Methods and systems for developing media for extracellular vesicle production

Abstract

The present invention relates generally to methods and systems for analyzing, developing, testing and / or optimizing media and / or culture conditions for extracellular vesicle production; culture media that use such assays to analyze, develop, test and / or optimize; and extracellular vesicles produced using such a culture medium and extracellular vesicle-containing compositions comprising extracellular vesicles produced using such a culture medium.

Description

Technical Field

[0001] The present invention generally relates to methods and systems for analyzing, developing, testing and / or optimizing culture media (and / or culture conditions) for extracellular vesicle production. The present invention also relates to culture media analyzed, developed, tested and / or optimized by such extracellular vesicle characterization assays; and extracellular vesicles produced using such culture media and compositions containing extracellular vesicles. Background Art

[0002] Cells (including cells cultured in vitro or in vitro) secrete a variety of molecules and biological factors (collectively referred to as secretomes) into the extracellular space. See Vlassov et al. (Biochim Biophys Acta, 2012; 940-948). As part of the secretome, various bioactive molecules are secreted from cells in membrane-bound extracellular vesicles (such as exosomes). Extracellular vesicles can change the physiology of other cells by signaling or by transporting their cargo (including, for example, proteins, lipids, and nucleic acids). The cargo of the extracellular vesicles is encapsulated in a membrane that allows, in particular, specific targeting (e.g., targeting cells) by specific markers on the membrane; and increased stability during transport in biological fluids, such as through the bloodstream or across the blood-brain barrier (BBB).

[0003] Exosomes play a series of important physiological functions, for example, as molecular messengers that transmit information between different cell types. For example, exosomes deliver proteins, lipids, and soluble factors (including RNA and microRNA), which, depending on their origin, participate in signal transduction pathways that can affect apoptosis, metastasis, angiogenesis, tumor progression, thrombosis, and immunity by directing T cells to immune activation, immunosuppression, growth, division, survival, differentiation, stress response, apoptosis, etc. See Vlassov et al. (Biochim Biophys Acta, 2012; 940-948). Extracellular vesicles may contain a combination of molecules that can work synergistically to exert specific biological effects. Exosomes combine a wide range of cytosolic components and membrane components that reflect the properties of the mother cell. Therefore, the term applied to the cell of origin can be used as a simple reference to secreted exosomes in some cases.

[0004] Extracellular vesicles (such as exosomes) have considerable potential for use as effective cell-free therapies (with attendant benefits such as improved convenience, stability, and operator handling) for the treatment of a variety of diseases, including, for example, cancer, heart disease, and inflammation. However, there is a need for methods to increase the yield and / or quality of extracellular vesicles from extracellular vesicle-producing cells; as well as methods to establish and optimize production and purification processes for therapeutic extracellular vesicles. Summary of the invention

[0005] The present invention addresses the above limitations in the art by providing methods and systems for analyzing, developing, testing and / or optimizing culture media (and / or culture conditions) for extracellular vesicle production; culture media analyzed, developed, tested and / or optimized using such assays; and extracellular vesicles produced using such culture media and compositions containing extracellular vesicles produced using such culture media.

[0006] For example, in some embodiments, the present invention provides for one or more high-throughput methods for cell culture, media exchange, vesicle formation, analysis of cell growth and / or viability, analysis of extracellular vesicle production and secretion, and characterization of extracellular vesicles, thereby providing improved methods and systems for analyzing, developing, testing and / or optimizing culture media (and / or culture conditions) for extracellular vesicle production (such as by allowing an increase in the number of samples tested and / or a decrease in the length of time).

[0007] Non-limiting embodiments of the present invention are as follows:

[0008] [1] A high-throughput method for analyzing, developing and / or optimizing a culture medium for extracellular vesicle production, the method comprising: (a) culturing cells in a first culture medium, wherein cell division occurs during the culturing, and wherein the culturing is performed in multiples; (b) after step (a), removing the first culture medium from the multiple cell cultures, adding different candidate vesicle formation culture media to different cell cultures in the multiple cell cultures, and further culturing the multiple cell cultures to produce a conditioned medium containing extracellular vesicles; (c) recovering the conditioned medium from the multiple cell cultures or the cells after culturing in step (b), or both; and (d) analyzing at least one property of the extracellular vesicles in the recovered conditioned medium or the recovered cells, or both, wherein at least one of steps (a) to (d) is at least partially automated.

[0009] [2] The method according to [1], wherein at least one of steps (a) to (c) is semi-automatic.

[0010] [3] The method according to [1], wherein at least one of steps (a) to (c) is fully automated.

[0011] [4] The method according to [1], wherein each of steps (a) to (c) is semi-automatic or fully automatic.

[0012] [5] The method according to any one of [1] to [4], wherein at least one of steps (a) to (c) is performed using an automated liquid handler.

[0013] [6] The method according to [5], wherein steps (a) to (c) are all performed using an automatic liquid handler.

[0014] [7] The method according to any one of [1] to [6], wherein the cell is an iPSC-derived cell.

[0015] [8] The method according to [7], wherein the culture is a two-dimensional cell culture.

[0016] [9] The method according to [8], wherein the two-dimensional cell culture comprises culturing the cells on a surface of a culture container.

[0017]

[10] The method according to [9], wherein the surface of the culture container is coated with a substance that promotes cell adhesion.

[0018]

[11] The method according to

[10] , wherein the substance that promotes cell adhesion is vitronectin or fibronectin.

[0019]

[12] The method according to any one of [1] to

[11] , wherein the multiple cell cultures are cultured in one or more multi-well plates or microplates.

[0020]

[13] The method according to any one of [1]-

[12] , wherein the method further comprises mixing or combining two or more culture media, and / or adding one or more additives or supplements to one or more culture media to produce a group of different candidate vesicle formation culture media.

[0021]

[14] The method of

[13] , wherein the set of different candidate vesicle formation media is generated using an automated liquid handler.

[0022]

[15] The method of

[14] , wherein the set of candidate vesicle formation culture media is generated by mixing or combining two or more culture media from at least five different culture media initially selected.

[0023]

[16] The method of

[15] , wherein the set of candidate vesicle formation culture media is generated by mixing or combining two or more culture media from at least ten different culture media initially selected.

[0024]

[17] The method of

[16] , wherein the set of candidate vesicle formation culture media is generated by mixing or combining two or more culture media from at least twenty different culture media initially selected.

[0025]

[18] The method of

[17] , wherein the set of candidate vesicle formation culture media is generated by mixing or combining two or more culture media from an initial selection of at least fifty different culture media.

[0026]

[19] The method according to any one of

[14] -

[18] , wherein the group of candidate vesicle formation culture media comprises at least ten candidate vesicle formation culture media.

[0027]

[20] The method according to

[19] , wherein the group of candidate vesicle formation culture media comprises at least twenty candidate vesicle formation culture media.

[0028]

[21] The method according to

[20] , wherein the group of candidate vesicle formation culture media comprises at least thirty candidate vesicle formation culture media.

[0029]

[22] The method according to

[21] , wherein the group of candidate vesicle formation culture media comprises at least fifty candidate vesicle formation culture media.

[0030]

[23] The method according to

[22] , wherein the group of candidate vesicle formation culture media comprises at least one hundred candidate vesicle formation culture media.

[0031]

[24] The method according to any one of [1] to

[23] , wherein the cells comprise progenitor cells.

[0032]

[25] The method according to any one of [1] to

[24] , wherein the cells have been previously frozen.

[0033]

[26] A method according to any one of [1] to

[25] , wherein at least one property of the recovered cells from step (c) analyzed is selected from the group consisting of cell number, cell viability, cell density, cell morphology, cell identity, cell karyotype, cell transcriptome, hypertrophy, cell health, cell adhesion, cell physiology and / or ATP content.

[0034]

[27] The method of

[26] , wherein at least one property is selected from the group consisting of cell number and cell viability.

[0035]

[28] The method according to

[27] , wherein the cell number and / or cell viability is measured using an automated cell counter.

[0036]

[29] The method according to

[27] or

[28] , wherein the cell number and / or cell viability is determined by staining the cells with at least one dye.

[0037]

[30] The method according to

[29] , wherein the cells are stained with acridine orange and / or propidium iodide.

[0038]

[31] The method according to any one of [1]-

[25] , wherein at least one property of the extracellular vesicles analyzed is the total number of extracellular vesicles, the number of extracellular vesicles per cell, the extracellular vesicle concentration, the extracellular vesicle size, the extracellular vesicle size distribution, the protein concentration, the protein profile concentration, the RNA profile, the potency or the marker expression.

[0039]

[32] The method according to

[31] , wherein at least one property of the extracellular vesicles analyzed is selected from the group consisting of total extracellular vesicles, number of extracellular vesicles per cell, extracellular vesicle size, and marker expression.

[0040]

[33] The method according to

[32] , wherein the total number of extracellular vesicles and / or the number of extracellular vesicles per cell is determined by measuring the expression of at least one marker present on the extracellular vesicles.

[0041]

[34] The method according to

[33] , wherein the marker is a tetratransmembrane protein.

[0042]

[35] The method according to

[34] , wherein the quadruple transmembrane protein is selected from the group consisting of CD9, CD63 and CD81.

[0043]

[36] The method according to

[35] , wherein the quadruple transmembrane protein is CD63.

[0044]

[37] The method according to any one of

[32] -

[36] , wherein the marker is detected using an immunoassay.

[0045]

[38] The method according to

[37] , wherein the immunoassay is ELISA.

[0046]

[39] The method according to

[38] , wherein the ELISA is a Tim4-capture ELISA.

[0047]

[40] The method according to

[39] , wherein the ELISA is a Tim4-capture ELISA for detecting CD63 expression.

[0048]

[41] A method according to any of

[26] -

[40] , wherein the measurement of at least one property is at least partially automated.

[0049]

[42] The method according to

[31] , wherein at least one property of the extracellular vesicles analyzed is analyzed at the level of a single extracellular vesicle.

[0050]

[43] The method according to

[42] , wherein at least one property analyzed at the level of single extracellular vesicles is marker expression.

[0051]

[44] The method of

[42] or

[43] , wherein the analysis is performed using super-resolution microscopy.

[0052]

[45] The method according to

[44] , wherein the super-resolution microscope is direct stochastic optical reconstruction microscopy (dSTORM).

[0053]

[46] The method according to any one of

[43] -

[45] , wherein the marker expression analyzed comprises analyzing the expression of at least one tetraspanin.

[0054]

[47] The method of

[46] , wherein the quadruple transmembrane protein is selected from the group consisting of CD9, CD63 and CD81.

[0055]

[48] ​​The method according to any one of

[42] ,

[44] and

[45] , wherein the size of single extracellular vesicles is analyzed.

[0056]

[49] The method according to any one of

[42] -

[48] , wherein the analysis is used to analyze extracellular vesicle subpopulations.

[0057]

[50] The method according to [1], wherein in the method, any one of steps (a) to (c) is semi-automated, using an automatic liquid handler; culturing comprises culturing the cells on the surface of a culture container; the culture container is a multi-well plate or a microplate; the method also includes mixing or combining two or more culture media together, from at least two different culture media initially selected, to produce a group of different candidate vesicle formation culture media; the method includes, in step (d), analyzing cell number and cell viability using an automated cell counter; the method also includes, in step (d), measuring the total number of extracellular vesicles and / or the number of extracellular vesicles per cell by measuring the expression of at least one marker present on extracellular vesicles by a high-throughput immunoassay; and the method also includes, in step (d), analyzing marker expression and / or vesicle size of individual extracellular vesicles using super-resolution microscopy.

[0058]

[51] The method of

[50] , wherein the immunoassay is a Tim4-capture ELISA.

[0059]

[52] The method according to

[51] , wherein the ELISA is a Tim4-capture ELISA for detecting CD63 expression.

[0060]

[53] The method according to any one of

[50] -

[52] , wherein the analysis comprises analyzing one or more of CD9, CD63 and CD81 expression using the super-resolution microscope.

[0061]

[54] The method of

[53] , wherein the analysis comprises analyzing CD9, CD63 and CD81 expression using the super-resolution microscope.

[0062]

[55] The method according to any one of

[53] and

[54] also includes using the super-resolution microscope to analyze the size of individual extracellular vesicles.

[0063]

[56] The method according to any one of [1]-

[55] , further comprising selecting a candidate vesicle formation culture medium based on the analysis results of step (d).

[0064]

[57] The method of

[56] , wherein the selected culture medium provides improvements in one or more of the following aspects: cell growth, cell viability, total extracellular vesicle number, number of extracellular vesicles per cell, marker expression on extracellular vesicles, and extracellular vesicle size compared to a control benchmark culture medium or unmixed culture medium.

[0065]

[58] A vesicle formation medium selected according to the method of

[56] or

[57] .

[0066]

[59] A system for implementing any one of the methods of [1]-

[57] , wherein the system comprises one or more of an automated liquid handler, an automated cell counter, an immunoassay kit, and a super-resolution microscope.

