Methods and systems for developing a medium for extracellular vesicle production

Abstract

The present disclosure generally relates to methods and systems for the analysis, development, testing, and / or optimization of media and / or culture conditions for extracellular vesicle production; media that have been analyzed, developed, tested, and / or optimized via such assays; and extracellular vesicles produced using such media and compositions comprising the extracellular vesicles.

Description

Technical Field

[0001] The present disclosure generally relates to methods and systems for the analysis, development, testing, and / or optimization of media (and / or culture conditions) for extracellular vesicle production. The present disclosure further relates to media that have been analyzed, developed, tested, and / or optimized via such extracellular vesicle characterization assays; and extracellular vesicles and extracellular vesicle-containing compositions produced using such media.

Background Art

[0002] Cells, such as cells in in vitro or ex vivo culture, secrete a diverse array of molecules and biological factors (collectively known as the secretome) 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 within membrane-bound extracellular vesicles such as exosomes. Extracellular vesicles have the ability to alter the biology of other cells, either through signaling or by delivery of their cargo (including, for example, proteins, lipids, and nucleic acids). The cargo of extracellular vesicles is encapsulated within membranes that allow for increased stability during transport in biological fluids, among other things, via specific targeting through particular markers on the membrane (e.g., to target cells); transport through the bloodstream, or passage through the blood-brain barrier (BBB).

[0003] Exosomes exert a wide range of important physiological functions, for example, by acting as molecular messengers that send information between different cell types. For example, exosomes can affect apoptosis, metastasis, angiogenesis, tumor progression, thrombosis, immunity by inducing T cells towards immune activation, immunosuppression, proliferation, division, survival, differentiation, stress response, apoptosis, etc. depending on their source, and are involved in signal transduction pathways that deliver proteins, lipids and soluble factors such as RNA and microRNA. See Vlassov et al. (Biochim Biophys Acta, 2012; 940 - 948). Extracellular vesicles may contain a combination of molecules that can act simultaneously to exert specific biological effects. Exosomes incorporate a wide range of cytoplasmic and membrane components that reflect the characteristics of the parent cell. Thus, the term applied to the originating cell can, in some cases, be used as a simple reference to the secreted exosomes.

Summary of the Invention

Problems to be Solved by the Invention

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

Means for Solving the Problems

[0005] The present disclosure addresses the above limitations in the art by providing methods and systems for the analysis, development, testing, and / or optimization of media (and / or culture conditions) for extracellular vesicle production; media that have been analyzed, developed, tested, and / or optimized via such assays; and extracellular vesicles produced using such media and extracellular vesicle-containing compositions comprising the extracellular vesicles.

[0006] For example, in some embodiments, the present disclosure provides high-throughput methods for one or more of cell culture; media exchange; vesiculation; 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 the analysis, development, testing, and / or optimization of media (and / or culture conditions) for extracellular vesicle production (e.g., by enabling an increase in the number of test samples and / or a decrease in the period).

[0007] Non-limiting embodiments of the present disclosure include the following:

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

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

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

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

[0012] [5] The method of 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 of [5], wherein each of steps (a) to (c) is performed using an automated liquid handler.

[0014] [7] The method of any one of [1] to [6], wherein the cells are iPSC-derived cells.

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

[0016] [9] The method of [8], wherein the two-dimensional cell culture includes culturing the cells on the surface of a culture vessel.

[0017]

[10] The method of [9], wherein the surface of the culture vessel is coated with a substance for promoting cell adhesion.

[0018]

[11] The method of

[10] , wherein the substance for promoting cell adhesion is vitronectin or fibronectin.

[0019]

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

[11] , wherein a plurality of cell cultures are cultured in one or more multi-well plates or micro-well plates.

[0020]

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

[12] , further comprising mixing or combining two or more media and / or adding one or more additives or supplements to one or more media to produce a panel of different candidate media for vesicle formation.

[0021]

[14] The method according to

[13] , wherein a panel of different candidate media for vesicle formation is produced using an automated liquid handler.

[0022]

[15] The method according to

[14] , wherein a panel of candidate media for vesicle formation is produced by mixing or combining two or more media from an initial selection of at least five different media.

[0023]

[16] The method according to

[15] , wherein a panel of candidate media for vesicle formation is produced by mixing or combining two or more media from an initial selection of at least ten different media.

[0024]

[17] The method according to

[16] , wherein a panel of candidate media for vesicle formation is produced by mixing or combining two or more media from an initial selection of at least twenty different media.

[0025]

[18] The method according to

[17] , wherein a panel of candidate media for vesicle formation is produced by mixing or combining two or more media from an initial selection of at least fifty different media.

[0026]

[19] The method according to any one of

[14] to

[18] , wherein a panel of candidate media for vesicle formation comprises at least ten candidate media for vesicle formation.

[0027]

[20] The method according to

[19] , wherein a panel of candidate media for vesicle formation comprises at least twenty candidate media for vesicle formation.

[0028]

[21] The method according to

[20] , wherein a panel of candidate media for vesicle formation comprises at least thirty candidate media for vesicle formation.

[0029]

[22] The method of

[21] , wherein the panel of candidate media for vesicle formation comprises at least 50 candidate media for vesicle formation.

[0030]

[23] The method of

[22] , wherein the panel of candidate media for vesicle formation comprises at least 100 candidate media for vesicle formation.

[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 are pre-frozen.

