.

[0099] In another aspect, a composition comprising the cells of the application is provided.

[0100] 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 configured for administration to an organ comprising cells of the same cell type as the cells of the application. In some embodiments, the composition is for use in ACT.

[0101] In another aspect, a method of treating a subject in need thereof is provided, the method comprising administering to the subject a composition of the application, thereby treating the subject.

[0102] In some embodiments, the subject has 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 mitochondria-related disease or condition. In some embodiments, the mitochondria-related disease or condition is a mitochondrial disease or condition. In some embodiments, the disease or condition is treatable by the compositions of the present application. 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 growth 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 neurophtalmic disease or condition. In some embodiments, the disease is an ophthalmic disease or condition. In some embodiments, the disease is an ocular disease or condition. Non-limiting examples of ophthalmic diseases and conditions include: Dominant 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), Kearns-Sayre Syndrome (KSS). In some embodiments, the ocular disease is LHON. In some embodiments, the ocular disease is MELAS. In some embodiments, the disease is a muscle disease or condition. In some embodiments, the disease is a disease treatable by mitochondrial enrichment 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 enrichment therapy. In some embodiments, the mitochondrial replacement and / or mitochondrial enrichment 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.

[0103] 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 nonalcoholic fatty liver disease (NAFLD). In some embodiments, the fatty liver disease is steatotic live disease. In some embodiments, the NAFLD is metabolic dysfunction associated steatotic live 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 seizure. 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 cardiac arrhythmia. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is 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 inherited mitochondrial disorder. In some embodiments, the inherited disorder is primary mitochondrial myopathy. In some embodiments, the inherited disorder is mitochondrial encephalopathy. In some embodiments, the inherited disorder is mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. In some embodiments, the inherited disorder is myoclonic epilepsy with ragged red fibers (MERRF). In some embodiments, the inherited disorder is neuropathy, ataxia, and retinitis pigmentosa (NARP) syndrome. In some embodiments, the inherited 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 improving at least one symptom of aging. 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, the cognitive aging comprises dementia.

[0104] In some embodiments, the disease is not a local disease. In some embodiments, the disease is a local 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 the myocardium. In some embodiments, the disease is not a disease that can be treated by mitochondria in a total amount of 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 application. In some embodiments, the disease is not a disease that can be treated by mitochondria in a total amount of 12 mg. In some embodiments, the disease is not a disease that can be treated by mitochondria in a total amount of 17 mg. In some embodiments, the disease is not a disease that can be treated by mitochondria in a total amount of 50 mg. In some embodiments, the disease is a disease that can only be treated by mitochondria in a total amount of 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 application. In some embodiments, the disease is a disease that can only be treated by mitochondria in a total amount of 16 mg. In some embodiments, the disease is a disease that can only be treated by mitochondria in a total amount of 100 mg. In some embodiments, the disease is a disease that can only be treated by mitochondria in a total amount of 200 mg. In some embodiments, only treatable is only effectively treatable. In some embodiments, only treatable is that a clinical benefit can only be achieved by the recited amount of mitochondria. In some embodiments, the total amount is the total amount of a single dose. In some embodiments, the total amount is the total amount of all doses.

[0105] 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.

[0106] 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.

[0107] In some embodiments, the T cells, B cells, NK cells, or HSCs are isolated from a blood sample. In some embodiments, the T cells, B cells, NK cells, or HSCs are isolated from hematopoietic cells. In some embodiments, the T cells are isolated from a blood sample. In some embodiments, the T cells are isolated from hematopoietic cells. Methods of T cell isolation 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).

[0108] In some embodiments, the mitochondrial extract is produced from a blood sample. In some embodiments, the mitochondrial extract is produced from hematopoietic cells. In some embodiments, the mitochondrial extract is produced by the methods of the present application. 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.