[0067]

[60] The system according to

[59] , wherein the immunoassay kit is an ELISA kit.

[0068]

[61] The method according to any one of

[15] -

[18] , wherein the set of candidate vesicle formation culture media is produced by mixing or combining three or more culture media.

[0069]

[62] The method according to any one of

[15] -

[18] , wherein the set of candidate vesicle formation culture media is produced by mixing or combining four or more culture media.

[0070]

[63] The method according to any one of

[50] -

[55] , wherein the two or more culture media mixed or combined together are from at least five different culture media initially selected.

[0071]

[64] The method of any one of

[50] -

[55] , wherein the analysis by super-resolution microscopy comprises immobilizing the extracellular vesicles on at least one of a coverslip and a microscope channel slide.

[0072]

[65] The method according to any one of

[50] -

[55] and

[64] , wherein the analysis by super-resolution microscopy comprises detecting at least one marker using a fluorescent antibody.

[0073] Incorporated by Reference

[0074] All patents, publications, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual patent, publication, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0076] Figure 1 A flow chart depicting the various stages of the exemplary assays described in Examples 1 and 2.

[0077] Figure 2 Viable cell density after culture with control and M1-M31 vesicle formation medium formulations described in Example 1 is depicted.

[0078] Figure 3A and Figure 3B Depicted are the extracellular vesicle production of cells after culture in vesicle formation medium, as measured by detecting the presence of CD63 on Tim4-captured extracellular vesicles using the extracellular vesicle ELISA (EV ELISA) assay described in Example 1. Figure 3A Total extracellular vesicle yield is depicted. Figure 3B Extracellular vesicle production per cell is depicted.

[0079] Figure 4 Depicted are the results of a comparison between manual processing of tissue culture fluid and semi-automated processing of tissue culture fluid as described in Example 1 regarding extracellular vesicle yield (measured by EV ELISA assay detecting the presence of CD63 on Tim4-captured extracellular vesicles).

[0080] Figure 5A Depicted are the total number of extracellular vesicles determined from single extracellular vesicle (dSTORM) analysis as described in Example 1.

[0081] Figure 5B Results of nanoparticle tracking analysis (NTA) are depicted. MC represents conditioned medium after vesicle formation and MV represents original medium control as described in Example 1.

[0082] Figure 5C Depicted are CD63 expression levels determined using the EV ELISA assay as described in Example 1.

[0083] Figure 6 Depicted are the results of a human cardiomyocyte survival assay performed as described in Example 1 to assess EV function.

[0084] Figure 7 Depicted are the results of a HUVEC scratch wound healing assay performed as described in Example 1 to assess EV function.

[0085] Figure 8Depicted are viable cell density (right vertical axis) after culture with control and M1-M46 vesicle formation medium formulations, and total extracellular vesicle production measured by the amount of CD63, CD9 and CD81 as described in Example 2 by EV ELISA assay (left vertical axis).

[0086] Fig. 9 Depicted are the extracellular vesicle production per cell calculated by dividing the signal from CD63, CD9 and CD81 EV ELISA assays by the viable cell count following culture with control and M1-M46 vesicle formation medium formulations described in Example 2.

[0087] Fig.10 Depicted are the results of single extracellular vesicle level analysis using super-resolution microscopy as described in Example 2.

[0088] Fig.11A and Fig. 11B Shown are the expression levels of CD63, CD9, and CD81 determined using the EV ELISA assay for vesicles generated manually in culture flasks ( Fig.11A ) and in multiwell plates ( Fig. 11B ) is generated in .

[0089] Fig.12 Depicted are the results of a HUVEC scratch wound healing assay performed as described in Example 2 to assess EV function.

[0090] Fig.13 An exemplary workflow for the development and / or optimization of vesicle formation culture medium is depicted. In this exemplary workflow, a design of experiments (DoE) approach is used to strategically plan culture medium mixing, and an automated liquid handler is used to semi-automatically perform cell culture liquid processing (e.g., culture medium mixing or mixing, culture medium distribution, culture medium replacement, and culture medium recovery). After culture, an automated cell counter is used to measure cell number and viability. A preliminary screening of extracellular vesicles in conditioned medium is performed using a Tim4-capture extracellular vesicle ELISA (using an antibody that binds to CD63 on extracellular vesicles as a detection antibody). Further analysis of extracellular vesicles is performed at the level of single extracellular vesicles by super-resolution microscopy. DETAILED DESCRIPTION

[0091] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope thereof. As used in this specification and 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 cell" includes one or more cells.

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although other methods and materials similar or equivalent to those described herein can be used in the present invention, preferred materials and methods are described herein.

[0093] As used herein, "subject", "individual" or "patient" are used interchangeably herein and refer to any member of the phylum Chordata, including but not limited to humans and other primates, including non-human primates, such as rhesus macaques, chimpanzees and other monkey and ape species; farm animals, such as cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats and guinea pigs; birds, including poultry, wild birds and game birds, such as chickens, turkeys and other quail, ducks and geese, etc. The term does not indicate a specific age or sex. Therefore, the term includes adult, youth and newborn animal individuals and male and female animals. In some embodiments, cells (such as stem cells, including pluripotent stem cells, progenitor cells or tissue-specific cells) are derived from a subject. In some embodiments, the subject is a non-human subject.

[0094] As used herein, "differentiation" refers to a process by which a non-specialized cell (such as a pluripotent stem cell or other stem cell) or a multipotent cell or an oligopotent cell, for example, acquires, initiates or directs specialized structural and / or functional characteristics of a more mature or fully mature cell. "Transdifferentiation" is the process of converting one differentiated cell type into another differentiated cell type.

[0095] As used herein, "embryoid bodies" refer to three-dimensional aggregates of pluripotent stem cells. These cells can differentiate into cells of the three germ layers, namely, endoderm, mesoderm, and ectoderm. The three-dimensional structure, including the establishment of complex cell adhesion and paracrine signals in the embryoid body microenvironment, enables differentiation and morphogenesis.

[0096] As used herein, "stem cell" refers to a cell with self-renewal ability, i.e., a cell that can undergo multiple cell division cycles while maintaining its non-terminal differentiation state. Stem cells can be totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, or unipotent stem cells. Stem cells can be, for example, embryonic, fetal, amniotic, adult, or induced pluripotent stem cells.

[0097] As used herein, "pluripotent stem cells" (PSC) refer to cells that have the ability to propagate themselves indefinitely and differentiate into any other cell type of an adult organism. In general, pluripotent stem cells are stem cells that can induce teratomas when transplanted into immunodeficient (SCID) mice; can differentiate into cell types of all three germ layers (e.g., can differentiate into ectoderm, mesoderm, and endoderm cell types); and express one or more characteristic markers of PSC. Examples of such markers expressed by PSC (such as embryonic stem cells (ESC) and iPSC) include Oct4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, and / or REX1.

[0098] As used herein, "induced pluripotent stem cells" (iPSC) refers to a class of pluripotent stem cells, which are artificially derived from non-pluripotent cells, typically somatic cells. In some embodiments, the somatic cells are human somatic cells. Examples of somatic cells include, but are not limited to, dermal fibroblasts, mesenchymal cells of bone marrow origin, human pluripotent stem cells (HPSc), hematopoietic cells, cardiomyocytes, keratinocytes, hepatocytes, gastric cells, neural stem cells, lung cells, kidney cells, spleen cells and pancreatic cells. Other examples of somatic cells include cells of the immune system, including but not limited to B cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes / macrophages, myeloid-derived suppressor cells, natural killer (NK) cells, T cells, thymocytes and hematopoietic stem cells. iPSC can be produced by somatic cell reprogramming, and somatic cells are reprogrammed by expressing or inducing the expression of a factor or factor combination (referred to herein as reprogramming factors) in somatic cells. iPSC can be produced using fetal, postnatal, neonatal, infancy or adult somatic cells. In some cases, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, OCT4 (OCT3 / 4), SOX2, c-MYC and KLF4, NANOG and LIN28. In some cases, somatic cells can be reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors or at least four reprogramming factors, so that somatic cells are reprogrammed into pluripotent stem cells. Reprogramming cells can be reprogrammed by introducing reprogramming factors using a vector, and the vector includes, for example, episomal vectors, non-viral vectors, slow viruses, retroviruses, adenoviruses and Sendai virus vectors. Alternatively, non-viral techniques for introducing reprogramming factors include, for example, mRNA transfection, miRNA infection / transfection, PiggyBac, microcircle vectors and episomal plasmids. iPSC can also be produced by, for example, introducing reprogramming factors or activating endogenous programming genes using CRISPR-Cas9-based technology.

[0099] As used herein, "embryonic stem cell" is an embryonic cell derived from embryonic tissue, preferably the inner cell mass of blastocyst or morula, optionally as a cell line and continuously passaged. The term includes cells separated from one or more blastomeres of an embryo, preferably without destroying the rest of the embryo. The term also includes cells produced by somatic cell nuclear transplantation. ESC can be produced from or derived from a mammalian embryo in a fertilized egg, blastomere or blastocyst stage, such as produced by the fusion, nuclear transplantation or parthenogenesis of a sperm and an egg cell. Human ESC includes but is not limited to MAO1, MAO9, ACT-4, No.3, H1, H7, H9, H14 and ACT30 embryonic stem cells. The pluripotent stem cells of the example include embryonic stem cells derived from the inner cell mass (ICM) of an embryo in the blastocyst stage, and embryonic stem cells derived from one or more blastomeres of an embryo in the cleavage stage or morula stage. These embryonic stem cells can be produced from embryonic materials produced by fertilization or asexual means, and asexual means include somatic cell nuclear transplantation (SCNT), parthenogenesis and androgenesis. When transplanted into the uterus, PSCs alone cannot develop into a fetus or adult animal because they lack the potential to contribute to all extraembryonic tissues (e.g., placenta in vivo or trophoblast in vitro).

[0100] As used herein, the term "progenitor cell" refers to the offspring of stem cells that can further differentiate into one or more specialized cells but cannot infinitely divide and propagate. That is, unlike stem cells (which have unlimited self-renewal ability), progenitor cells only have limited self-renewal ability. Progenitor cells can be specialized, oligopotent or unipotent, and are usually classified according to the type of specialized cells they can differentiate into. For example, "cardiomyocyte progenitor cells" are progenitor cells derived from stem cells with the ability to differentiate into cardiomyocytes. Similarly, "cardiac progenitor cells" can differentiate into a variety of specialized cells that constitute cardiac tissue, including, for example, cardiomyocytes, smooth muscle cells and endothelial cells. In addition, "cardiovascular progenitor cells" have the ability to differentiate into, for example, heart and vascular lineage cells.

[0101] As used herein, "expansion" or "proliferation" may refer to a process in which the number of cells in a cell culture increases due to cell division. A culture in which such expansion occurs may be referred to as, for example, an "expansion culture".

[0102] "Multipotent" means that the cell is able to give rise, through its progeny, to several different cell types found in an adult animal.

[0103] "Pluripotent" means that a cell is able to give rise to all cell types that make up an adult animal through its progeny, including germ cells. Embryonic stem cells, induced pluripotent stem cells, and embryonic germ cells are pluripotent cells under this definition.

[0104] As used herein, the term "autologous cells" refers to donor cells that are genetically identical to the recipient.

[0105] As used herein, the term "allogeneic cells" refers to cells derived from different, genetically non-identical individuals of the same species.

[0106] As used herein, the term "totipotent" may refer to a cell that produces a live-bearing animal. The term "totipotent" may also refer to a cell that produces all cells in a particular animal. When a totipotent cell is used in the process of developing an embryo from one or more nuclear transfer steps, it can produce all cells of the animal.

[0107] As used herein, the term "extracellular vesicle" refers collectively to biological particles derived from cells, examples of which include exosomes, ectosomes, exovesicles, microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles, membranous vesicles, shedding vesicles, membrane particles, shedding microvesicles, oncosomes, exomeres and apoptotic bodies, but are not limited to these.

[0108] Extracellular vesicles can be, for example, classified according to size. For example, as used herein, the term "small extracellular vesicle" refers to an extracellular vesicle with a diameter between about 50-200nm. In contrast, extracellular vesicles with a diameter greater than about 200nm but less than 400nm can be referred to as "medium extracellular vesicles", while extracellular vesicles with a diameter greater than about 400nm can be referred to as "large extracellular vesicles". As used herein, the term "small extracellular vesicle component" ("sEV") refers to a portion, extract or component of a secretome or conditioned medium, which is a concentrated and / or enriched small extracellular vesicle with a diameter of about 50-200nm. In the context of extracellular vesicle production, cells that produce extracellular vesicles can be referred to as "production cells".

[0109] As used herein, the term "exosome" refers to extracellular vesicles released from cells upon fusion of multivesicular bodies (MVBs), intermediate endocytic compartments, with the plasma membrane.

[0110] "Exosome-like vesicles" that share a common origin with exosomes are generally described as having a size and sedimentation properties that distinguish them from exosomes, particularly the lack of lipid raft microdomains. As used herein, "ectosomes" are generally neutrophil- or monocyte-derived microvesicles.