[0033]

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

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

[0034]

[27] The method of

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

[0035]

[28] The method of

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

[0036]

[29] The method of

[27] or

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

[0037]

[30] The method of

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

[0038]

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

[25] , wherein at least one characteristic of the extracellular vesicles to be 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 concentration of the protein profile, the RNA profile, the potency, or the marker expression.

[0039]

[32] The method according to

[31] , wherein at least one characteristic of the extracellular vesicles to be analyzed is selected from the total number of extracellular vesicles, the number of extracellular vesicles per cell, the extracellular vesicle size, and the 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 tetraspanin.

[0042]

[35] The method according to

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

[0043]

[36] The method according to

[35] , wherein the tetraspanin is CD63.

[0044]

[37] The method according to any one of

[32] to

[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] The method of any one of

[26] to

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

[0049]

[42] The method of

[31] , wherein at least one characteristic of the extracellular vesicles to be analyzed is analyzed at the single extracellular vesicle level.

[0050]

[43] The method of

[42] , wherein at least one characteristic analyzed at the single extracellular vesicle level is marker expression.

[0051]

[44] The method of

[42] or

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

[0052]

[45] The method of

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

[0053]

[46] The method of any one of

[43] to

[45] , wherein the marker expression to be analyzed includes the analysis of the expression of at least one tetraspanin.

[0054]

[47] The method of

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

[0055]

[48] The method of any one of

[42] ,

[44] or

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

[0056]

[49] The method of any one of

[42] to

[48] , wherein an extracellular vesicle subpopulation is analyzed using the analysis.

[0057] In the method of

[50] [1], each of steps (a) to (c) is semi-automated using an automated liquid handler; the culturing includes culturing the cells on the surface of a culture vessel that is a multi-well plate or a microwell plate; the method further includes mixing or combining two or more media from an initial selection of at least two different media to produce a panel of different candidate media for vesicle formation; the method includes, in step (d), analyzing the cell number and cell viability using an automated cell counter; the method further 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 the extracellular vesicles by a high-throughput immunoassay; and the method further includes, in step (d), analyzing the marker expression and / or the vesicle size of individual extracellular vesicles by a super-resolution microscope, the method of [1].

[0058]

[51] The method of

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

[0059]

[52] The method of

[51] , wherein the ELISA is a Tim4-capture ELISA that detects CD63 expression.

[0060]

[53] The method according to any one of

[50] to

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

[0061]

[54] The method of

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

[0062]

[55] The method according to any one of

[53] or

[54] , further including analyzing the size of individual extracellular vesicles by the super-resolution microscope.

[0063] A method according to any one of [1] to

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

[0064]

[57] The method of

[56] , wherein the selected medium results in an improvement when compared to a control benchmark medium or a non-mixed medium in one or more of cell proliferation, cell viability, total number of extracellular vesicles, number of extracellular vesicles per cell, marker expression on extracellular vesicles, and extracellular vesicle size.

[0065]

[58] A medium for vesicle formation selected by the method of

[56] or

[57] .

[0066]

[59] A system for performing any one of the methods of [1] to

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

[0067]

[60] The system of

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

[0068]

[61] The method according to any one of

[15] to

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

[0069]

[62] The method according to any one of

[15] to

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

[0070]

[63] The method according to any one of

[50] to

[55] , wherein mixing or combining two or more media together is derived from an initial selection of at least five different media.

[0071]

[64] The method according to any one of

[50] to

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

[0072] A method according to any one of

[50] to

[55] and

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

[0073] Incorporation by reference All patents, publications, and patent applications cited herein are incorporated herein by reference as if each individual patent, publication, or patent application were specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0074] The patent or application file contains at least one drawing executed in color. A copy of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fees. BRIEF DESCRIPTION OF THE DRAWINGS

[0075]

Figure 1

[0076]

Figure 2

[0077]

Figure 3

[0078]

Figure 4

[0079]

Figure 5A

[0080]

Figure 5B

[0081]

Figure 5C

[0082]

Figure 6

[0083]

Figure 7

[0084]

Figure 8

[0085]

Figure 9

[0086]

Figure 10

[0087]

Figure 11

[0088]

Figure 12

[0089]

Figure 13

Mode for Carrying Out the Invention

[0090] It should be understood that the terms used in this specification are not intended to be limited to specific embodiments and are not intended to be limiting. 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, a reference to "a cell" includes one or more cells.

[0091] Unless otherwise defined, all scientific and technical terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Other methods and materials similar to or equivalent to those described herein may be useful in the practice of the present invention, but the preferred materials and methods are described herein.

[0092] As used herein, the terms "subject", "individual", or "patient" are used interchangeably herein and refer to any member of the phylum Chordata, such as, but not limited to, humans and other primates, such as non-human primates, such as monkeys, chimpanzees, and other monkeys and ape species; domestic animals, such as cows, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, such as rabbits, mice, rats, and guinea pigs; birds, such as domestic birds, wild birds, and game birds, such as chickens, turkeys, and other galliformes birds, ducks, and geese. The term does not denote a particular age or sex. Thus, the term includes adult, juvenile, and neonatal individuals, as well as males and females. In some embodiments, cells (e.g., stem cells, e.g., pluripotent stem cells, progenitor cells, or tissue-specific cells) are derived from a subject. In some embodiments, the subject is a non-human subject.

[0093] As used herein, "differentiation" refers to the process by which unspecialized cells (e.g., pluripotent stem cells, or other stem cells), or pluripotent or oligopotent cells acquire, are stimulated to acquire, or are induced to acquire specialized structural and / or functional characteristics characteristic of, for example, more mature cells, or fully mature cells. "Differentiation conversion" is the process of transforming one differentiated cell type into another differentiated cell type.