[0109] In some embodiments, the produced mitochondrial extract is administered to a subject. In some embodiments, the administration is systemic. In some embodiments, the administration is intravenous. In some embodiments, the administration is local. In some embodiments, the administration is intravenous. In some embodiments, the administration is intraocular. In some embodiments, the administration is to the site of disease. Those skilled in the art will appreciate that by using the subject's own blood cells as a source of mitochondria after expansion, a cheap, non-immunogenic and inexhaustible source of high quality mitochondria is found. In the case of a subject having a genetic mitochondrial disease or in the case of a subject whose all mitochondria are affected, it will be necessary to produce the extract from donor blood cells. However, many diseases characterized by mitochondrial dysfunction are characterized only by dysfunction in a subset of the subject's diseased cells (e.g., liver cells, pancreatic cells), while the mitochondria in the subject's blood cells will be healthy and can be used for treatment.

[0110] As used herein, the terms "treatment" or "treating" of a disease, disorder, or condition includes alleviating at least one symptom, reducing the severity, or inhibiting the progression thereof. Treatment does not necessarily indicate complete cure or complete resolution of the disease, disorder, or condition. To be therapeutically effective, 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 improve the quality of life of the patient or subject.

[0111] As used herein, the term "about," when used in conjunction with a value, means plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) means a length of 1000 nm+100 nm.

[0112] 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, reference to "a polynucleotide" includes a plurality of such polynucleotides, and reference to "a polypeptide" includes one or more polypeptides known to one skilled in the art, and equivalents thereof, and so forth. It should also be noted that the claims can be drafted to exclude any optional element. Thus, for example, in the claims, the use of "a" or "an" can be construed to mean "one or more." By using "a" or "an" it is intended that the claims include at least one, but it does not exclude more than one. In this document, the term "essentially" to refer to inclusions, exclusions, or options within the specified limits or ranges such that other instances outside of those limits are not typically used.

[0113] In those instances using coordination of similar to "at least one of A, B, and C," such a structure typically intends that either A or B or C be present but not a combination of these items, unless explicitly defined otherwise or allowed by context. In those instances using coordination of similar in the sense of "at least one of A, B, and C, or any combination thereof," such a structure means that one or more of A or B or C is present. In those instances using coordination of similar in the sense of "one or more of A, B, and C, or any combination thereof," such a structure means that one or more of A or B or C is present.

[0114] It should be understood that certain features of the application, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the application, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable

[0115] Additional objects, advantages, and novel features of the application will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, as described above and as claimed below, each of the various embodiments and aspects of the application are supported experimentally in the following examples.

[0116] As described above and as claimed below, each of the various embodiments and aspects of the application are supported experimentally in the following examples.

[0117] Example Generally, the nomenclature used herein and the laboratory procedures in the application include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" 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 (1998); methodologies as set forth in U.S. Pat. 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, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al.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.

[0118] Materials and Methods Isolation of PBMC from blood samples : The blood sample bag was washed with ethanol and 13 mL blood was transferred into 17 mL RPMI medium and mixed. 13 mL Ficoll was added to a new tube and the blood mixture was added to this tube while keeping it at 45°C. At room temperature (RT, 25°C) the Ficoll blood tube was centrifuged at 800 g for 30 min at slow acceleration and then break OFF. The PBMC layer (a whitish layer formed in the interphase between plasma and Ficoll) was transferred to a new 50 mL tube and the tube was filled to the 45 mL mark with 37°C RPMI. The PBMC were centrifuged at 330 g for 10 min at RT, the supernatant was discarded, the cells were resuspended by tapping the tube until no clumps were visible, 5 mL RPMI was added, the cells were counted and resuspended in freezing medium (freezing with) at a concentration of 50 X 10Λ6 cells / mL or resuspended in AutoMacs Running buffer (Miltenyi) for T cell isolation.

[0119] Isolation of T cells from PBMC : Isolation of CD3 positive T cells from PBMC was performed according to the protocol of the human Pan T cell isolation kit (Pan T Cell Isolation Kit human, Miltenyi) with several adaptations.