[0111] "Microparticles" as used herein are typically about 100-1000 nm in diameter and are derived from the plasma membrane. "Extracellular membrane structures" also include, for example, linear or folded membrane fragments from necrotic death, as well as membrane structures from other cellular sources, including secreted lysosomes and nanotubes.

[0112] As used herein, "apoptotic vesicles or apoptotic bodies" are typically about 1 to 5 μm in diameter and are released as vesicles from cells undergoing apoptosis (ie, diseased, unwanted, and / or abnormal cells).

[0113] In the category of extracellular vesicles, an important component is the "exosome" itself, which can be between about 40 to 50 nm and about 200 nm in diameter and is a membranous vesicle of endocytic origin, i.e., a vesicle surrounded by a phospholipid bilayer, which comes from the exocytic fusion or "exocytosis" of multivesicular bodies (MVBs). In some cases, the diameter of the exosome can be between about 40 to 50 nm and up to about 200 nm, such as 60 nm to 180 nm.

[0114] As used herein, the terms "secretome" and "secretome composition" interchangeably refer to one or more molecules and / or biological factors secreted by a cell into the extracellular space (such as secreted into the culture medium). The secretome or secretome composition may include, but is not limited to, extracellular vesicles (e.g., exosomes, microparticles, etc.), proteins, nucleic acids, cytokines, and / or other molecules secreted by a cell into the extracellular space (such as secreted into the culture medium). The secretome or secretome composition may not be purified or further processed (e.g., components of the secretome or secretome composition may be present in a culture medium, such as a conditioned medium; or, components of the secretome or secretome composition may be purified, isolated, and / or enriched from a culture medium or an extract, portion, or component thereof). The secretome or secretome composition may also include one or more substances that are not secreted from the cell (e.g., culture medium, additives, nutrients, etc.). Alternatively, the secretome or secretome composition does not include (or includes only trace amounts of) one or more substances that are not secreted from the cell (e.g., culture medium, additives, nutrients, etc.).

[0115] As used herein, the term "conditioned medium" refers to a culture medium (or an extract, part or component thereof) in which one or more cells of interest are cultured. Preferably, the conditioned medium is separated from the cultured cells before use and / or before further processing. Culturing cells in a culture medium may result in the secretion and / or accumulation of one or more molecules and / or biological factors (which may include, but are not limited to, extracellular vesicles (e.g., exosomes, microparticles, etc.), proteins, nucleic acids, cytokines, and / or other molecules secreted by cells into the extracellular space); a culture medium containing one or more molecules and / or biological factors is a conditioned medium. Examples of methods for preparing conditioned medium have been described, for example, in U.S. Pat. No. 6,372,494, which is incorporated herein by reference in its entirety.

[0116] As used herein, the term "cell culture" refers to cells grown under controlled conditions outside the natural environment of the cell. For example, the cell can be propagated completely outside its natural environment (in vitro), or can be removed from its natural environment and cultured (ex vivo). During the cell culture period, the cell can survive in a non-replicating state, or can replicate and grow in quantity, depending on, for example, specific culture medium, culture conditions and cell type. The in vitro environment can be any culture medium suitable for maintaining cells in vitro known in the art, for example, such as a suitable liquid culture medium or agar.

[0117] As used herein, the term "cell line" may refer to cultured cells that can be passaged at least once without termination.

[0118] As used herein, the term "suspension" may refer to cell culture conditions in which cells are not attached to a solid support. Cells proliferating in suspension culture may be agitated while proliferating using equipment well known to those skilled in the art.

[0119] As used herein, the term "monolayer" may refer to cells attached to a solid support while proliferating under appropriate culture conditions. A small portion of cells proliferating in monolayer culture under appropriate growth conditions may be attached to the monolayer but not to the solid support.

[0120] As used herein, the term "plated" or "plating" with respect to cells may refer to establishing a cell culture in vitro. For example, cells may be diluted in a cell culture medium and then added to a cell culture plate, dish, or flask. Cell culture plates are generally known to those of ordinary skill in the art. Cells may be plated at a variety of concentrations and / or cell densities.

[0121] The term "cell plating" can also be extended to the term "cell passaging". Cells can be passaged using cell culture techniques well known to those skilled in the art. The term "cell passaging" can refer to a technique comprising the steps of: (1) releasing cells from a solid support or matrix and dissociating these cells, and (2) diluting the cells in a medium suitable for further cell proliferation. Cell passaging can also refer to removing a portion of the liquid culture medium containing the cultured cells and adding liquid culture medium to the original culture container to dilute the cells and allow the cells to further proliferate. In addition, the cells can also be added to a new culture container, which is supplemented with a medium suitable for further cell proliferation.

[0122] As used herein, the terms "culture medium," "growth medium," or "medium" are used interchangeably and refer to compositions intended to support the growth and survival of an organism. Although culture media are typically in liquid form, other physical forms may be used, such as, for example, solid, semisolid, gel, suspension, etc. As used herein, the term "vesicle formation medium" refers to a medium in which the purpose and / or result of culturing cells is to produce extracellular vesicles, such as to produce conditioned medium containing extracellular vesicles.

[0123] As used herein, in the context of culture medium or growth medium, the term "serum-free" refers to a culture medium or growth medium in which serum is not present. Serum generally refers to the liquid component of blood that coagulates after the removal of coagulation factors (e.g., fibrinogen and prothrombin) by clot formation. Serum, such as fetal bovine serum, is commonly used as a component of cell culture medium in the art because the various proteins and growth factors therein are particularly useful for the survival, growth and division of cells.

[0124] As used herein, the term "basal medium" refers to an unsupplemented synthetic medium that may contain a buffer, one or more carbon sources, amino acids, and salts. Depending on the application, the basal medium may be supplemented with growth factors and supplements, including but not limited to additional buffers, amino acids, antibiotics, proteins, and useful growth factors, e.g., growth factors for promoting growth or maintaining or altering the differentiation state of a particular cell type (e.g., fibroblast growth factor (bFGF), also known as fibroblast growth factor 2 (FGF-2)).

[0125] As used herein, the terms "wild type", "naturally occurring" and "unmodified" are used herein to refer to a typical (or most common) form, appearance, phenotype or strain that occurs in nature; for example, a typical existing form of a cell, organism, polynucleotide, protein, macromolecular complex, gene, RNA, DNA or genome, and can be isolated from a natural source. The wild type form, appearance, phenotype or strain serves as the original parent before intentional modification. Therefore, mutants, variants, engineered, recombinant and modified forms are not wild type forms.

[0126] As used herein, the term "isolated" refers to material that has been removed from its original environment and thus altered from its natural state "by the hands of man."

[0127] As used herein, the term "enrichment" refers to selectively concentrating or increasing the amount of one or more components in a composition relative to one or more other components. For example, enrichment can include reducing or lowering (e.g., removing or eliminating) the amount of unwanted material; and / or can include specifically selecting or isolating a desired material from a composition.

[0128] The terms "engineered", "genetically engineered", "genetically modified", "recombinant", "modified", "non-naturally occurring" and "non-natural" refer to intentional manipulation of the genome of an organism or cell by humans. These terms encompass methods of genome modification, including genome editing as defined herein, as well as techniques for altering gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, codon optimization, and the like. Methods for genetic engineering are known in the art.

[0129] As used herein, the terms "nucleic acid sequence", "nucleotide sequence" and "oligonucleotide" all refer to a polymeric form of nucleotides. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides, which, when in linear form, has a 5' end and a 3' end, and may contain one or more nucleic acid sequences. Nucleotides may be deoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof, or combinations thereof, and may be of any length. Polynucleotides may perform any function and may have a variety of secondary and tertiary structures. The term encompasses known analogs of natural nucleotides and nucleotides modified at bases, sugars and / or phosphate moieties. Analogs of specific nucleotides have the same base pairing specificity (e.g., analogs of A pair with T bases). Polynucleotides may include one modified nucleotide or multiple modified nucleotides. Examples of modified nucleotides include fluorinated nucleotides, methylated nucleotides, and nucleotide analogs. The nucleotide structure may be modified before or after polymer assembly. After polymerization, polynucleotides may be further modified by, for example, conjugating with a labeling component or a target binding component. The nucleotide sequence may include non-nucleotide components. The term also encompasses nucleic acids that contain modified backbone residues or bonds that are synthetic, naturally occurring, and / or non-naturally occurring and that have similar binding properties to a reference polynucleotide (e.g., DNA or RNA). Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), TM ) (Exiqon Corporation, Woburn, MA) Nucleosides, glycol nucleic acids, bridged nucleic acids, and morpholino structures. Peptide nucleic acids (PNA) are synthetic homologues of nucleic acids in which the phosphate-sugar backbone of the polynucleotide is replaced by a flexible pseudopeptide polymer. The nucleobases are linked to the polymer. PNA has the ability to hybridize with complementary sequences of RNA and DNA with high affinity and specificity. Unless otherwise indicated, polynucleotide sequences are shown herein in the conventional 5' to 3' orientation.

[0130] As used herein, "sequence identity" generally refers to the percentage of identity of the nucleotide bases or amino acids of a first polynucleotide or polypeptide and a second polynucleotide or polypeptide using an algorithm with various weighting parameters. The sequence identity between two polynucleotides or two polypeptides can be determined using sequence alignments by various methods and computer programs (e.g., Exonerate, BLAST, CS-BLAST, FASTA, HMMER, L-ALIGN, etc.), which can be obtained through the World Wide Web website, including but not limited to GENBANK (www.ncbi.nlm.nih.gov / genbank / ) and EMBL-EBI (www.ebi.ac.uk.). The sequence identity between two polynucleotides or two polypeptide sequences is usually calculated using the standard default parameters of various methods or computer programs. A high degree of sequence identity between two polynucleotides or two polypeptides is typically about 90% identity to 100% identity over the length of a reference polynucleotide or polypeptide or query sequence, e.g., about 90% identity or higher, about 91% identity or higher, about 92% identity or higher, about 93% identity or higher, about 94% identity or higher, about 95% identity or higher, about 96% identity or higher, about 97% identity or higher, about 98% identity or higher, or about 99% identity or higher over the length of a reference polynucleotide or polypeptide or query sequence. Sequence identity can also be calculated for the overlapping region of two sequences, where only a portion of the two sequences can be aligned.

[0131] An intermediate degree of sequence identity between two polynucleotides or two polypeptides is typically about 80% identity to about 90% identity over the length of a reference polynucleotide or polypeptide or query sequence, e.g., about 80% identity or more, about 81% identity or more, about 82% identity or more, about 83% identity or more, about 84% identity or more, about 85% identity or more, about 86% identity or more, about 87% identity or more, about 88% identity or more, or about 89% identity or more, but less than 90% identity over the length of a reference polynucleotide or polypeptide or query sequence.

[0132] A low degree of sequence identity between two polynucleotides or two polypeptides is typically about 50% identity to 75% identity over the length of a reference polynucleotide or polypeptide or query sequence, for example, about 50% identity or more, about 60% identity or more, about 70% identity or more, but less than 75% identity over the length of a reference polynucleotide or polypeptide or query sequence.

[0133] As used herein, "binding" refers to non-covalent interactions between macromolecules (e.g., between a protein and a polynucleotide, between a polynucleotide and a polynucleotide, or between a protein and a protein, etc.). Such non-covalent interactions are also referred to as "association" or "interaction" (e.g., if a first macromolecule interacts with a second macromolecule, the first macromolecule binds to the second macromolecule in a non-covalent manner). Some portions of the binding interactions may be sequence-specific (the terms "sequence-specific binding," "sequence-specific binding," "site-specific binding," and "site-specific binding" are used interchangeably herein). The binding interactions may be identified by the dissociation constant (Kd). "Binding affinity" refers to the strength of the binding interaction. Increased binding affinity is associated with a lower Kd.

[0134] As used herein, "gene" refers to a polynucleotide sequence comprising exons and associated regulatory sequences. A gene may also comprise introns and / or untranslated regions (UTRs).

[0135] As used herein, "expression" refers to the transcription of a polynucleotide from a DNA template to produce, for example, messenger RNA (mRNA) or other RNA transcripts (e.g., non-coding RNA, such as structural or scaffold RNA). The term also refers to the process by which transcribed mRNA is translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0136] A "coding sequence" or a sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' terminus and a translation stop codon at the 3' terminus. A transcription termination sequence may be located at the 3' end of the coding sequence.

[0137] As used herein, for example, a "different" or "altered" level of a feature or property is a measurable, different difference, and preferably is statistically significant (e.g., not attributable to the standard error of the assay). In some embodiments, for example, a difference compared to a control or reference sample can be, for example, a difference of greater than 10%, a difference of greater than 20%, a difference of greater than 30%, a difference of greater than 40%, a difference of greater than 50%, a difference of greater than 60%, a difference of greater than 70%, a difference of greater than 80%, a difference of greater than 90%, a difference of greater than 2-fold; a difference of greater than 5-fold; a difference of greater than 10-fold; a difference of greater than 20-fold; a difference of greater than 50-fold; a difference of greater than 75-fold; a difference of greater than 100-fold; a difference of greater than 250-fold; a difference of greater than 500-fold; a difference of greater than 750-fold; a difference of greater than 1000-fold; a difference of greater than 250-fold; a difference of greater than 500-fold; a difference of greater than 750-fold; or a difference of greater than 1000-fold.