[0094] As used herein, "embryoid body" refers to a three-dimensional aggregate of pluripotent stem cells. These cells can undergo differentiation into cells of the three germ layers, such as the endoderm, mesoderm, and ectoderm. The three-dimensional structure, including the establishment of complex cell adhesion and paracrine signaling within the embryoid body microenvironment, enables differentiation and morphogenesis.

[0095] As used herein, "stem cell" refers to a cell having the ability of self-renewal, i.e., the ability to maintain their undifferentiated state while undergoing a number of cell division cycles. Stem cells can be totipotent, pluripotent, multipotent, oligopotent, or unipotent. Stem cells can be, for example, embryonic, fetal, amniotic, adult, or induced pluripotent stem cells.

[0096] As used herein, "pluripotent stem cell" (PSC) refers to a cell having the ability to proliferate indefinitely and to differentiate into any other cell type of an adult organism. Generally, pluripotent stem cells have the ability to induce teratomas when transplanted into immunodeficient (SCID) mice; have the ability to differentiate into cell types of all three germ layers (e.g., can differentiate into cell types of the ectoderm, mesoderm, and endoderm); and are stem cells that express one or more markers characteristic of PSCs. Examples of markers expressed by PSCs 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.

[0097] As used herein, "induced pluripotent stem cells" (iPSCs) refer to a type of pluripotent stem cells that are artificially induced 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, skin fibroblasts, bone marrow-derived mesenchymal cells, HPSc, hematopoietic, cardiomyocytes, keratinocytes, hepatocytes, gastric cells, neural stem cells, lung cells, kidney cells, spleen cells, and pancreatic cells. Additional examples of somatic cells include, but are not limited to, cells of the immune system, including B cells, dendritic cells, granulocytes, natural lymphoid cells, megakaryocytes, monocytes / macrophages, bone marrow-derived suppressor cells, natural killer (NK) cells, T cells, thymocytes, and hematopoietic stem cells. iPSCs can be generated by reprogramming somatic cells to express or induce the expression of one or a combination of factors (referred to herein as reprogramming factors) in the somatic cells. iPSCs can be generated using fetal, postnatal, neonatal, juvenile, 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, and the somatic cells can be reprogrammed into pluripotent stem cells. The cells can be reprogrammed, for example, by introducing reprogramming factors using vectors including episomal vectors, non-viral vectors, lentiviral vectors, retroviral vectors, adenoviral vectors, and Sendai viral vectors. Alternatively, non-viral techniques for introducing reprogramming factors include, for example, mRNA transfection, miRNA infection / transfection, PiggyBac, minicircle vectors, and episomal plasmids. iPSCs can also be generated, for example, by introducing reprogramming factors or activating endogenous programming genes using CRISPR-Cas9-based technologies.

[0098] As used herein, "embryonic stem cell" optionally refers to an embryonic cell derived from an embryonic tissue, preferably the inner cell mass of a blastocyst or morula, that is continuously passaged as a cell line. The term includes cells isolated 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 transfer. ESCs can be produced or derived from, for example, a zygote, blastomere, or blastocyst-stage mammalian embryo produced by the fusion of sperm and egg cells, nuclear transfer, or parthenogenesis. Examples of human ESCs include, but are not limited to, MAO1, MAO9, ACT-4, No.3, H1, H7, H9, H14, and ACT30 embryonic stem cells. Exemplary pluripotent stem cells include embryonic stem cells derived from the inner cell mass (ICM) of a blastocyst-stage embryo, as well as embryonic stem cells derived from one or more blastomeres of a cleavage-stage embryo or morula-stage embryo. These embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, such as somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. PSCs, when transplanted alone into the uterus, lack the ability to contribute to all extraembryonic tissues (e.g., the placenta in vivo or the trophoblast in vitro) and thus cannot develop into a fetus or adult animal.

[0099] As used herein, the term "progenitor cell" refers to the progeny of a stem cell that has the ability to further differentiate into one or more specialized cells but cannot divide and regenerate indefinitely. That is, unlike stem cells (which have an unlimited ability for self-renewal), progenitor cells have only a limited ability for self-renewal. Progenitor cells can be multipotent, oligopotent, or unipotent, but are typically classified according to the type of specialized cells into which they can differentiate. For example, a "cardiomyocyte progenitor cell" is a progenitor cell derived from a stem cell with the ability to differentiate into cardiomyocytes. Similarly, a "cardiac progenitor cell" can differentiate into multiple specialized cells that make up cardiac tissue, including, for example, cardiomyocytes, smooth muscle cells, and endothelial cells. In addition, a "cardiovascular progenitor cell" has the ability to differentiate, for example, into cells of the heart and vascular system.

[0100] As used herein, "expanding" or "proliferating" can refer to the process by which the number of cells under cell culture increases due to cell division. The culture in which this expansion occurs may be recognized, for example, as an "expansion culture".

[0101] "Multipotent" means that a cell has the ability to give rise to several different cell types found in an adult animal through its progeny.

[0102] "Pluripotent" means that a cell has the ability to give rise to all cell types, including embryonic cells, through its progeny, including those of an adult animal. Embryonic stem cells, induced pluripotent stem cells, and embryonic germ cells are pluripotent cells under this definition.

[0103] The term "autologous cell" as used herein refers to donor cells that are genetically identical to the recipient.