[0120] To prepare the MACSiBead particles 200 μL CD2-biotin, 200 μL CD3-biotin and 200 μL CD28-biotin were added to the tube and mixed, then 1 mL anti-biotin MACSiBead particles and 400 uL AutoMacs Running buffer (Miltenyi) were added and incubated for 2 hours at 4°C with gentle rotation.

[0121] To isolate T cells, suspend PBMC in AutoMacs Running Buffer (40 μΐ Buffer / 10 X 10Λ6 total cells) and add Pan T Cell Biotin-antibody cocktail (10 μΐ / 10 X 10Λ6 total cells) followed by a 5 minute cold incubation (2-8°C). Next, add Pan T Cell Microbead cocktail (20 μΐ / 10 X 10Λ6 total cells) and incubate for 10 minutes cold (2-8°C).

[0122] Place the LS column with the mesh on top of the magnetic field of a suitable MACS separator, the top of the tube, and rinse with 3 mL buffer, then change to a new tube. Apply the cell suspension to the mesh, and collect the flow-through containing the unlabeled cells, representing the enriched T cells. This is followed by a wash of the column with 9 mL buffer and collect the flow-through into the same tube. Remove the column from the separator and place it on a new tube, then pipette 5 mL buffer and flush the magnetically labeled non-T cells by pushing the plunger into the column.

[0123] T cell activation and expansion : Plate cells in G-REX 24-well plates or G-REX 6 plates in suspension cell media (4Cell NutriStem® media (Gibco)) with or without beads coated with anti-human CD2, CD3, and CD28 antibodies to mimic antigen presenting cells, and with IL-2 as an activator. Incubate cells in a 37°C incubator with 5% CO2for 14 days. Update media with IL-2 (6 mL in 8 mL) on day 4 and update and top up to 8 mL on days 7, 9, and 11. In G-REX 6-well plates, seed 1-5 x 10Λ6 cells per well and add 100 mL media. Incubate cells in a 37°C incubator with 5% CO2. Do not change media and add IL-2 to the wells every 3 days. Perform cell counts on days 7, 9, 11, and 14.

[0124] Use the Human T Cell Activation / Expansion Kit (Miltenyi Biotec, Cat. No. 130-091-441) and MACS Buffer (Miltenyi, Cat. No. 130-091-221) to prepare MACSiBead particles (beads coated with anti-human CD2, CD3, and CD28 antibodies to mimic antigen presenting cells). Follow the manufacturer's instructions for this procedure.

[0125] Amount of mitochondria : Isolate T cells by using the Pan T Cell Isolation Kit, human (Miltenyi Biotec, Cat. No. 130-095-976) according to the manufacturer's instructions. BCAReagent test mitochondrial purified fraction protein concentration to determine relative amount of mitochondria, normalized to total number of cells used to produce mitochondria (initial cell plating).

[0126] Western blot Total protein was extracted by lysing cells with RIPA lysis buffer supplemented with protease inhibitors, incubated at 4°C for 30 min, then centrifuged at 14,000 g and 4°C for 10 min, and the supernatant was collected. Isolated mitochondria were used as mitochondrial sub-fractions. The supernatant above the mitochondrial pellet was used as the cytosolic sub-fraction. Protein concentration was determined by Bradford assay. Samples were separated by SDS-PAGE gels, then proteins were electrotransferred to Immobilon-P transfer membranes. The membranes were then blotted with anti-pyruvate dehydrogenase El alpha and anti-alpha-tubulin.

[0127] ATP test ATP in expanded T cells was tested with the ATPlite 1-step kit (PerkinElmer) according to the manufacturer’s instructions. Briefly, cells were moved into a 96-well plate in a total volume of 100 uL, and 100 uL of kit reagent was added. Luminescence of whole cell lysates was measured with a plate reader.