[0138] As used herein, the term "between" includes both ends of a given range (eg, the length is between about 1 and about 50 nucleotides, inclusive).

[0139] As used herein, the term "amino acid" refers to natural and synthetic (non-natural) amino acids, including amino acid analogs, modified amino acids, peptidomimetics, glycine, and D or L optical isomers.

[0140] As used herein, the terms "peptide", "polypeptide" and "protein" are interchangeable and refer to polymers of amino acids. A polypeptide can be of any length. It can be branched or linear, can be interrupted by non-amino acids, and can contain modified amino acids. The term also refers to amino acid polymers that have been modified by, for example, acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, pegylation, biotinylation, cross-linking and / or conjugation (e.g., conjugation with a labeling component or ligand). Unless otherwise indicated, polypeptide sequences are shown herein in a conventional N-terminal to C-terminal direction. Polypeptides and polynucleotides can be prepared using conventional techniques in the field of molecular biology.

[0141] As used herein, "moiety" refers to a portion of a molecule. The moiety can be a functional group or describe a portion of a molecule having multiple functional groups (e.g., having common structural aspects). The terms "moiety" and "functional group" are often used interchangeably; however, "functional group" can more specifically refer to a portion of a molecule that contains some common chemical behavior.

[0142] "Part" is often used as a structural description.

[0143] The term "effective amount" (eg, an "effective amount" of a composition or product) refers to an amount of a composition or product sufficient to provide a desired response.

[0144] As used herein, "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, regardless of the method used for insertion. For example, transformation can be performed by direct uptake, transfection, infection, etc. The exogenous polynucleotide can be maintained as a non-integrated vector (e.g., an episomal vector), or can be integrated into the host genome.

[0145] As used herein, the term "hypoxia" or "low oxygen" refers to a condition in which the concentration of oxygen (O2) is lower than the atmospheric O2 concentration (usually 20-21%). In some embodiments, hypoxia refers to a condition in which the O2 concentration is between 0% and 19%, between 2% and 18%, between 3% and 17%, between 4% and 16%, between 5% and 15%, between 5% and 10%, or less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0146] As used herein, the term "normoxia" refers to normal atmospheric oxygen concentration, typically about 20% to 21% O2.

[0147] As used herein, with respect to processes for the development, testing and / or optimization of culture media (and / or culture conditions) for extracellular vesicle production, the term "high throughput" refers to processes that allow for the processing of an increased number of samples and / or a reduction in experimental time, and typically both. Typically, but not necessarily, a high throughput process will employ one or more steps or assays with a certain level of automation (by a machine, device, computer, etc.), e.g., semi-automatic or fully automated steps or assays.

[0148] As used herein, the term "automatic" in the context of a process or step refers to a process or step in which some or no part of the process or step is performed manually, assuming that not all processes or steps are performed manually (e.g., it includes the concepts of semi-automatic and fully automatic).

[0149] As used herein, the term "semi-automated" in the context of a process or step refers to a process or step in which some aspects, portions, or parts of the process or step are performed manually (such as, for example, without the assistance of a liquid handler), provided that not all of the process or step is performed manually.

[0150] As used herein, the term "fully automated" in the context of a process or step means that no part (or substantially no part) of the process or step is performed manually.

[0151] Cells that secrete extracellular vesicles

[0152] The present invention relates in part to methods and systems for analyzing, developing, testing and / or optimizing culture media (and / or culture conditions) for extracellular vesicle production. Cells that can be used for extracellular vesicle production in this method and system include, but are not limited to, stem cells, progenitor cells, and differentiated cells (including terminally differentiated or partially differentiated cells). Such cells can be obtained by, for example, isolation from a subject or tissue; from an in vitro cell line or culture; or by in vitro differentiation induction (e.g., progenitor cells can be generated from pluripotent stem cells, such as embryonic stem (ES) cells or induced pluripotent stem cells (iPSC)).

[0153] Generation of iPSC cells

[0154] iPSC cells can be obtained, for example, from somatic cells, including human somatic cells. Somatic cells can be derived from humans or non-human animals, including, for example, humans and other primates, including non-human primates, such as rhesus monkeys, chimpanzees and other monkey and ape species; farm animals, such as cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats and guinea pigs; birds, including poultry, wild birds and game birds, such as chickens, turkeys and other quail, ducks and geese, etc.

[0155] In some embodiments, somatic cells are selected from keratinized epithelial cells, mucosal epithelial cells, exocrine gland epithelial cells, endocrine cells, hepatocytes, epithelial cells, endothelial cells, fibroblasts, muscle cells, blood cells and immune system cells, nervous system cells (including neurons and glial cells), pigment cells and progenitor cells (including hematopoietic stem cells). Somatic cells can be fully differentiated (specialized) or incompletely differentiated. For example, undifferentiated progenitor cells that are not PSCs can be used, including adult stem cells and terminally differentiated mature cells. Somatic cells can come from animals of any age, including adult and fetal cells.

[0156] Somatic cells can be mammalian in origin. If, for example, the secretory group (or extracellular vesicle) from its progenitor cells is used for in vivo administration, allogeneic or autologous stem cells can be used. In some embodiments, iPSC is not the MHC / HLA matched with the subject. In some embodiments, iPSC is the MHC / HLA matched with the subject. In embodiments, for example, when iPSC is used to produce PSC-derived cells (to obtain secretory groups or extracellular vesicles), such as progenitor cells, somatic cells may be from the subject to be treated, or from another subject with the same or substantially the same HLA type as the subject. For example, somatic cells can be cultured before nuclear reprogramming, or can be reprogrammed without being cultured after separation.

[0157] In order to introduce reprogramming factors into somatic cells, for example, viral vectors can be used, including, for example, from viruses such as SV40, adenovirus, vaccinia virus, adeno-associated virus, herpes virus (including HSV and EBV), Sindbis virus, alphavirus, human herpes virus vector (HHV) (such as HHV-6 and HHV-7), and retroviral vectors. Slow viruses include but are not limited to human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), bovine immunodeficiency virus (BIV), sheep myelin shedding virus (VISNA) and caprine arthritis-encephalitis virus (CAEV). Slow virus vectors can infect non-dividing cells and can be used for in vivo and in vitro gene transfer and nucleic acid sequence expression. By connecting viral proteins (such as envelope proteins) with binding agents (such as antibodies) or specific ligands (for targeting receptors or proteins on or within, for example, specific cell types), viral vectors can be targeted to specific cell types.

[0158] In some embodiments, viral vectors, such as lentiviral vectors, can be integrated into the genome of the host cell. The genetic material thus transferred is then transcribed and may be translated into protein in the host cell. In other embodiments, viral vectors that are not integrated into the host cell genome are used.

[0159] The viral gene delivery system can be an RNA-based or DNA-based viral vector. The episomal gene delivery system can be, for example, a plasmid, an episomal vector based on Epstein-Barr virus (EBV), a yeast-based vector, an adenovirus-based vector, an episomal vector based on simian virus 40 (SV40), a bovine papilloma virus (BPV)-based vector, or a lentiviral vector.

[0160] Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to those skilled in the art. Those skilled in the art can easily prepare induced pluripotent stem cells, see, for example, published U.S. Patent Application No. 2009 / 0246875, published U.S. Patent Application No. 2010 / 0210014; published U.S. Patent Application No. 2012 / 0276636; U.S. Patent No. 8,058,065, 8,129,187; and U.S. Patent No. 8,268,620, all of which are incorporated herein by reference.

[0161] Generally, reprogramming factors that can be used to generate induced pluripotent stem cells, either alone, in combination, or as a fusion with a transactivation domain, include, but are not limited to, one or more of the following genes: Oct4 (Oct3 / 4, Pou5f1), Sox (e.g., Sox1, Sox2, Sox3, Sox18 or Sox15), Klf (e.g., Klf4, Klf1, Klf3, Klf2 or Klf5), Myc (e.g., c-myc, N-myc or L-myc), nanog or LIN28. As examples of sequences of these genes and proteins, the following accession numbers are provided: mouse MyoD: M84918, NM_010866; mouse Oct4 (POU5F1): NM_013633; mouse Sox2: NM_011443; mouse Klf4: NM_010637; mouse c-Myc: NM_001177352, NM_001177353, NM_001177354; mouse Nanog: NM_0280 16; mouse Lin28: NM_145833; human MyoD: NM_002478; human Oct4 (POU5F1): NM_002701, NM_203289, NM_001173531; human Sox2: NM_003106; human Klf4: NM_004235; human c-Myc: NM_002467; human Nanog: NM_024865; and / or human Lin28: NM_024674. Also contemplated are sequences similar thereto, including those having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are used. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are used.

[0162] Exemplary reprogramming factors for generating iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced by Sox1, Sox3, Sox15, Sox17 or Sox18; Klf4 can be replaced by Klf1, Klf2 or Klf5); (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, SV40 large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, human papillomavirus (HPV) 16E6; (4) Oct3 / 4, Klf4, Sox2 , L-Myc, TERT, HPV16E7; (5) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16E6, HPV16E7; (6) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmi1; (7) Oct 3 / 4, Klf4, Sox2, L-Myc, Lin28; (8) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, SV40LT; (9) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT; ( 10) Oct3 / 4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3 / 4, Esrrb, Sox2, L-Myc (Esrrb can be replaced by Esrrg); (12) Oct3 / 4, Klf4, Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, HPV16E6; (15) Oct3 / 4, Klf4, Sox2, TERT, HPV16E7; (16) Oct3 / 4, Klf4, Sox2 , TERT, HPV16E6, HPV16E7; (17) Oct3 / 4, Klf4, Sox2, TERT, Bmi1; (18) Oct3 / 4, Klf4, Sox2, Lin28 (19) Oct3 / 4, Klf4, Sox2, Lin28, SV40LT; (20) Oct3 / 4, Klf4, Sox2, Lin28, TERT, SV40LT; (21) Oct3 / 4, Klf4, Sox2, SV40LT; or (22) Oct3 / 4, Esrrb, Sox2 (Esrrb can be replaced by Esrrg).

[0163] iPSCs typically exhibit the characteristic morphology of human embryonic stem cells (hESCs) and express the pluripotency factor NANOG. Embryonic stem cell-specific surface antigens (SSEA-3, SSEA-4, TRA1-60, TRA1-81) can also be used to identify fully reprogrammed human cells. In addition, at the functional level, PSCs (such as ESCs and iPSCs) have also demonstrated the ability to differentiate into lineages from all three embryonic germ layers and form teratomas in vivo (e.g., in SCID mice).

[0164] PSC differentiation

[0165] The present invention further contemplates differentiating PSCs, including ESCs and iPSCs, to produce extracellular vesicle-producing cells that have a more differentiated state than PSCs. For example, PSCs can be differentiated into terminally differentiated (specialized) cells that can be used to produce extracellular vesicles; or differentiated into progenitor cells that can be used to produce extracellular vesicles.

[0166] Progenitor cells of the present invention include, for example, hematopoietic progenitor cells, bone marrow progenitor cells, neural progenitor cells; pancreatic progenitor cells, cardiac progenitor cells, cardiomyocyte progenitor cells, cardiovascular progenitor cells, renal progenitor cells, skeletal muscle myoblasts, satellite cells, intermediate progenitor cells formed in the subventricular zone, radial glial cells, bone marrow interstitial cells, periosteal cells, endothelial progenitor cells, blast cells, border cap cells and mesenchymal stem cells. Methods for differentiating pluripotent stem cells into progenitor cells and culturing and maintaining progenitor cells are known in the art, such as those described in U.S. Provisional Patent Application No. 63 / 243,606 entitled "Methods for the Production of Committed Cardiac Progenitor Cells", the entire contents of which are incorporated herein by reference.

[0167] Specialized cells of the invention include, for example, fibroblasts, muscle cells, keratinocytes, hepatocytes, gastric cells, neural cells, lung cells, kidney cells, spleen cells, endothelial cells and pancreatic cells; and cells of the immune system, including but not limited to B cells, dendritic cells, granulocytes, innate lymphocytes, megakaryocytes, monocytes / macrophages, myeloid-derived suppressor cells, natural killer (NK) cells and T cells.

[0168] Analyze, develop, test and / or optimize media and / or culture conditions for extracellular vesicle production

[0169] The present invention provides methods and systems for analyzing, developing, testing and / or optimizing culture media (and / or culture conditions) for extracellular vesicle production. For example, in some embodiments, the present invention provides methods and systems for analyzing, developing, testing and / or optimizing culture media (and / or culture conditions) for extracellular vesicle production (such as by allowing an increase in the number of test samples and / or a decrease in the length of time) for one or more high-throughput methods for cell culture, liquid handling (including, for example, culture media distribution, culture media replacement and culture media harvesting), vesicle formation, analysis of cell growth and / or viability, analysis of extracellular vesicle production and secretion, and characterization of extracellular vesicles.