[0104] As used herein, the term "allogeneic cell" refers to cells derived from different genetically non-identical individuals of the same species.

[0105] The term "totipotent" as used herein can refer to a cell that gives rise to a live-born animal. The term "totipotent" can also refer to a cell that gives rise to all of the cells of a particular animal. Totipotent cells can give rise to all of the cells of an animal when utilized in a procedure for generating an embryo from one or more nuclear transfer steps.

[0106] As used herein, the term "extracellular vesicle" collectively refers to biological particles derived from cells, examples of which include, but are not limited to, exosomes, ectosomes, exovesicles, microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles, membrane vesicles, shedding vesicles, membrane particles, shedding microvesicles, oncosomes, exomeres, and / or apoptotic bodies.

[0107] Extracellular vesicles can be categorized, for example, according to size. For example, as used herein, the term "small extracellular vesicle" refers to an extracellular vesicle having a diameter between about 50 and 200 nm. In contrast, extracellular vesicles having a diameter greater than about 200 nm but less than 400 nm may be referred to as "medium extracellular vesicles", and extracellular vesicles having a diameter greater than about 400 nm may be referred to as "large extracellular vesicles". As used herein, the term "small extracellular vesicle fraction" ("sEV") refers to a part, extract, or fraction of a secretome or conditioned medium that is concentrated and / or enriched for small extracellular vesicles having a diameter between about 50 and 200 nm. In the context of extracellular vesicle production, cells that produce extracellular vesicles may be known as "producer cells".

[0108] The term "exosome", as used herein, refers to an extracellular vesicle released from a cell upon fusion of a multivesicular body (MVB) (intermediate endocytic compartment) with the plasma membrane.

[0109] "Exosome-like vesicles" having a common origin with exosomes are typically described as having size and sedimentation characteristics that distinguish them from exosomes, in particular as lacking lipid raft microdomains. "Ectosomes", as used herein, are typically microvesicles derived from neutrophils or monocytes.

[0110] "Microparticles", as used herein, typically have a diameter of about 100 to 1000 nm and are derived from the plasma membrane. Also, "extracellular membrane structures" include, for example, linear or folded membrane fragments from necrosis, as well as membrane structures from other cellular sources including secreted lysosomes and nanotubes.

[0111] As used herein, "apoptotic blebs or bodies" typically have a diameter of about 1 to 5 μm and are blebs of cells undergoing apoptosis, i.e., released as diseased, unwanted, and / or abnormal cells.

[0112] An important component within the class of extracellular vesicles is the "exosome" itself, which may have a diameter of about 40 nm to 50 nm and about 200 nm, and is a membrane vesicle of endocytic origin obtained from the extracellular fusion or "exocytosis" of multivesicular bodies (MVBs), i.e., a vesicle covered by a phospholipid bilayer. In some cases, exosomes can have a diameter of about 40 to 50 nm, up to about 200 nm, for example 60 nm to 180 nm.

[0113] As used herein, the terms "secretome" and "secretome composition" refer interchangeably to one or more molecules and / or biofactors secreted by a cell into the extracellular space (e.g., the culture medium). Secretomes or secretome compositions may include, but are 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 (e.g., the culture medium). Secretomes or secretome compositions may remain unpurified or may be further processed (e.g., the components of the secretome or secretome composition may be present in the culture medium, e.g., a conditioned medium; or alternatively, the components of the secretome or secretome composition may be purified, isolated, and / or enriched from the culture medium or an extract, portion, or fraction thereof). Secretomes or secretome compositions may further include one or more substances not secreted by the cell (e.g., culture medium, additives, nutrients, etc.). Alternatively, secretomes or secretome compositions may not include (or only contain trace amounts of) one or more substances not secreted by the cell (e.g., culture medium, additives, nutrients, etc.).

[0114] As used herein, the term "conditioned medium" refers to a medium (or an extract, portion, or fraction thereof) in which one or more target cells are being cultured. Preferably, the conditioned medium is separated from the cultured cells before use and / or further processing. Culturing cells in the medium may result in the secretion and / or accumulation of one or more molecules and / or biological factors (including, but not limited to, extracellular vesicles (e.g., exosomes, microparticles, etc.), proteins, nucleic acids, cytokines, and / or other molecules secreted by the cells into the extracellular space); a medium containing one or more molecules and / or biological factors is a conditioned medium. Examples of methods for preparing conditioned medium are described, for example, in U.S. Patent No. 6,372,494, which is hereby incorporated by reference in its entirety.

[0115] As used herein, the term "cell culture" refers to cells that grow under controlled conditions outside of their natural environment. For example, cells can grow completely outside of their natural environment (in vitro), or they can be removed from their natural environment and then cultured (ex vivo). During cell culture, cells can survive in a non-replicating state, or they can replicate and grow in number, depending, for example, on the specific medium, culture conditions, and cell type. The in vitro environment can be any medium known in the art suitable for maintaining cells in vitro, such as a suitable liquid medium or agar.

[0116] The term "cell line" as used herein can refer to cultured cells that can be passaged at least once without end.

[0117] The term "suspension" as used herein can refer to cell culture conditions in which the cells are not attached to a solid support. In cell growth in suspension, it is possible to grow while stirring, using devices well known to those skilled in the art.

[0118] As used herein, the term "monolayer" can refer to cells that are attached to a solid support while growing under suitable culture conditions. A small portion of the cells growing in a monolayer under suitable growth conditions may attach to the cells in the monolayer but not to the solid support.