[0128] Oxygen consumption rate Oxygen consumption of expanded T cells was tested with the MitoXpress Xtra Oxygen Consumption Assay Kit (Aligent) according to the manufacturer’s instructions. Briefly, cells were moved into a 96-well plate, then kit reagent was added, the wells were sealed with oil, and fluorescence (Ex / Em 380 / 650) was recorded with a plate reader. Assays were performed on isolated mitochondria or intact cells.

[0129] Membrane potential Membrane potential was tested using the JC-1 reagent (Sigma) according to the manufacturer’s instructions. Briefly, JC-1 was added to cells (5 uM) for 30 min, then washed 3 times, and fluorescence of cells was recorded with a plate reader at Ex / Em 550 / 600 and 485 / 535. Assays were also performed on isolated mitochondria.

[0130] Citrate synthase activity Determination of mitochondrial citrate synthase (CS) activity in samples (isolated mitochondria or whole cell / cell lysates) was performed via an immuno-capture based assay. The principle of the assay is to capture the enzyme within the wells of a microplate, then determine activity by recording TNB color development, which is generated from DTNB produced in the citrate synthesis reaction. The entire reaction product TNB absorbs at 412 nm. The reaction was performed as follows: Oxaloacetate + Acetyl CoA + H20 -> Citrate + CoA-SH + H+ CoA-SH + DTNB -> TNB + CoA-S-S-TNB (412 nm absorbance increase) Antibody ab 119692 specifically immunocaptured only native citrate synthase from the applied test sample. In general, this immuno-capture based activity assay allows for the measurement of citrate synthase activity with simple sample preparation without the need for mitochondrial isolation.

[0131] Isolation of mitochondria : Cells were centrifuged at 300 X g for 10 minutes, washed with PBS, re-centrifuged, resuspended in mitochondrial isolation buffer (320 mM sucrose, 5 mM Tris-HCl, pH 7.4, 2 mM EGTA, with or without BSA (0.5%) without fatty acids, protease inhibitor cocktail, and PMSF) and homogenized with a Dounce homogenizer, sheared through a 30 G needle (10 passes of cells through the needle) or homogenized by nitrogen cavitation. Nitrogen cavitation was performed using a disruption vessel (Parr Instruments) at 800 psi for 20 minutes. The cell homogenate was centrifuged at 3000 X g for 5 minutes, the supernatant was collected and centrifuged again at 3000 X g for 5 minutes, and the supernatant was then centrifuged at 12,000 X g for 10 minutes. The mitochondrial pellet was resuspended in mitochondrial isolation buffer. Mitochondrial concentration was determined by Bradford assay.

[0132] 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 with the kit isolation buffer, anti-TOM22 microbeads were added, and the suspension was incubated for 1 hour with gentle rocking in the refrigerator (2-8°). The suspension was then applied to the kit column, washed with isolation buffer, and mitochondria were obtained by removing the column from the isolator, adding mitochondrial isolation buffer, and pushing the plunger into the column. Mitochondrial concentration was determined by Bradford assay.

[0133] Liver analysisLiver samples from 36 mice were harvested, fixed in 4% formaldehyde and stored for 48 hours for further fixation. Then, the tissues were trimmed, put in embedding cassettes and processed routinely for paraffin embedding. Two cassettes were prepared per animal. Paraffin sections (4 pm thick) were cut, mounted on glass slides and stained with hematoxylin & eosin (H&E) for general histological examination. In addition, cryostat sections (8 pm thick) of liver samples (n = 36) were prepared using a cryostat and stained with oil red O for detection of triglycerides. The slides were evaluated histopathologically by a pathologist from PATHO-LOGICA Ltd.

[0134] Photographs were taken using an Olympus microscope (BX60, serial number 7D04032) together with the microscope's camera (Olympus DP73, serial number OH05504) at objective magnifications of X1.25, X4 and X10.