[0170] In some embodiments, one or more types of extracellular vesicle-producing cells (i.e., cells from which extracellular vesicles can be obtained, also referred to as producer cells) first undergo one or more culture expansion steps prior to the vesicle formation step.

[0171] In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 different types of producer cells are cultured in parallel and subsequently used for extracellular vesicle production.

[0172] The one or more production cells can be, for example, recently isolated or differentiated cells (e.g., from stem cells). Alternatively, in some embodiments, previously refrigerated, frozen, and / or cryopreserved cells can be used in the culture method. In some embodiments, the cells are thawed from a cryopreserved state (e.g., -80°C or colder) prior to use. In some embodiments thereof, the cells are thawed in a thawing medium. In some embodiments, the thawing medium can include a liquid culture medium containing one or more supplements. In some embodiments, the cells can be thawed in a thawing device, such as, for example, a water bath or a waterless thawing system (e.g., Biolife ThawSTAR TM Automatic defrost system).

[0173] Although not required, each type of production cell cultured is preferably cultured in large quantities. For example, depending on the number of vesicle formation medium compositions and / or culture conditions to be analyzed, each type of production cell can be cultured in duplicate, triplicate, quadruplicate, quintuple, sextuple, septuplicate, octaple, etc. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 50000 or at least 100000 production cells (of the same species or different cells) are cultured in parallel. Cell culture can be adherent or non-adherent (e.g., suspension) cell culture. The cell culture may be a two-dimensional or three-dimensional cell culture.

[0174] For example, in some embodiments, the culture container for culturing can be a culture flask, a tissue culture flask (e.g., T25, T75), a super cell culture flask (e.g., CellBind surface Corning; Reference No. 10024) or ultra-large cell culture chambers (e.g., 12 or 36 chambers, Corning, Reference Numbers: 10012, 10036, 10013, 10037), dish, culture dish, tissue culture dish, multidish, microplate, microwell plate, multiplate, multiwell plate, microslide, chamber slide, test tube, tray, The cells can be cultured in a volume of at least or about 0.2 mL, 0.5 mL, 1 mL, 2 mL, 5 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, 1 L, 5 L, 10 L, 50 L, 100 L, 1000 L, 5000 L, or 10000 L.

[0175] In some embodiments, multiple cell cultures are preferably cultured in different compartments or wells of a tissue culture container. For example, in some embodiments, multiple cell cultures are cultured adherently in different wells or parts of a tissue culture container, such as a microplate or multiwell plate (e.g., a plate containing at least 6 wells, at least 12 wells, at least 24 wells, at least 48 wells, at least 96 wells, at least 128 wells, at least 256 wells, at least 384 wells, at least 500 wells, at least 1000 wells, at least 1500 wells, at least 1536 wells, at least 2000 wells, at least 5000 wells, or at least 10000 wells.

[0176] In embodiments where the culture comprises two-dimensional cell culture, for example, when cultured on the surface of a culture vessel, the culture surface (to which the cells are intended to adhere) may be coated with one or more substances that promote cell adhesion. Such substances that can be used to enhance attachment to a solid support include, for example, collagen types I, II, and IV, concanavalin A, chondroitin sulfate, fibronectin, fibronectin-like polymers, gelatin, laminin, poly-D- and poly-L-lysine, artificial basement membrane (Matrigel), thrombospondin, osteopontin, poly-D-lysine, human extracellular matrix, Cell-Tak TM Cell and tissue adhesives, Corning Peptide hydrogels and / or vitronectin.

[0177] In some embodiments, when cell culture is carried out with adherent culture, for example, when cells adhere to a solid support, cells can be inoculated at 25000-250000 cells per square centimeter, 50000-200000 cells per square centimeter, 75000-175000 cells per square centimeter, or 100000-150000 cells per square centimeter. In some embodiments, when cell culture is carried out with adherent culture, cells can be inoculated onto a solid support under gravity. In other embodiments, cells can be inoculated onto a solid support under centrifugation.

[0178] In some embodiments, the inoculation and expansion culture of multiple cell cultures are preferably at least partially automated and / or high throughput. In some embodiments, the inoculation and culture of multiple production cell cultures are semi-automatic. In some embodiments, the inoculation and culture of multiple production cell cultures are fully automated.

[0179] The expansion culture can be carried out for different lengths of time. For example, the expansion culture can be carried out for a period of 6-96 hours, 12-72 hours, 36-60 hours, 42-56 hours, or about or at least 12 hours, about or at least 18 hours, about or at least 24 hours, about or at least 30 hours, about or at least 36 hours, about or at least 42 hours, about or at least 48 hours, about or at least 54 hours, about or at least 60 hours, about or at least 66 hours, about or at least 72 hours, about or at least 78 hours, about or at least 84 hours, about or at least 90 hours, about or at least 96 hours, about or at least 120 hours, about or at least 144 hours, about or at least 168 hours, about or at least 192 hours, about or at least 1 week, about or at least 2 weeks, about or at least 3 weeks, or about or at least 4 weeks.

[0180] In some embodiments, all or part of the expansion culture is carried out under hypoxic conditions. In some embodiments, the hypoxic condition is an O2 concentration between 0% and 15%, between 0% and 10%, or less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0181] In some embodiments, all or part of the expansion culture steps are performed under normoxic conditions. In some embodiments, at least the last 6-72 hours, the last 10-48 hours, or the last 12-36 hours of the culture step are performed under normoxic conditions. In some embodiments, normoxic conditions are O2 concentrations between 20% and 21%.

[0182] Any culture medium suitable for cultivating production cells can be used, including, for example, known and commercially available cell culture medium. In some embodiments, a basal medium containing a buffer, one or more carbon sources, an amino acid, and a salt can be used. In some embodiments, the basal medium can be supplemented with growth factors and supplements, including but not limited to additional buffers, amino acids, antibiotics, proteins, and useful growth factors, for example, growth factors (e.g., fibroblast growth factor (bFGF), also referred to as fibroblast growth factor 2 (FGF-2)) for promoting growth or maintaining or changing the differentiation state of a specific cell type. The basal medium can be any basal medium suitable for the cell type to be cultured, including, for example, a medium containing, consisting of, or comprising Dulbecco's modified Eagle's medium (DMEM), DMEM F12 medium, Eagle's minimum essential medium (MEM), α-MEM, F-12K medium, Iscove's modified Dulbecco's medium (IMDM), Knockout DMEM, RPMI-1640 medium, F-10 medium, Glasgow's modified essential medium (GMEM), McCoy 5A medium, basal medium Eagle (BME), 199 medium, or variations, combinations, or modifications thereof.

[0183] Additional supplements can also be added to the culture medium to provide cells with trace elements for optimal growth and amplification. Such supplements include, for example, insulin, transferrins, sodium selenium, Hanks balanced salt solution, Earle salt solution, antioxidant supplements, MCDB-201, phosphate buffered saline (PBS), N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES), nicotinamide, ascorbic acid and / or ascorbic acid-2-phosphate, and other amino acids and combinations thereof. These amino acids include but are not limited to L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-inositol, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

[0184] Optionally, hormones may also be used for cell culture, including but not limited to D-aldosterone, diethylstilbestrol (DES), dexamethasone, β-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine and L-thyroxine. β-Mercaptoethanol may also be supplemented to the cell culture medium.

[0185] Depending on the cell type, lipids and lipid carriers may also be used to supplement the cell culture medium. Such lipids and carriers may include, but are not limited to, cyclodextrins, cholesterol, linoleic acid coupled to albumin, linoleic acid and oleic acid coupled to albumin, uncoupled linoleic acid, linoleic acid-oleic acid-arachidonic acid coupled to albumin, uncoupled and coupled to albumin oleic acid, and the like.

[0186] In certain embodiments, albumin, such as human albumin, is present in culture medium. Albumin, including human albumin, can be, for example, isolated, synthesized, recombinant and / or modified. The amount of albumin can be adjusted according to required culture conditions and / or needs. In some embodiments, albumin can be present in an amount of 0.1 μg / mL-50 mg / mL, in an amount of 1 μg / mL-25 mg / mL, in an amount of 10 μg / mL-20 mg / mL, in an amount of 100 μg / mL-10 mg / mL, in an amount of 0.5 mg / mL-5 mg / mL, in an amount of 1 mg / mL-3 mg / mL, or in an amount of about 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL.

[0187] In some embodiments, the culture medium further comprises one or more selected from the group consisting of glutamine, biotin, DL α tocopherol acetate, DL α-tocopherol, vitamin A, catalase, insulin, transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, progesterone, putrescine, sodium selenite, triiodothyronine, amino acids, sodium pyruvate, lipoic acid, vitamin B12, nucleosides and ascorbic acid. The culture medium can also supplement one or more carbon sources. One or more carbon sources can be selected from, for example, carbon sources such as glycerol, glucose, galactose, sucrose, fructose, mannose, lactose or maltose. The culture medium can contain serum, such as fetal calf serum (fetal calfserum) or fetal bovine serum (fetal bovine serum), or the culture medium can be a serum-free culture medium.

[0188] After the expansion culture of the production cells, the culture medium used for the expansion cell culture is preferably removed and replaced with a vesicle formation medium; after the production cells are cultured in the vesicle formation medium, a conditioned medium is produced. Preferably, the conditioned medium contains extracellular vesicles.

[0189] In some embodiments, where the expanded production cells are used to analyze, develop, test and / or optimize vesicle formation medium, multiple different vesicle formation medium formulations can be tested in parallel (using multiple production cell cultures). In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 50000 or at least 100000 different vesicle formation medium formulations can be tested in parallel or continuously.

[0190] For example, by mixing or combining two or more different types of culture media, a variety of vesicle formation medium formulations can be produced. In some embodiments, one or more culture media used for mixing or combining are known in the art and / or commercially available. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 or more culture media are mixed or combined to produce a specific vesicle formation medium formulation. The culture medium used for mixing can be any basic culture medium suitable for the cell type to be cultured, including, for example, a culture medium containing, consisting of or comprising the following substances: Dulbecco's modified Eagle medium (DMEM), DMEM F12 medium, Eagle's minimum essential medium (MEM), α-MEM, F-12K medium, Iscove's modified Dulbecco's medium (IMDM), Knockout DMEM, RPMI-1640 medium, F-10 medium, Glasgow's modified essential medium (GMEM), McCoy 5A medium, basal medium Eagle (BME), 199 medium or their variants, combinations or modifications.

[0191] The culture medium for mixing may contain one or more supplements or additives. In some embodiments, the supplements or additives may be one or more growth factors. In some embodiments, the one or more growth factors may be selected from adrenomedullin, angiogenin, autotaxin, bone morphogenetic protein (BMPs), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), epidermal growth factor (EGF), ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3, erythropoietin (Erythr opoietin)(EPO), fibroblast growth factor 1(FGF-1), fibroblast growth factor 2(FGF-2), fibroblast growth factor 3(FGF-3), fibroblast growth factor 4(FGF-4), fibroblast growth factor 5(FGF-5), fibroblast growth factor 6(FGF-6), fibroblast growth factor 7(FGF-7), fibroblast growth factor 8(FGF-8), fibroblast growth factor 9(FGF-9), fibroblast growth factor 10(FGF-10), fibroblast growth factor 11(FGF-11), fibroblast growth factor 12(FGF-1 2), Fibroblast growth factor 13 (FGF-13), Fibroblast growth factor 14 (FGF-14), Fibroblast growth factor 15 (FGF-15), Fibroblast growth factor 16 (FGF-16), Fibroblast growth factor 17 (FGF-17), Fibroblast growth factor 18 (FGF-18), Fibroblast growth factor 19 (FGF-19), Fibroblast growth factor 20 (FGF-20), Fibroblast growth factor 21 (FGF-21), Fibroblast growth factor 22 (FG-F22), Fibroblast growth factor 23 (FGF-23), Fetal bovine growth hormone (FBS) , glial cell line-derived neurotrophic factor (GDNF), neurotrophic factors, Persephin, Artemin, growth differentiation factor-9 (GDF-9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, keratinocyte growth factor (KGF), migration stimulating factor (MSF), macrophage stimulating protein (MSP), myostatin (GDF-8), neuregulin 1 (NRG1),Neuregulin 2 (NRG2), Neuregulin 3 (NRG3), Neuregulin 4 (NRG4), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), placental growth factor (PGF), platelet-derived growth factor (PDGF), renal enzyme (RNLS), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF).

[0192] In some embodiments, one or more growth factors can each independently be present in an amount of 0.001 μg / mL-1000 μg / mL, in an amount of 0.01 μg / mL-100 μg / mL, in an amount of 0.1 μg / mL-10 μg / mL, in an amount of 0.05 μg / mL-5 μg / mL, in an amount of 0.5 μg / mL-2.5 μg / mL, or in an amount of about 0.5 μg / mL, an amount of about 1 μg / mL, an amount of about 2 μg / mL, an amount of about 3 μg / mL, an amount of about 4 μg / mL, or an amount of about 5 μg / mL.