[0119] As used herein, the term "seeded" or "seeding" in relation to cells can refer to establishing a cell culture in vitro. For example, cells can be diluted in 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 can be seeded at various concentrations and / or cell densities.

[0120] The term "cell seeding" can also be extended to the term "cell passage". Cells can be passaged using cell culture techniques well known to those of ordinary skill in the art. The term "cell passage" can refer to a technique that includes (1) releasing the cells from a solid support or substrate and dissociating these cells, and (2) diluting the cells in a medium suitable for further cell growth. Cell passage can also refer to removing a portion of the liquid medium containing the cultured cells, adding the liquid medium to the original culture vessel to dilute the cells and allow for further cell growth. In addition, the cells can be added to a new culture vessel to which a medium suitable for further cell growth has been added.

[0121] As used herein, the terms "culture medium", "growth medium", or "medium" are used interchangeably and refer to a composition intended to support the growth and survival of cells. While many are in liquid form, other physical forms such as solid, semi-solid, gel, suspension, etc. may be used. The term "medium for vesicle formation" as used herein refers to a medium in which cells are cultured by virtue of the intention and / or result that the cells produce extracellular vesicles, for example, a conditioned medium containing extracellular vesicles.

[0122] As used herein, the term "serum-free" in the context of a medium or growth medium refers to a culture or growth medium that is free of serum. Serum typically refers to the liquid component of clotted blood after the clotting factors (e.g., fibrinogen and prothrombin) have been removed by clot formation. Serum such as fetal bovine serum is routinely used in the art as a component of cell culture media because the various proteins and growth factors therein are particularly useful for cell survival, proliferation, and division.

[0123] As used herein, the term "basal medium" refers to an unsupplemented synthetic medium that may contain buffers, one or more carbon sources, amino acids, and salts. Depending on the application, growth factors and supplements (e.g., basic fibroblast growth factor (bFGF), also known as fibroblast growth factor 2 (FGF-2)) may be added to the basal medium, including, but not limited to, additional buffers, amino acids, antibiotics, proteins, and growth factors that are useful for promoting the growth of a particular cell type or maintaining or altering the differentiated state.

[0124] As used herein, the terms "wild-type," "naturally occurring," and "unmodified" refer to the typical (or most common) form, appearance, phenotype, or strain that occurs in nature; e.g., a cell, organism, polynucleotide, protein, macromolecular complex, gene, RNA, DNA, or genome in its typical form, as it exists and can be isolated from its natural source. The wild-type form, appearance, phenotype, or strain serves as the original parent prior to intentional modification. Thus, mutants, variants, modifications, recombinants, and modified forms are not wild-type forms.

[0125] As used herein, the term "isolated" refers to a material that has been removed from its original environment and has thus been "manually" altered from its natural state.

[0126] As used herein, the term "enriched" means selectively concentrating or increasing the amount of one or more components in a composition relative to one or more other components. For example, enrichment may include reducing or decreasing (e.g., removing or eliminating) the amount of an undesirable material; and / or specifically selecting or isolating a desired material from the composition.

[0127] "Modified," "genetically modified," "genetically recombinant," "recombinant," "engineered," "non-naturally occurring," and "non-natural" indicate intentional human manipulation of the genome of an organism or cell. The terms encompass methods of genome modification, including genome editing, as defined herein, as well as techniques that alter gene expression or inactivation, enzyme modification, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, codon optimization, and the like. Methods for genetic engineering are known in the art.

[0128] 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 that, when in linear form, has one 5' end and one 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 can perform any function and may have various secondary and tertiary structures. The term encompasses known analogs of natural nucleotides, as well as nucleotides modified in the base, sugar, and / or phosphate moieties. Certain nucleotide analogs have the same base pairing specificity (e.g., an analog of A base pairs with T). A polynucleotide may contain 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 construction. After polymerization, the polynucleotide may be further modified, for example, via conjugation with a labeling component or a target-binding component. Non-nucleotide components may be incorporated into the nucleotide sequence. The term also encompasses nucleic acids containing modified backbone residues or linkages that are synthetic, naturally occurring, and / or non-naturally occurring and have binding properties similar to those of a reference polynucleotide (e.g., DNA or RNA). Examples of such analogs include, but are not limited to, phosphorothioate, phosphoroamidate, methylphosphonate, chiral methylphosphonate, 2-O-methyl ribonucleotide, peptide nucleic acid (PNA), locked nucleic acid (LNA™) (Exiqon, Inc., Woburn, MA) nucleosides, glycol nucleic acid, bridged nucleic acid, and morpholino structures. Peptide nucleic acid (PNA) is a synthetic homolog of nucleic acids in which the polynucleotide phosphate-sugar backbone 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.Polynucleotide sequences are presented herein in the conventional 5' to 3' directionality, unless otherwise indicated.