[0135] H&E stained sections were examined, described and scored by the study pathologist using a semi-quantitative grading scale (5-point scale) to determine the severity of histopathological changes (the scale has been published in Schafer et al., "Use of severity grades to characterize histopathologic changes", Toxicologic Pathology 2018, 46:256-265, the content of which is incorporated herein by reference in its entirety). The scale was as follows: Grade 0 - the tissue appeared normal with no changes; Grade 1 - minimal pathologic findings; Grade 2 - mild pathologic findings; Grade 3 - moderate pathologic findings; Grade 4 - severe pathologic findings.

[0136] Oil red O staining of lipid droplets in hepatocytes was scored as follows: Grade 0 = normal liver with no lipid accumulation; Grade 1 = very mild accumulation of lipid droplets; Grade 2 = mild accumulation of lipid; Grade 3 = moderate accumulation of lipid; Grade 4 = marked accumulation of lipid.

[0137] Statistics Statistical analysis was performed using one-way ANOVA followed by Tukey HSD. p<0.05 was considered significant.

[0138] Measurement of membrane potential by 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 a potential-dependent accumulation in mitochondria, manifested as a shift from green fluorescence (monomer) to red fluorescence (aggregate). Mitochondria were isolated from T cells and resuspended in isolation buffer at a protein concentration of 1 mg / ml, determined by BCA assay.

[0139] Isolated mitochondria were incubated with JC-1 dye at a final concentration of 0.2 μg / ml for 30 minutes at 37°C in assay buffer to allow for dye uptake and equilibration. Following incubation, the fluorescence intensity of JC-1 monomer and aggregate 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.

[0140] Example 1: Expansion of primary T cells for mitochondrial isolation To produce large quantities of functional, therapeutic mitochondria from cells grown in culture, it was decided to optimize the production / extraction of mitochondria from primary T cells. In immortalized cells, the viability and integrity of mitochondria is compromised, and mitochondrial damage is a hallmark of cancer cells, thus primary cells were used, although these cells are generally much more difficult to culture and expand. Hematopoietic cells such as T cells, natural killer (NK cells), B cells, and hematopoietic stem cells (HSCs) have the benefit that they grow in suspension and do not attach to plates. This allows for culturing much larger numbers of cells in the same amount of culture medium as would be possible for adherent cells.

[0141] T cells were isolated from human PBMCs as described above (Materials and Methods). Flow cytometry was performed to confirm the homogeneity of the T cell population, and cultures were seeded with greater than 90% T cells. Isolated T cells were then expanded in Gas-Permeable Rapid Expansion (G-REX) plates for 2 weeks. These plates are designed specifically for suspension cells. The G-REX setup provides unlimited access to nutrients without mixing (reducing contact with cells, which can cause mitochondrial damage). Specifically, oxygen is accessed essentially unlimitedly due to the gas-permeable membrane increasing diffusion of oxygen throughout the culture medium. Flow cytometry was also performed after expansion, and the population was found to be greater than 97% pure.

[0142] Cell number and viability were monitored over the two weeks Figure 1). Cell numbers were found to peak at 10 days, with 400 X 10Λ6 cells per 6 well present. This is an increase of about 80-fold when the initial plating concentration was 5 X 10Λ6 cells / 6 well. Cell viability, as assessed by trypan blue staining, was also still high at day 10, but dropped dramatically by day 14.

[0143] Example 2: Mitochondrial evaluation and isolation Next, mitochondria were isolated from the expanded T cells. It was important that the isolation process not be overly harsh, as this would risk damaging / breaking the mitochondria. Two methods of cell disruption were tested: Dounce homogenization and nitrogen cavitation. Both methods are known in the art, and are generally considered to be equivalent for adherent cells. However, adherent cells must first be trypsinized from the plate, and thus are already somewhat damaged / broken. Therefore, it was not clear whether these procedures for adherent cells would be equivalent to those for suspension cells. Results are summarized in Table 1.