[0193] Other supplements or additives can include, for example, a carbon source (such as glycerol, glucose, galactose, sucrose, fructose, mannose, lactose or maltose), albumin, biotin, DL alpha tocopheryl acetate, DL alpha-tocopherol, vitamin A, catalase, superoxide dismutase, corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, putrescine, sodium selenite, triiodothyronine, sodium pyruvate, lipoic acid, vitamin B12, nucleosides, β-mercaptoethanol, insulin, transferrin, sodium selenium, Hanks balanced salt solution, Earle's salt solution, antioxidant supplements, MCDB-201, phosphate buffered saline (PBS), N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES), nicotinamide, ascorbic acid and / or ascorbic acid-2-phosphate, and other amino acids and combinations thereof. These amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-inositol, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. Optionally, hormones may be added, including, but not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, β-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, and L-thyroxine.

[0194] Lipids and lipid carriers may also be used as additives or supplements. Such lipids and carriers may include, but are not limited to, cyclodextrins, cholesterol, linoleic acid coupled to albumin, linoleic acid and oleic acid coupled to albumin, uncoupled linoleic acid, linoleic acid-oleic acid-arachidonic acid coupled to albumin, uncoupled and coupled albumin oleic acid, and the like.

[0195] In some embodiments, the set of vesicle formation medium preparations is generated by mixing or combining two or more different types of culture media from initially selected different culture media. In some embodiments, the initially selected different culture media comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, or at least 100 different culture media.

[0196] In some embodiments, the initial selection of different culture media is based on the suitability of a particular culture medium for a particular type of extracellular vesicle-secreting cell, culture conditions, desired outcomes, and other considerations. In some embodiments, the initial selection of different culture media is performed by screening different culture media with desired characteristics, for example, by culturing extracellular vesicle-secreting cells in the culture medium to obtain conditioned medium, and analyzing one or more properties of extracellular vesicles in the recovered conditioned medium, the recovered cells, or both.

[0197] In some embodiments, a panel of vesicle formation medium formulations is generated by mixing or combining two or more different types of culture media together without requiring any initial selection or screening of different culture media.

[0198] Alternatively, multiple vesicle formation medium formulations can be generated by adding one or more supplements, additives, etc. to an existing culture medium (or to a mixture of different types of culture media, as described above), for example, in different combinations and / or at different concentrations.

[0199] In some embodiments, the mixing or combination of different culture media; and / or the addition of one or more additives or supplements to prepare the vesicle formation medium formulation can be assisted or strategized using one or more statistical methods. In some embodiments, a design of experiments (DoE) method is used. In some embodiments, the mixing or combination of different culture media; and / or the addition of one or more additives or supplements to prepare the vesicle formation medium formulation can be assisted or strategized using software (e.g., StatEase's Design Expert software).

[0200] In some embodiments, the supplement or additive may be one or more growth factors. In some embodiments, the one or more growth factors may be selected from adrenomedullin, angiogenin, autotaxin, bone morphogenetic proteins (BMPs), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), epidermal growth factor (EGF), ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3, erythropoietin (EPO), fibroblast growth factor 1 (FGF-1) , fibroblast growth factor 2 (FGF-2), fibroblast growth factor 3 (FGF-3), fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5), fibroblast growth factor 6 (FGF-6), fibroblast growth factor 7 (FGF-7), fibroblast growth factor 8 (FGF-8), fibroblast growth factor 9 (FGF-9), fibroblast growth factor 10 (FGF-10), fibroblast growth factor 11 (FGF-11), fibroblast growth factor 12 (FGF-12), fibroblast growth factor 13 (FGF-13), fibroblast growth factor 14 (FGF-14 ), fibroblast growth factor 15 (FGF-15), fibroblast growth factor 16 (FGF-16), fibroblast growth factor 17 (FGF-17), fibroblast growth factor 18 (FGF-18), fibroblast growth factor 19 (FGF-19), fibroblast growth factor 20 (FGF-20), fibroblast growth factor 21 (FGF-21), fibroblast growth factor 22 (FG-F22), fibroblast growth factor 23 (FGF-23), fetal bovine growth hormone (FBS), glial cell line-derived neurotrophic factor (GDNF), neurotrophic factors, Persephin, Artemin, growth differentiation factor GDF-9, hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, keratinocyte growth factor (KGF), migration stimulating factor (MSF), macrophage stimulating protein (MSP), myostatin (GDF-8), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), neuregulin 4 (NRG4), brain-derived neurotrophic factor (BDNF),Nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), placental growth factor (PGF), platelet-derived growth factor (PDGF), renal enzyme (RNLS), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF).

[0201] In some embodiments, one or more growth factors can each independently be present in an amount of 0.001 μg / mL-1000 μg / mL, in an amount of 0.01 μg / mL-100 μg / mL, in an amount of 0.1 μg / mL-10 μg / mL, in an amount of 0.05 μg / mL-5 μg / mL, in an amount of 0.5 μg / mL-2.5 μg / mL, or in an amount of about 0.5 μg / mL, an amount of about 1 μg / mL, an amount of about 2 μg / mL, an amount of about 3 μg / mL, an amount of about 4 μg / mL, or an amount of about 5 μg / mL.

[0202] Other supplements or additives can include, for example, a carbon source (such as glycerol, glucose, galactose, sucrose, fructose, mannose, lactose or maltose), albumin, biotin, DL alpha tocopheryl acetate, DL alpha-tocopherol, vitamin A, catalase, superoxide dismutase, corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, putrescine, sodium selenite, triiodothyronine, sodium pyruvate, lipoic acid, vitamin B12, nucleosides, β-mercaptoethanol, insulin, transferrin, sodium selenium, Hanks balanced salt solution, Earle's salt solution, antioxidant supplements, MCDB-201, phosphate buffered saline (PBS), N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES), nicotinamide, ascorbic acid and / or ascorbic acid-2-phosphate, and other amino acids and combinations thereof. These amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-inositol, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. Optionally, hormones may be added, including, but not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, β-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, and L-thyroxine.

[0203] Lipids and lipid carriers may also be used as additives or supplements. Such lipids and carriers may include, but are not limited to, cyclodextrins, cholesterol, linoleic acid coupled to albumin, linoleic acid and oleic acid coupled to albumin, uncoupled linoleic acid, linoleic acid-oleic acid-arachidonic acid coupled to albumin, uncoupled and coupled albumin oleic acid, and the like.

[0204] Vesicle formation culture can be carried out for different lengths of time. For example, the culture can be carried out for a period of 6-96 hours, 12-72 hours, 36-60 hours, 42-56 hours, or about or at least 6 hours, about or at least 12 hours, about or at least 18 hours, about or at least 24 hours, about or at least 36 hours, about or at least 48 hours, about or at least 60 hours, about or at least 72 hours, about or at least 84 hours, about or at least 96 hours, about or at least 120 hours, about or at least 144 hours, about or at least 168 hours, about or at least 192 hours, about or at least 1 week, about or at least 2 weeks, about or at least 3 weeks, or about or at least 4 weeks.

[0205] In some embodiments, all or part of the vesicle formation culture is carried out under hypoxic conditions. In some embodiments, the last 6-72 hours, the last 10-48 hours, or the last 12-36 hours of the culture are carried out under hypoxic conditions. In some embodiments, the hypoxic condition is an O2 concentration between 0% and 15%, between 0% and 10%, or less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0206] In some embodiments, all or part of the vesicle formation culturing step is performed under normoxic conditions. In some embodiments, at least the last 6-72 hours, the last 10-48 hours, or the last 12-36 hours of the culturing step is performed under normoxic conditions. In some embodiments, normoxic conditions are O2 concentrations between 20% and 21%.

[0207] In some embodiments, removal of expansion medium from expansion cell cultures, washing of cell cultures, preparation of vesicle formation medium formulations, addition of vesicle formation medium formulations to cell cultures, culturing of cell cultures (expansion and / or vesicle formation culturing), and / or harvesting of conditioned medium are at least partially automated (e.g., semi-automated or fully automated), and / or high-throughput. For example, an automated liquid handler, such as manufactured by Beckman Coulter, may be used. Automated liquid handler.

[0208] During and / or after culturing the producer cells in the vesicle formation medium (the vesicle formation phase during which conditioned medium containing extracellular vesicles is produced), one or more properties of the cultured cells can be examined (including, for example: total cell number, cell density, number of live cells, percentage of cell viability; cell morphology; cell identity; cell karyotype; cell transcriptome, hypertrophy, cell health, cell adhesion, cell physiology and / or ATP content).

[0209] In some embodiments, the cultured production cells are analyzed by counting (e.g., by determining cell density) and / or using a cell viability assay. In some embodiments, during and / or after culturing the production cells in the vesicle formation medium, the cells are stained with a DNA labeling dye and / or a nuclear staining dye and counted (e.g., using a cell counter). In some embodiments thereof, the DNA labeling dye or the nuclear staining dye is a fluorescent dye.

[0210] In some embodiments, during and / or after culturing the production cells in the vesicle formation medium, the production cells are stained with acridine orange and propidium iodide and counted using a cell counter. In some embodiments, the counting of live and / or non-live cells is at least partially automated (e.g., semi-automatic or fully automated) and / or high-throughput. For example, the Cellaca cell counter manufactured by Nexcelom can be used.

[0211] Additionally, or alternatively, one or more assays (including, for example, particle number; particle concentration; particle size distribution; protein concentration; protein profile concentration; RNA profile; potency; marker expression; host cell protein assessment; residual DNA quantification and / or characterization; appearance; pH; osmotic pressure, etc.) can be used to determine one or more properties of the extracellular vesicles.

[0212] In some embodiments, the conditioned medium is analyzed to estimate or determine the number and / or type of extracellular vesicles in the conditioned medium. In some embodiments, the number and / or type of extracellular vesicles in the conditioned medium is estimated or determined using an affinity-based assay such as an immunoassay. In some embodiments, the immunoassay can be a high-throughput immunoassay. In some embodiments, the immunoassay can be, for example, an enzyme-linked immunosorbent assay (ELISA), a competitive binding assay, an immunoassay, a radioimmunoassay (RIA), a fluorescent immunoassay (FIA), a chemiluminescent immunoassay (CLIA), a counting immunoassay (CIA) or a flow cytometry or FACS.

[0213] In some embodiments, the extracellular vesicles in the conditioned medium are captured using an agent (such as a protein) that has an affinity for the extracellular vesicles. In some embodiments, the agent is a Tim4 protein (or a variant or derivative thereof) that has an affinity for phosphatidylserine displayed on the surface of the extracellular vesicles. In some embodiments, the captured extracellular vesicles can then be detected using a different agent that binds or detects the extracellular vesicles, such as, for example, an agent that binds or detects any one or more markers present on the extracellular vesicles. Such a marker can be any marker present in an extracellular vesicle. Such markers can be selected from, for example, tetraspanins (e.g., CD9, CD63, and CD81), ceramides, MHC class I, MHC class II, integrins, adhesion molecules, phosphatidylserine, sphingomyelin, cholesterol, cytoskeletal proteins (e.g., actin, gelsolin, myosin, tubulin), enzymes (e.g., catalase, GAPDH, nitric oxide synthase, LT synthase), nucleic acids (e.g., RNA, miRNA), heat shock proteins (e.g., HSC70, HSP60, HSP70, HSPA5, CCT2, and HSP90), exosome biogenesis proteins (ALIX, Tsg101), LT, prostaglandins, and S100 proteins.Other exemplary extracellular vesicle markers that can be analyzed and / or detected include, for example, CD3, vesicle proteins (e.g., vesicle proteins-1, vesicle proteins-2), TSG101 (tumor susceptibility gene 101), CD4, CD19, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CD1c, CD25, CD49e, ROR1 (receptor tyrosine kinase-like orphan receptor 1), CD209, SSEA-4 (stage-specific embryonic antigen-4), HLA-ABCG, CD40, CD 62P, CD11c, MCSP (melanoma-associated chondroitin sulfate proteoglycan), CD146, CD41b, CD42a, CD24, CD86, CD44, CD326, CD133 / 1, CD29, CD69, CD142, CD45, CD31, CD20, CD14, Rab-5b, TSG101, annexins (e.g., annexin 2, annexin 5), programmed cell death 6 interacting protein (Alix), fibronectin 1, galectin 3 binding protein, α2 macrophage Globulin, hemoglobin β subunit, gelsolin, β-actin, β2 microglobulin, human erythrocyte integral membrane protein, moesin, peroxiredoxin 2, RAP1B (RAS oncogene family member), filamin A, integrin, selectin, multifunctional intracellular adaptor protein (such as multifunctional intracellular adaptor protein-1), syndecan binding protein (SDCBP), 14-3-3 protein, IMMT (mitochondrial protein), Disco interacting protein 2 homolog B (DIP2B), four transmembrane proteins One or more of superfamily members TSPAN6 and TSPAN3, inhibitory domain-containing protein 11 (ARRDC1), immunoglobulin superfamily member 8 (IGSF8), CD82, TfR2, LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, NT5E, complement binding proteins (such as CD55 and CD59), glycophorin A, AChE-E, β-amyloid A4 / APP, ESCRT-I / II / III, and caveolins.