[0129] As used herein, "sequence identity" generally refers to the percent identity of nucleotide bases or amino acids when a first polynucleotide or polypeptide is compared to a second polynucleotide or polypeptide using algorithms having various weighting parameters. Sequence identity between two polynucleotides or between two polypeptides can be determined using, without limitation, sequence alignments (e.g., Exonerate, BLAST, CS-BLAST, FASTA, HMMER, L-ALIGN, etc.) by various methods and computer programs available through the World Wide Web at sites including GENBANK (www.ncbi.nlm.nih.gov / genbank / ) and EMBL-EBI (www.ebi.ac.uk.). Sequence identity between two polynucleotide sequences or between two polypeptide sequences is generally calculated using the standard default parameters of various methods or computer programs. High sequence identity between two polynucleotides or between two polypeptides is between about 90% identity and 100% identity over the length of the reference polynucleotide or polypeptide or query sequence, e.g., over the length of the reference polynucleotide or polypeptide or query sequence, about 90% or greater identity, about 91% or greater identity, about 92% or greater identity, about 93% or greater identity, about 94% or greater identity, about 95% or greater identity, about 96% or greater identity, about 97% or greater identity, about 98% or greater identity, or about 99% or greater identity. Sequence identity can also be calculated for overlapping regions of two sequences when only a portion of the two sequences is alignable.

[0130] Moderate sequence identity between two polynucleotides or two polypeptides is between about 80% identity and about 90% identity, e.g., about 80% or more identity, about 81% or more identity, about 82% or more identity, about 83% or more identity, about 84% or more identity, about 85% or more identity, about 86% or more identity, about 87% or more identity, about 88% or more identity, or about 89% or more identity, but often less than 90%, over the length of the reference polynucleotide or polypeptide or query sequence.

[0131] Low sequence identity between two polynucleotides or two polypeptides is between about 50% identity and 75% identity, e.g., about 50% or more identity, about 60% or more identity, about 70% or more identity, but often less than 75%, over the length of the reference polynucleotide or polypeptide or query sequence.

[0132] As used herein, "binding" refers to non-covalent interaction between macromolecules (e.g., between a protein and a polynucleotide, between polynucleotides, or between proteins, etc.). Such non-covalent interactions are also referred to as "associated" or "interacting" (e.g., when a first macromolecule interacts with a second macromolecule and the first macromolecule binds non-covalently to the second macromolecule). Some portions of the binding interaction can be sequence-specific (the terms "sequence-specific binding", "binds sequence-specifically", "site-specific binding", and "binds site-specifically" are used interchangeably herein). The binding interaction can be characterized by a dissociation constant (Kd). "Binding affinity" refers to the strength of the binding interaction. An increase in binding affinity correlates with a lower Kd.

[0133] "Gene", as used herein, refers to a polynucleotide sequence that includes exons and related regulatory sequences. A gene may further include introns and / or untranslated regions (UTRs).

[0134] As used herein, "expression" refers to, for example, the transcription of a polynucleotide from a DNA template that results in a messenger RNA (mRNA) or other RNA transcript (e.g., non-coding, e.g., structural or scaffold RNA). The term further refers to the process by which the transcribed mRNA is translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may collectively be referred to as a "gene product". Expression may include splicing of mRNA in eukaryotic cells when the polynucleotide is derived from genomic DNA.

[0135] A "coding sequence" or a sequence that "encodes" a selected polypeptide is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo. The boundaries of the coding sequence are determined by the start codon at the 5' end and the translation stop codon at the 3' end. A transcription termination sequence may be located 3' of the coding sequence.

[0136] As used herein, for example, a "different" or "altered" level of a feature or characteristic is a difference that is measurably different, preferably statistically significant (e.g., not attributable to the standard error of the assay). In some embodiments, for example, the difference when comparing to a control or reference sample may be a difference of more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, for example, a fold change of more than 2; a fold change of more than 5; a fold change of more than 10; a fold change of more than 20; a fold change of more than 50; a fold change of more than 75; a fold change of more than 100; a fold change of more than 250; a fold change of more than 500; a fold change of more than 750; or a fold change of more than 1,000.

[0137] As used herein, the term "between" includes the end values within a given range (e.g., a nucleotide length between about 1 and about 50 includes 1 nucleotide and 50 nucleotides).

[0138] As used herein, the term "amino acid" refers to natural and synthetic (non-natural) amino acids, such as amino acid analogs, modified amino acids, peptidomimetics, glycine, and D- or L-enantiomers.

[0139] As used herein, the terms "peptide", "polypeptide", and "protein" are interchangeable and refer to polymers of amino acids. The polypeptide may be of any length. It may be branched or linear, it may be interrupted by non-amino acids, and it may contain modified amino acids. The term also refers to amino acid polymers that have been modified, for example, through acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, pegylation, biotinylation, cross-linking, and / or conjugation (e.g., with a labeling component or ligand). Polypeptide sequences are represented herein in the conventional N-terminal to C-terminal direction, unless otherwise indicated. Polypeptides and polynucleotides can be made using conventional techniques in the field of molecular biology.

[0140] "Moiety", as used herein, refers to a portion of a molecule. A moiety can be a functional group or can describe a part of a molecule having multiple functional groups (e.g., sharing a common structural aspect). The terms "moiety" and "functional group" are typically used interchangeably, although "functional group" can more specifically refer to a part of a molecule that includes some common chemical behaviors. "Moiety" is often used as a structural descriptor.

[0141] For example, the term "effective amount" of a composition or product refers to an amount sufficient for the composition or product to produce a desired response.

[0142] 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 occur via direct uptake, transfection, infection, etc. The exogenous polynucleotide may be maintained as a non-integrating vector, e.g., an episome, or alternatively, may be integrated into the host genome.

[0143] As used herein, the terms "hypoxia" or "hypoxic" refer to a state where the oxygen (O 2 ) concentration is below the atmospheric O 2 concentration (typically 20 - 21%). In some embodiments, hypoxia refers to a state having an O 2 concentration 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%.