[0144] Table 1: Summary of mitochondrial isolation from expanded T cells

[0145] Both methods produced robust yields of intact mitochondria. These yields were far higher than those produced from adherent cells. The ratio of even 220 ug of mitochondria from 1 million starting primary suspension cells is not known in the art, and 90 or 110 ug certainly far exceeds what can currently be produced from such a small starting number of cells. Of course, these amounts of mitochondria cannot be produced when seeding similar initial amounts of primary adherent cells. The cultures were also performed in bioreactors, rather than in tissue culture cells. From just 10Λ6 starting T cells, the bioreactor yields reached and exceeded 1000 ug of protein.

[0146] The quality of the mitochondria from the expanded T cells was evaluated. This included measuring oxygen consumption, membrane potential, and citrate synthase (CS) activity. The mitochondria isolated from the T cells were found to be functional, and within expected ranges for all of these assays. CS activity was measured at three time points during T cell expansion, and was found to be equivalent at all time points, indicating that prolonged expansion did not negatively impact mitochondrial quality (Table 2). Figure 2 and Table 2).

[0147] Table 2: Average CS activity over time Culture day (T cells) CS activity (nmol / min / μL) 7 0.032 10 0.029 13 0.028 By isolating such a large number of mitochondria, it is feasible to produce therapeutic compositions with very high concentrations of mitochondria. Mitochondria were isolated from proliferating T cells at day 8 post-seeding. Isolated mitochondria were tested for oxygen consumption rate (OCR) at four concentrations: 16 ug / mL, 8 ug / mL, 4 ug / mL, and 2 ug / mL in the same total volume. Results are presented in Figure 3 Throughout the experiment, the substrate glutamate / malate levels were kept constant, which allows for a direct comparison of mitochondrial activity based on quantity alone. Basal respiration levels were recorded initially. Addition of ADP triggers oxidative phosphorylation - evidenced by an increase in OCR - indicating that mitochondria are producing ATP. This response is concentration dependent - with the 16 ug / mL preparation showing the most significant increase - indicating that increasing mitochondrial density results in higher metabolic activity.

[0148] Introduction of oligomycin resulted in a decrease in OCR at all concentrations, consistent with its role as an ATP synthase inhibitor. This indicates that the previous 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 inner mitochondrial membrane, resulting in maximal electron transport chain activity without ATP production. The peak OCR - especially at the highest mitochondrial concentration - verifies electron transport capacity and indicates robust inner membrane integrity and function. Finally, addition of the complex I inhibitor rotenone resulted in a decrease in OCR in all samples, confirming the role of complex I in the measured respiratory activity.

[0149] Example 3: Mitochondria retain their functionality after freezing The following isolated mitochondria were tested for membrane potential using the JC-1 dye: freshly isolated mitochondria stored directly at 4°C after isolation, isolated mitochondria stored at 4°C for 4 days, isolated mitochondria stored at -80°C for 4 days. Fresh mitochondria intentionally damaged by several freeze / thaw cycles were used as a negative control.

[0150] The ratio of red to green fluorescence intensity was calculated for each sample, which serves as an indication of mitochondrial membrane potential. An increase in the red / green fluorescence ratio indicates that JC-1 aggregation within the mitochondria results in an increase in membrane potential, while a decrease in this ratio indicates that the mitochondria are depolarized, associated with a loss of membrane potential. It can be seen from Figure 4A that when mitochondria are stored at 4°C for 4 days, the membrane potential is greatly reduced, however, 4 days of freezing does not have a significant effect on the membrane potential of mitochondria after thawing. Intentionally damaged mitochondria do not show substantial membrane potential.