[0214] In some embodiments, the reagent that binds or detects extracellular vesicles is an antibody. In some embodiments, the reagent (such as an antibody) binds or detects CD9, CD63 or CD81. In some embodiments, the reagent is an antibody that binds CD63.

[0215] In some embodiments, the inoculation and expansion of multiple cell cultures are preferably at least partially automated and / or high throughput. In some embodiments, the inoculation and cultivation of multiple cell cultures are semi-automatic. In some embodiments, the inoculation and cultivation of multiple cell cultures are fully automated.

[0216] In some embodiments, the conditioned medium is analyzed using a high-throughput ELISA to estimate or determine the number and / or type of extracellular vesicles therein, the extracellular vesicles are captured using Tim4 protein (or its variant or derivative), and the captured extracellular vesicles are detected using an anti-CD63 antibody.

[0217] In some embodiments, the extracellular vesicles in the conditioned medium are characterized at the level of single extracellular vesicles. In some embodiments, single extracellular vesicles are analyzed for the presence of one or more markers, and / or their size is determined. In some embodiments, single extracellular vesicles are analyzed using microscopy techniques. In some embodiments, the microscopy technique is a super-resolution microscopy technique. In some embodiments, the super-resolution microscope is a direct stochastic optical reconstruction microscope (dSTORM).

[0218] In some embodiments, the presence of one or more markers on the extracellular vesicle is analyzed at the level of a single extracellular vesicle. Such a marker can be any marker present in an extracellular vesicle. Such a marker can be selected from four transmembrane proteins (e.g., CD9, CD63, and CD81), ceramides, MHC class I, MHC class II, integrins, adhesion molecules, phosphatidylserine, sphingomyelin, cholesterol, cytoskeletal proteins (e.g., actin, gelsolin, myosin, tubulin), enzymes (e.g., catalase, GAPDH, nitric oxide synthase, LT synthase), nucleic acids (e.g., RNA, miRNA), heat shock proteins (e.g., HSC70, HSP60, HSP70, HSPA5, CCT2, and HSP90), exosome biogenesis proteins (ALIX, Tsg101), LT, prostaglandins, and S100 proteins.Other exemplary extracellular vesicle markers that can be analyzed and / or detected include, for example, CD3, vesicle proteins (e.g., vesicle proteins-1, vesicle proteins-2), TSG101 (tumor susceptibility gene 101), CD4, CD19, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CD1c, CD25, CD49e, ROR1 (receptor tyrosine kinase-like orphan receptor 1), CD209, SSEA-4 (stage-specific embryonic antigen-4), HLA-ABCG, CD 40, CD62P, CD11c, MCSP (melanoma-associated chondroitin sulfate proteoglycan), CD146, CD41b, CD42a, CD24, CD86, CD44, CD326, CD133 / 1, CD29, CD69, CD142, CD45, CD31, CD20, CD14, Rab-5b, TSG101, annexins (e.g., annexin 2, annexin 5), programmed cell death 6 interacting protein (Alix), fibronectin 1, galectin 3 binding protein, α2 macroglobulin, hemoglobin β subunit, gelsolin, β-actin, β2 microglobulin, human erythrocyte integral membrane protein, moesin, peroxiredoxin 2, RAP1B (RAS oncogene family member), filamin A, integrin, selectin, multifunctional intracellular adaptor protein (such as multifunctional intracellular adaptor protein-1), syndecan binding protein (SDCBP), 14-3-3 protein, IMMT (mitochondrial protein), Disco interacting protein 2 homolog B (DIP2B), One or more of the four-transmembrane protein superfamily members TSPAN6 and TSPAN3, inhibitory domain-containing protein 11 (ARRDC1), immunoglobulin superfamily member 8 (IGSF8), CD82, TfR2, LAMP1 / 2, heparan sulfate proteoglycans, EMMPRIN, ADAM10, NT5E, complement binding proteins (such as CD55 and CD59), glycophorin A, AChE-E, β-amyloid A4 / APP, ESCRT-I / II / III, and caveolin.

[0219] In some embodiments, the presence of one or more of CD9, CD63, and CD81 of the extracellular vesicles is analyzed at the level of single extracellular vesicles. In some embodiments, the presence of CD9, CD63, and CD81 of single extracellular vesicles is analyzed.

[0220] In some embodiments, prior to analyzing extracellular vesicles at the level of single extracellular vesicles, extracellular vesicles are captured from conditioned medium prior to labeling with one or more antibodies that bind to a marker of interest. In some embodiments, prior to labeling with one or more antibodies that bind to a marker of interest, extracellular vesicles are captured on a solid phase, such as a chip or a cartridge. In some embodiments, the antibody is labeled with a fluorescent group.

[0221] In some embodiments, the conditioned medium may undergo one or more further processing steps. For example, the conditioned medium and / or the extracellular vesicles in the conditioned medium may be removed, recovered, concentrated, enriched, separated, purified, refrigerated, frozen, cryopreserved, lyophilized, sterilized, etc. The conditioned medium may also be pre-cleaned or clarified by one or more centrifugation and / or filtration techniques. For example, the extracellular vesicles may be enriched, purified or further concentrated by, for example, centrifugation, ultracentrifugation, filtration, ultrafiltration, gravity, ultrasound, density gradient ultracentrifugation, tangential flow filtration, size exclusion chromatography, ion exchange chromatography, affinity capture, polymer-based precipitation or organic solvent precipitation.

[0222] experiment

[0223] The following examples illustrate non-limiting embodiments of the present invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., quantity, concentration, percentage change etc.), but some experimental errors and deviations should be taken into account. It should be understood that these embodiments are only provided in an illustrative manner, and are not intended to limit the scope of the various embodiments of the present invention that the inventors consider. Not all of the following steps listed in each embodiment are necessary, and the order of steps in each embodiment is not necessarily as presented.

[0224] Example 1

[0225] Development of a high-throughput culture medium for extracellular vesicle production by iPSC-derived cells

[0226] iPSC-derived cells were used as a model for extracellular vesicle production in a high-throughput assay for testing multiple vesicle formation medium formulations. This high-throughput, semi-automated assay allowed for the production of multiple different vesicle formation medium formulations and then testing them simultaneously, also reducing experimental time. Figure 1 A flow chart showing the various stages of an exemplary assay performed.

[0227] In order to prepare a variety of different vesicle formation medium formulations for analysis, the medium mixing was strategized using the experimental design (DoE) method using Design Expert software (StatEase). Six different types of culture media (including commercially available culture media) were used as mixed starting media. These six different types of culture media were mixed using an automatic liquid handler (Biomek i7 automatic liquid handler manufactured by Beckman Coulter) to produce 31 different vesicle formation medium mixtures (M1-M31). The resulting 31 groups of culture medium mixtures were then tested as follows.

[0228] After iPSC-derived cells were expanded and cultured in multiwell plates, the expansion medium was removed from the cell culture wells and replaced with a reference control medium ("reference") or a vesicle formation medium mixture (M1-M31). Duplicate wells were used for control and vesicle formation medium mixtures. The cells were then cultured to produce conditioned medium containing extracellular vesicles. Expansion medium removal, vesicle formation medium replacement, vesicle formation culture, and conditioned medium removal were all performed semi-automatically (using a Biomek i7 automated liquid handler manufactured by Beckman Coulter).

[0229] After collecting the conditioned medium, the cell density and cell viability of the cultured cells were assessed in a semi-automated manner. Briefly, live and dead cells were stained with AOPI (acridine orange and propidium iodide) and counted using an automated Cellacca cell counter manufactured by Nexcelom. The results are shown in Figure 2 , the graph depicts viable cell density after incubation with control and M1-M31 vesicle formation medium formulations. Figure 2 As can be seen, this procedure allows for the efficient determination of the effects of different vesicle formation medium formulations on cell viability and the identification of vesicle formation medium formulations that promote cell growth.

[0230] In addition, the recovered conditioned medium was then analyzed to determine the extracellular vesicle yield using different vesicle formation media. Extracellular vesicle yield was determined using a Tim4-based high-throughput extracellular vesicle ELISA (EV ELISA) assay, where Tim4 was used to capture extracellular vesicles in the conditioned medium (ELISA kit #297-79201, FUJIFILM Wako Pure Chemical Industries, Ltd.). The captured extracellular vesicles were detected using an anti-CD63 antibody. The results are shown in Figure 3A and Figure 3B shown. Figure 3A Data depicting total extracellular vesicle production, Figure 3B Extracellular vesicle production per cell is depicted. Total extracellular vesicle counts were estimated based on the amount of CD63 on the surface of extracellular vesicles. Figure 3A and Figure 3B As can be seen, this procedure allows for the efficient determination of the effects of different vesicle formation medium formulations on extracellular vesicle yields and the identification of vesicle formation medium formulations that promote extracellular vesicle production. Figure 3A and Figure 3B It was also shown that formulation M17 produced more extracellular vesicles than the benchmark control medium and more than the other vesicle formation medium formulations tested.

[0231] To determine the accuracy of the automated liquid handling steps described above, extracellular vesicle production using the automated liquid handling process was compared to an equivalent step performed by manual processing. The comparison was made using the Tim4-based extracellular vesicle ELISA assay described above. The results are shown in Figure 4 The CD63 level in the baseline culture medium was set as 100%, and the X and Y axes showed the relative EV production levels. Figure 4 As shown, the extracellular vesicle yield using the automated liquid handler correlated closely with that using manual processing, confirming the accuracy and robustness of the high-throughput, semi-automated extracellular vesicle production process.

[0232] Next, super-resolution microscopy (direct stochastic optical reconstruction microscopy (dSTORM)) was used to characterize the extracellular vesicles in the conditioned medium at the level of single extracellular vesicles. Briefly, for each sample, 10 μL of conditioned medium was used as the injection sample. The extracellular vesicles in the injection sample were captured on the chip using the EV Profiler Kit (manufactured by ONi) and labeled with CD63, CD81, and CD9 fluorophore-coupled antibodies included in the kit. The individual fluorophores were then located using a Nanoimager S (manufactured by ONi). The images obtained were then processed and analyzed using CODI software (https: / / oni.bio / applications / ) and R.

[0233] like Figure 5A , Figure 5B and Figure 5C The total number of extracellular vesicles ( Figure 5A ) and nanoparticle tracking analysis (NTA) ( Figure 5B ) was closely correlated with CD63 expression levels determined using the EV ELISA assay ( Figure 5C ).

[0234] Figure 6Results of a human cardiomyocyte survival assay performed to evaluate EV function are presented. EVs purified from conditioned medium and mock control medium using ultracentrifugation were used as injection samples. Testing of EV efficacy in a cardiomyocyte survival assay was described in International Patent Application Publication No. WO2022106890A1. EVs produced by candidate vesicle formation media were functional in a staurosporine CM2 activity assay.

[0235] Figure 7 Results of a HUVEC scratch wound healing assay performed to assess EV function are shown. EVs purified from conditioned media and mock control media using ultracentrifugation were used as injection samples. Essen BioSciences ELISA kit was used according to the manufacturer’s instructions. Developed scratch wound healing assay. Results show that EVs generated by candidate vesicle-forming cultures are functional in the wound healing assay.

[0236] The above experiments demonstrate that using iPSC-derived cells as a model, vesicle-forming media that promote cell growth and stimulate extracellular vesicle secretion can be identified by this high-throughput approach compared to a benchmark control medium. These experiments further demonstrate the surprising effect of culture media on the cell microenvironment, affecting not only cell expansion and cell quality, but also the yield, quality, and purity of extracellular vesicles.

[0237] Example 2

[0238] Development of a high-throughput culture medium for the production of extracellular vesicles from primary MSCs

[0239] Primary mesenchymal stromal stem cells (MSCs) were used as a model for extracellular vesicle production in a high-throughput assay for testing multiple vesicle formation medium formulations. This high-throughput, semi-automated assay allows for multiple different vesicle formation medium formulations to be produced and then tested simultaneously, also reducing experimental time. Figure 1 A flow chart showing the various stages of an exemplary assay performed.

[0240] In order to prepare a variety of different vesicle formation medium formulations for analysis, the design of experiments (DoE) method was used to strategize the medium mixing using Design Expert software (StatEase). Six different types of culture media (including commercially available culture media) were used as mixed starting media. These six different types of culture media were mixed using an automatic liquid handler (Biomek i7 automatic liquid handler manufactured by Beckman Coulter) to produce 46 different vesicle formation medium mixtures (M1-M46). The resulting 46 groups of culture media mixtures were then tested as follows.