[0144] As used herein, the term "normoxia" refers to normal atmospheric concentrations of oxygen, typically about 20% - 21% O 2 .

[0145] As used herein, the term "high-throughput" as related to methods for the development, testing, and / or optimization of media (and / or culture conditions) for extracellular vesicle production refers to methods that allow for an increase in the number of samples and / or a decrease in the experimental period, and often both. Often, but not always, high-throughput methods utilize one or more steps or assays with some degree of automation (via machines, devices, computers, etc.), e.g., semi-automated or fully automated steps or assays.

[0146] As used herein, the term "automated" in connection with a method or step refers to a method or step in which some part of the method or step is performed manually, or is not performed manually as part of it, provided that not all of the method or step is performed manually (e.g., it encompasses the concepts of semi-automation and full automation).

[0147] As used herein, the term "semi-automated" in connection with a method or step refers to a method or step in which some aspects, parts, or portions of the method or step are performed manually, provided that not all of the method or step is performed manually (e.g., without the assistance of a liquid handler).

[0148] As used herein, the term "fully automated" in connection with a method or step refers to a method or step in which no part (or substantially no part) of the method or step is performed manually.

[0149] Extracellular vesicle-secreting cells

[0150] The present disclosure relates, in part, to methods and systems for the analysis, development, testing, and / or optimization of media (and / or culture conditions) for extracellular vesicle production. Cells that may be used for extracellular vesicle production in such methods and systems include, but are not limited to, stem cells, progenitor cells, and differentiated cells (including terminally differentiated or partially differentiated cells). Such cells may be obtained, for example, by isolation from a subject or tissue; by isolation from an in vitro cell line or culture; or through in vitro induction of differentiation (e.g., progenitor cells can be generated from pluripotent stem cells, such as embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs)).

[0151] Generation of iPSC cells

[0152] iPSC cells may be obtained from somatic cells, for example, human somatic cells. Somatic cells may be derived from human or non-human animals, including, for example, humans and other primates, such as non-human primates, for example, rhesus monkeys, chimpanzees, and other monkeys and ape species; livestock, such as cows, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, such as rabbits, mice, rats, and guinea pigs; birds, such as domestic birds, wild birds, and game birds, for example, chickens, turkeys, and other galliformes birds, ducks, and geese.

[0153] In some embodiments, the 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, cells of the blood and immune system, cells of the nervous system including neurons and glial cells, pigment cells, and progenitor cells, such as hematopoietic stem cells. The somatic cells may be fully differentiated (specialized) or may not have reached full differentiation. For example, undifferentiated progenitor cells that are not PSCs, including somatic stem cells, and terminally differentiated mature cells can be used. The somatic cells may be obtained from animals of any age, for example, adult cells and fetal cells.

[0154] The somatic cells may be of mammalian origin. Allogeneic or autologous stem cells can be used, for example, when the secretome (or extracellular vesicles) derived from their progenitor cells is used for in vivo administration. In some embodiments, the iPSCs are not MHC / HLA compatible with the subject. In some embodiments, the iPSCs are MHC / HLA compatible with the subject. In embodiments, for example, when iPSCs are to be used to produce PSC-derived cells, such as progenitor cells (to obtain the secretome, or extracellular vesicles), the somatic cells may be obtained from the subject to be treated or from another subject having the same or substantially the same HLA type as that subject. The somatic cells can be cultured, for example, before nuclear reprogramming, or can be reprogrammed without culturing after isolation.

[0155] To introduce reprogramming factors into somatic cells, for example, viral vectors can be used, including SV40, adenovirus, vaccinia virus, adeno-associated virus, herpes viruses including HSV and EBV, Sindbis viruses, alphaviruses, human herpesvirus vectors (HHV) such as HHV-6 and HHV-7, and viral-derived vectors such as retroviruses. Lentiviruses 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 Anaemia Virus (EIAV), Bovine Immunodeficiency Virus (BIV), Visna Virus of sheep (VISNA), and Caprine Arthritis-Encephalitis Virus (CAEV). Lentiviral vectors have the ability to infect non-dividing cells and can be used for gene transfer and expression both in vivo and in vitro. Viral vectors can be targeted to specific cell types by binding to viral proteins, such as envelope protein binders, such as antibodies, or specific ligands (e.g., when targeting specific cell types or internal receptors or proteins).

[0156] In some embodiments, a viral vector, such as a lentiviral vector, can integrate into the genome of a host cell. Thus, the introduced genetic material can then be transcribed and potentially translated into proteins inside the host cell. In other embodiments, viral vectors that do not integrate into the genome of the host cell are used.

[0157] 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 Epstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, an adenovirus-based vector, a simian virus 40 (SV40)-based episomal vector, a bovine papillomavirus (BPV)-based vector, or a lentiviral vector.

[0158] Somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) using methods known to those of skill in the art. Those of skill in the art can readily generate induced pluripotent stem cells. See, for example, U.S. Patent Application Publication No. 2009 / 0246875; U.S. Patent Application Publication No. 2010 / 0210014; U.S. Patent Application Publication No. 2012 / 0276636; U.S. Patent No. 8,058,065; U.S. Patent No. 8,129,187; and U.S. Patent No. 8,268,620, all of which are incorporated herein by reference.

[0159] Generally, reprogramming factors that can be used to create 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 for 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_028016; 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 that are similar thereto, e.g., 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 3, or at least 4 of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc and Klf4 are utilized.