[0151] For controls, mitochondria were treated with the potassium ionophore, valinomycin (0.5 µM) to dissipate the membrane potential prior to the addition of the JC-1 dye. This served as a depolarized positive control, and as expected, all valinomycin-treated mitochondria showed little to no membrane potential. Oligomycin (1 µg / ml) is an ATP synthase inhibitor used to inhibit proton flow back into the mitochondria and served as a control to assess the coupling efficiency of the electron transport chain. Blocking ATP synthase results in an increase in the proton gradient (i.e., an increase in membrane potential). Fresh mitochondria treated with oligomycin did show the expected increase in membrane potential, while mitochondria stored at 4°C for 4 days were essentially unresponsive to oligomycin, indicating that these mitochondria were non-functional. Importantly, mitochondria stored at -80°C and then thawed also showed an increase in membrane potential upon oligomycin treatment, indicating that -80°C storage did not significantly impact membrane potential maintenance.

[0152] Next, to confirm the functionality of the isolated mitochondria stored at -80°C for 4 days, the oxygen consumption rate was measured in a 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.

[0153] 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 under basal non-phosphorylating conditions (State 4). High RCR values indicate efficient coupling of electron transport to ATP synthesis, reflecting healthy mitochondrial function, while low RCR values indicate mitochondrial dysfunction or uncoupling. The RCR was calculated to be 5 for fresh mitochondria, while mitochondria held at 4°C had an RCR of only 1.6. In contrast, frozen mitochondria had an RCR of 4.2, indicating that they still had high functionality and activity.

[0154] Example 4: Treatment of Diabetes and NAFLD in a Mouse Model Type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) are associated with mitochondrial dysfunction and oxidative stress. In diabetic patients, hyperglycemia enhances the production of reactive oxygen species (ROS) in mitochondria, triggering oxidative damage and impaired insulin signaling. Mitochondrial biogenesis also regulates energy balance, and excess ROS production under high glucose can exacerbate vascular complications. In non-alcoholic fatty liver disease (NAFLD), mitochondrial dysfunction drives abnormal hepatic lipid metabolism and oxidative stress. Lifestyle interventions such as diet, exercise, and antioxidants provide limited protection because mitochondrial DNA and proteins are irreversibly damaged. Replacing and / or fortifying dysfunctional mitochondria with healthy ones could be a promising approach to treat these diseases. Thus, the ability of a mitochondrial composition produced by T cells to treat both diabetes and NAFLD was tested in a mouse model.

[0155] Healthy male C57BL / 6J mice weighing 18-22 g were used in the study. The animals were housed under standard cage conditions with access to standard laboratory mouse chow and water. All animal experiments were performed in accordance with the guidelines.

[0156] C57BL / 6J mice were randomly divided into 6 groups (n = 6 each). The experimental groups are described in Table 3. Mice in groups A and D were fed a standard chow diet (approximately 6% fat content) and served as normal controls, while the other three groups (B, C, E) of mice received intragastric administration of a high lard fat and high cholesterol diet (where 60% of the total calories come from fat).

[0157] Table 3: Mouse experimental groups

[0158] After 8 weeks, mice in groups D and E were injected intravenously with a healthy mitochondrial preparation isolated from human T cells (0.5 mg / kg body weight, e.g., >9 mg mitochondria per injection), once every three days for a total of six administrations. Mice in group C received only half the treatment, with one dose administered every three days, but only for a total of 3 treatments. These mice represent the best possible with a more limited source of mitochondria, which was not completely dosed. For groups A and B, mice were administered an equal volume of saline intravenously. After the last mitochondrial treatment, all mice were fasted for 12 h and then euthanized by an overdose of sodium pentobarbital. Mouse serum and liver tissue were collected.

[0159] Groups A and D (healthy control animals) exhibited normal morphology with no pathological changes or lipid deposition (pathology severity score and lipid deposition were 0 for all animals) Figures 5A-5B ).

[0160] Group B (high fat diet, control) showed a pathological severity score and lipid deposition of 2.16 (p < 0.05) (Fig. 2). Figure 5C In addition to the large and microvesicular changes in hepatocytes, hepatocyte necrosis was also observed. H&E staining of the heart was also performed as a control, and all animals presented a pathological score of zero. Group E (high fat diet, mitochondria treatment) showed a significant improvement, with a clear reduction in the number and size of the vesicles within the hepatocytes and areas showing hepatocyte regeneration (p < 0.05) (Fig. 2). Figure 5D Oil red O staining strongly supported these findings. The pathological severity grade (H&E) and oil red O staining were 1.5 and 1.33, respectively. When only a half-dosing regimen was used (Group C), no improvement was observed (p < 0.05) (Fig. 2). Figure 5E Statistical tests showed a significant change between the pathological livers of Group B and E, but not between Group B and C.