[0241] After MSCs were expanded and cultured in multi-well plates, the expansion medium was removed from the cell culture wells and replaced with a reference control medium ("reference") or a vesicle formation medium mixture (M1-M46). Duplicate wells were used for controls and some vesicle formation medium mixtures. The cells were then cultured to produce conditioned medium containing extracellular vesicles. Expansion medium removal, vesicle formation medium replacement, vesicle formation culture, and conditioned medium removal were all performed in a semi-automatic manner (using a Biomek i7 automated liquid handler manufactured by BeckmanCoulter).

[0242] After collecting the conditioned medium, the cell density of the cultured cells was assessed in a semi-automated manner. Briefly, live cells were analyzed using PrestoBlue TM Cell viability reagent (Thermo Fisher Scientific) was used for staining, and the fluorescence intensity was measured by a plate reader. The results are shown in Figure 8 , the graph depicts viable cell density after incubation with control and M1-M46 vesicle formation medium formulations. Figure 8 As can be seen, this procedure allows for the efficient determination of the effects of different vesicle formation medium formulations on cell viability and the identification of vesicle formation medium formulations that promote cell growth.

[0243] In addition, the recovered conditioned medium was then analyzed to determine the extracellular vesicle yield using different vesicle formation media. Extracellular vesicle yield was determined using a Tim4-based high-throughput extracellular vesicle ELISA (EV ELISA) assay, in which Tim4 was used to capture extracellular vesicles in the conditioned medium and anti-CD63, anti-CD9, and anti-CD81 antibodies were used to detect the captured extracellular vesicles. The results are shown in Figure 8 and Fig. 9 In, it depicts the data of total extracellular vesicle production and extracellular vesicle production per fixed cell, respectively. The total extracellular vesicle count was estimated based on the amount of CD63, CD9 and CD81 on the surface of extracellular vesicles. Figure 8 and Fig. 9 As can be seen, this procedure allows for the efficient determination of the effects of different vesicle formation medium formulations on extracellular vesicle yields and the identification of vesicle formation medium formulations that promote extracellular vesicle production. Figure 8 and Fig. 9 It was also shown that some formulations, including formulation M3, produced more extracellular vesicles than the benchmark control medium and more than the other vesicle formation medium formulations tested.

[0244] Next, super-resolution microscopy (direct stochastic optical reconstruction microscopy (dSTORM)) was used to characterize the extracellular vesicles in the conditioned medium at the level of single extracellular vesicles. Briefly, for each sample, 4.5 μL of purified EVs were used as the injection sample. The extracellular vesicles in the injection sample were captured on the chip using the EV Profiler Kit (manufactured by ONi) and labeled with CD63, CD81, and CD9 antibodies before analysis. The individual fluorophores were then localized using a Nanoimager S (manufactured by ONi). The images obtained were then processed and analyzed using CODI software (https: / / oni.bio / applications / ) and R. The results are presented in Fig.10 middle.

[0245] Then, the samples were used for primary MSC cell culture and vesicle formation using T-150. The collected extracellular vesicles were analyzed using EV ELISA assay.

[0246] Fig.11A and Fig. 11B Shown are the expression levels of CD63, CD9, and CD81 determined using the EV ELISA assay for vesicles generated manually in culture flasks ( Fig.11A ) and in multiwell plates ( Fig. 11B ). By comparison Fig.10 , Fig.11A and Fig. 11B As can be seen in the results, the total number of extracellular vesicles determined from single extracellular vesicle (dSTORM) analysis correlated closely with the expression levels of CD63, CD9, and CD81 determined using the EV ELISA assay.

[0247] HUVEC plate assay was performed to evaluate EV function. EVs purified from conditioned medium and mock medium using ultracentrifugation were used as injection samples. Fig.12 As shown, EVs purified from conditioned media stimulated HUVEC cell growth.

[0248] The above experiments demonstrate that using MSCs as a model, vesicle-forming media that promote cell growth and stimulate extracellular vesicle secretion can be identified by this high-throughput approach compared to a benchmark control medium. These experiments further demonstrate the surprising effect of culture media on the cell microenvironment, affecting not only cell expansion and cell quality, but also the yield, quality, and purity of extracellular vesicles.

Claims

1. A high-throughput method for analyzing, developing and / or optimizing a culture medium for extracellular vesicle production, the method comprising: (a) culturing cells in a first culture medium, wherein cell division occurs during the culturing, and wherein the culturing is performed in multiples; (b) after step (a), removing the first culture medium from the multiple cell cultures, adding different candidate vesicle formation culture media to different cell cultures in the multiple cell cultures, and further culturing the multiple cell cultures to produce conditioned medium containing extracellular vesicles; (c) recovering the conditioned medium from the multiple cell cultures or the cells after culturing in step (b), or both; and (d) analyzing at least one property of the extracellular vesicles in the recovered conditioned medium or the recovered cells, or both, wherein steps (a) to (c) are each semi-automated using an automated liquid.

2. The method of claim 1, wherein at least one of steps (a) to (c) is fully automated.

3. The method according to claim 1, wherein each of steps (a) to (c) is semi-automatic or fully automatic.

4. The method according to any one of claims 1-3, wherein the cells are iPSC-derived cells and / or primary stem cells.

5. The method according to any one of claims 1 to 4, wherein the culture is a two-dimensional cell culture. The method according to claim 5 , wherein the two-dimensional cell culture comprises culturing the cells on a surface of a culture container. The method according to claim 6 , wherein the surface of the culture container is coated with a substance that promotes cell adhesion. The method according to claim 7 , wherein the substance promoting cell adhesion is vitronectin or fibronectin.

9. The method according to any one of claims 1 to 8, wherein the multiple cell cultures are cultured in one or more multi-well plates or microplates.

10. The method according to any one of claims 1 to 9, wherein the different candidate vesicle formation culture media in step (b) are obtained by mixing or combining two or more culture media, and / or adding one or more additives or supplements to one or more culture media, to produce a group of different candidate vesicle formation culture media.

11. The method of claim 10, wherein the set of different candidate vesicle formation media is generated using an automated liquid handler.

12. The method of claim 11, wherein the set of candidate vesicle formation culture media is generated by mixing or combining two or more different culture media from at least five different culture media initially selected.

13. The method of claim 11, wherein the set of candidate vesicle formation culture media is generated by mixing or combining two or more culture media from at least ten different culture media initially selected.

14. The method of claim 11, wherein the set of candidate vesicle formation culture media is generated by mixing or combining two or more culture media from at least twenty different culture media initially selected.

15. The method of claim 11, wherein the set of candidate vesicle formation culture media is generated by mixing or combining two or more culture media from an initially selected set of at least fifty different culture media.

16. The method of any one of claims 11-15, wherein the set of candidate vesicle formation culture media comprises at least ten candidate vesicle formation culture media.

17. The method of claim 16, wherein the set of candidate vesicle formation culture media comprises at least twenty candidate vesicle formation culture media.

18. The method of claim 16, wherein the panel of candidate vesicle formation culture media comprises at least thirty candidate vesicle formation culture media.

19. The method of claim 16, wherein the set of candidate vesicle formation media comprises at least fifty candidate vesicle formation media.

20. The method of claim 16, wherein the panel of candidate vesicle formation media comprises at least one hundred candidate vesicle formation media.

21. The method of any one of claims 1-20, wherein the cells comprise progenitor cells.

22. The method of any one of claims 1-21, wherein the cells have been previously cryopreserved or frozen.

23. The method of any one of claims 1-22, wherein at least one property of the recovered cells from step (c) analyzed is selected from the group consisting of cell number, cell viability, cell density, cell morphology, cell identity, cell karyotype, cell transcriptome, hypertrophy, cell health, cell adhesion, cell physiology and / or ATP content.

24. The method of claim 23, wherein at least one property is selected from the group consisting of cell number and cell viability.

25. The method of claim 24, wherein cell number and / or cell viability is measured using an automated cell counter.

26. The method according to claim 24 or 25, wherein the cell number and / or cell viability is determined by staining the cells with at least one dye.

27. The method of claim 26, wherein the cells are stained with acridine orange and / or propidium iodide.

28. The method of any one of claims 1-22, wherein at least one property of the extracellular vesicles analyzed is the total number of extracellular vesicles, the number of extracellular vesicles per cell, the extracellular vesicle concentration, the extracellular vesicle size, the extracellular vesicle size distribution, the protein concentration, the protein profile concentration, the RNA profile, the potency, or the marker expression.

29. The method of claim 28, wherein at least one property of the extracellular vesicles analyzed is selected from the group consisting of total number of extracellular vesicles, number of extracellular vesicles per cell, extracellular vesicle size, and marker expression.

30. The method of claim 29, wherein the total number of extracellular vesicles and / or the number of extracellular vesicles per cell is determined by measuring the expression of at least one marker present on the extracellular vesicles.

31. The method of claim 30, wherein the marker is a tetraspanin.

32. The method of claim 31, wherein the quadruple transmembrane protein is selected from the group consisting of CD9, CD63, and CD81.

33. The method of claim 31, wherein the quadruple transmembrane protein is CD63.

34. The method of any one of claims 29-33, wherein the marker is detected using an immunoassay.

35. The method of claim 34, wherein the immunoassay is an ELISA.

36. The method of claim 35, wherein the ELISA is a Tim4-capture ELISA.

37. The method of claim 36, wherein the ELISA is a Tim4-capture ELISA that detects CD63 expression.

38. A method according to any one of claims 23-37, wherein the measuring of at least one property is at least partially automated.

39. The method of claim 28, wherein at least one property of the extracellular vesicles analyzed is analyzed at the level of a single extracellular vesicle.

40. The method of claim 39, wherein at least one property analyzed at the level of single extracellular vesicles is marker expression.

41. The method of claim 39 or 40, wherein the analysis is performed using super-resolution microscopy.

42. The method of claim 41, wherein the super-resolution microscope is direct stochastic optical reconstruction microscopy (dSTORM).

43. The method of any one of claims 40-42, wherein the marker expression analyzed comprises analyzing the expression of at least one tetratransmembrane protein.

44. The method of claim 43, wherein the quadruple transmembrane protein is selected from the group consisting of CD9, CD63, and CD81.

45. The method of any one of claims 39, 41 and 42, wherein the size of single extracellular vesicles is analyzed.

46. ​​The method of any one of claims 42-45, wherein the analysis is used to analyze an extracellular vesicle subpopulation.

47. The method of claim 1, wherein in said method, Cultivating includes culturing the cells on the surface of a culture container, wherein the culture container is a multi-well plate or a micro-well plate, wherein the different candidate vesicle formation culture media in step (b) are obtained by mixing or combining two or more culture media, from at least two different culture media initially selected, to produce a group of different candidate vesicle formation culture media, The method comprises, in step (d), analyzing the cell number and cell viability using an automated cell counter, The method further comprises, in step (d), measuring the total number of extracellular vesicles and / or the number of extracellular vesicles per cell by measuring the expression of at least one marker present on the extracellular vesicles using a high throughput immunoassay, and The method further comprises, in step (d), analyzing marker expression and / or vesicle size of single extracellular vesicles using super-resolution microscopy.

48. The method of claim 47, wherein the immunoassay is a Tim4-capture ELISA.

49. The method of claim 48, wherein the ELISA is a Tim4-capture ELISA detecting CD63 expression.

50. The method of any one of claims 47-49, wherein analyzing comprises analyzing one or more of CD9, CD63, and CD81 expression using said super-resolution microscopy.

51. The method of claim 50, wherein analyzing comprises analyzing CD9, CD63, and CD81 expression using said super-resolution microscopy.

52. The method of any one of claims 50 and 51 , further comprising analyzing the size of individual extracellular vesicles using the super-resolution microscope.

53. The method of any one of claims 1-52, further comprising selecting a candidate vesicle formation medium based on the analysis results of step (d).

54. The method of claim 53, wherein the selected medium provides improvements in one or more of: cell growth, cell viability, total extracellular vesicle number, number of extracellular vesicles per cell, marker expression on extracellular vesicles, and extracellular vesicle size compared to a control baseline medium or an unmixed medium.

55. A vesicle formation medium selected according to the method of claim 53 or 54.

56. A system for implementing the method of any one of claims 1-55, wherein the system comprises one or more of an automated liquid handler, an automated cell counter, an immunoassay kit, and a super-resolution microscope.

57. The system of claim 56, wherein the immunoassay kit is an ELISA kit.

58. The method of any one of claims 47-57, wherein the set of candidate vesicle formation culture media is produced by mixing or combining three or more different culture media.

59. The method of any one of claims 47-57, wherein the set of candidate vesicle formation culture media is produced by mixing or combining four or more different culture media.

60. The method of any one of claims 47-59, wherein the two or more culture media that are mixed or combined together are from at least five different culture media initially selected.

61. The method of any one of claims 47-59, wherein analysis by super-resolution microscopy comprises immobilizing the extracellular vesicles on at least one of a coverslip and a microscope channel slide.

62. The method of any one of claims 47-61, wherein analysis by super-resolution microscopy comprises detecting at least one marker using a fluorescent antibody.

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