[0161] When administered intravenously, high total amounts of mitochondria are necessary to produce a systemic effect. However, due to the incomplete recovery of lipid levels and histology to normal, even higher doses of mitochondria can be needed. To date, such large amounts of therapeutic mitochondria are not isolatable. 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 also not suitable for therapeutic administration. Prior to the methods of producing mitochondria provided herein, the only suitable method for producing therapeutic mitochondria was to grow adherent primary cells in culture or to use direct tissue biopsy to produce mitochondria. As outlined above, primary adherent cell culture produced cells are too few to produce such high concentrations and total amounts of mitochondria. Bharadwaj et al. teach the use of percutaneous needle biopsy to produce skeletal muscle tissue for therapeutic mitochondrial isolation (see 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 by reference in their entirety herein). As can be seen from Bharadwaj’s Figure 5, the highest total mitochondrial yield from a single biopsy was approximately 2 mg. The amount produced was even less (<1.5 mg) most of the time.

[0162] In the above experiment, 0.5 mg / kg mitochondria were administered 6 times. This is a 35 mg dose of mitochondria for a person of average 70 kg body weight. A person with diabetes or NAFLD can be much heavier. A single muscle needle biopsy cannot yield enough mitochondria for even one therapeutic dose, much less 6 (210 mg). Furthermore, if the subject’s diet does not change, it can be necessary to repeat such treatments. Thus, it is clear that muscle biopsy is not a viable source of therapeutic mitochondria. In fact, other than the T cell isolation method of the present invention, there is no robust source that yields enough mitochondria to produce the therapeutic effects demonstrated herein.

[0163] While the application has been described in connection with specific embodiments thereof, it will be understood that a number of alternatives, modifications and variations can be made thereto without departing from the spirit and scope of the application. Accordingly, it is intended to embrace all such alternatives, modifications and variations as fall within the spirit and broad scope of the appended claims.

Claims

1. A method for producing a mitochondrial extract, the method comprising: a. providing a suspension of primary human hematopoietic cells; b. expanding the primary human hematopoietic cells for a time sufficient to produce an expanded population of 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.

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

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 the group consisting of 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. The method of claim 5, wherein the primary human immune cells are selected from the group consisting of 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 time is from 7 to 14 days.

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

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

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

12. The method of claim 11, wherein the isolated mitochondria comprise at least 50 micrograms of protein per 1 million primary human hematopoietic cells provided.

13. The method of claim 12, wherein the isolated mitochondria comprise at least 80 micrograms of protein per 1 million primary human hematopoietic cells provided.

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

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 the activity of an APC, a factor secreted by an activated T cell, and any combination thereof.

16. The method of claim 15, wherein the provided primary human hematopoietic cells are primary T cells, and wherein the 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. The method of claim 16, wherein 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.

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

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

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

21. The method of any one of claims 1-20, wherein the isolating comprises lysing cells to produce 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.

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

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

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

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

26. The method of any one of claims 21-23, comprising centrifuging the lysate at about 3000 g to remove cell debris and produce a supernatant and centrifuging the supernatant at about 12,000 g to produce a mitochondria pellet.

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

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

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

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

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

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

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

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

36. Recombinant cells comprising the mitochondrial extract according to any one of claims 27 to 31.

37. The recombinant cell according to claim 36, wherein the endogenous mitochondria are depleted.

38. The recombinant cell according to claim 36 or 37, wherein the cell is a non-hematopoietic cell.

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

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

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

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

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

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

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

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

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