[0160] Exemplary reprogramming factors for producing iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 is interchangeable with Sox1, Sox3, Sox15, Sox17 or Sox18; Klf4 is interchangeable with 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 papilloma virus (HPV) 16 E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E7; (5) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7; (6) Oct3 / 4, Klf4, Sox2, L-Myc, Bmi1; (7) Oct3 / 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 is interchangeable with Esrrg); (12) Oct3 / 4, Klf4, Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6; (15) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E7; (16) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7; (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 is interchangeable with Esrrg).

[0161] iPSCs typically exhibit a morphology characteristic of human embryonic stem cells (hESCs) and express the pluripotency factor NANOG. It is also possible to identify fully reprogrammed human cells using surface antigens specific to embryonic stem cells (SSEA-3, SSEA-4, TRA1-60, TRA1-81). Additionally, at the functional level, PSCs such as ESCs and iPSCs can differentiate into cell lineages from all three embryonic germ layers and also show the ability to form teratomas in vivo (e.g., in SCID mice).

[0162] Differentiation of PSCs

[0163] The present disclosure further contemplates differentiating PSCs including ESCs and iPSCs to generate extracellular vesicle-producing cells having 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 into progenitor cells that can be used to produce extracellular vesicles.

[0164] Examples of progenitor cells of the present disclosure include hematopoietic progenitor cells, bone marrow progenitor cells, neural progenitor cells; pancreatic progenitor cells, cardiac progenitor cells, cardiomyocyte progenitor cells, cardiovascular progenitor cells, kidney progenitor cells, skeletal myoblasts, satellite cells, intermediate progenitor cells formed in the subventricular zone, radial glial cells, bone marrow stromal cells, periosteal cells, endothelial progenitor cells, blast cells, boundary 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 and include, for example, the methods described in U.S. Provisional Patent Application No. 63 / 243,606 entitled "Methods for the Production of Committed Cardiac Progenitor Cells", which is hereby incorporated by reference in its entirety.

[0165] As specialized cells of the present disclosure, for example, fibroblasts, muscle cells, keratinocytes, hepatocytes, gastric cells, nerve cells, lung cells, kidney cells, spleen cells, endothelial cells, and pancreatic cells; and cells of the immune system, for example, but not limited to, B cells, dendritic cells, granulocytes, natural lymphoid cells, megakaryocytes, monocytes / macrophages, bone marrow-derived suppressor cells, natural killer (NK) cells, and T cells.

[0166] Analysis, development, testing, and / or optimization of media and / or culture conditions for extracellular vesicle production

[0167] The present disclosure provides methods and systems for the analysis, development, testing, and / or optimization of media (and / or culture conditions) for extracellular vesicle production. For example, in some embodiments, the present disclosure provides high-throughput methods for cell culture; liquid handling (including, for example, media suspension, media exchange, and media recovery); vesicle formation; analysis of cell proliferation and / or viability; analysis of extracellular vesicle production and secretion; and characterization of extracellular vesicles, thereby providing improved methods and systems for the analysis, development, testing, and / or optimization of media (and / or culture conditions) for extracellular vesicle production (for example, by enabling an increase in the number of test samples and / or a decrease in the period).

[0168] 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.

[0169] 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.

[0170] One or more producer cells can be, for example, cells that have been recently isolated or differentiated (e.g., from stem cells). Alternatively, in some embodiments, cells that have been previously refrigerated, frozen, and / or cryopreserved may be used in the culturing method. In some embodiments, the cells are thawed from the cryopreserved state (e.g., -80°C or below) before use. In some of those embodiments, the cells are thawed in a thawing medium. In some embodiments, the thawing medium may include a liquid medium containing one or more supplements. In some embodiments, the cells may be thawed using a thawing device such as, for example, a water bath or a dry thawing system (e.g., ThawSTAR™ Automated Thawing System, Biolife Solution®).

[0171] Each type of producer cell to be cultured is preferably, but not necessarily, cultured in plurality. For example, each type of producer cell may be cultured, for example, in duplicates, triplicates, quadruplicates, quintuplicates, sextuplicates, septuplicates, octuplicates, depending on the number of vesicle-forming medium preparations to be analyzed, and / or the culturing conditions, 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 10,000, at least 50,000, or at least 100,000 producer cell cultures (of the same or different cells) are cultured in parallel. The cell culture may be adherent or non-adherent (e.g., suspension) cell culture. The culturing may be two-dimensional or three-dimensional cell culture.

[0172] In some embodiments, the culture vessel used for culturing may be, for example, a flask, a tissue culture flask (e.g., T25, T75), a hyperflask (e.g., CellBind surface HYPERFlask®; Corning, Ref: 10024) or a hyperstack (e.g., 12 or 36 chambers, HYPERStacks®, Corning, Refs: 10012, 10036, 10013, 10037), a dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multi-plate, a multiwell plate, a microscope slide, a chamber slide, a tube, a tray, a CellSTACK® chamber (e.g., 1ST, 2ST, 5ST, 10ST; Corning, Refs: 3268, 3269, 3313, 3319), a culture bag, a roller bottle, a bioreactor, a stirred culture vessel, a spinner flask, a microcarrier, or a vertical wheel bioreactor. The cells may be cultured, for example, in a volume of at least or approximately 0.2, 0.5, 1, 2, 5, 10, 15, 20, 30, 40, 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 10,000 L.

[0173] In some embodiments, the plurality of cell cultures are preferably cultured in different compartments or wells of a tissue culture vessel. For example, in some embodiments, the plurality of cell cultures are adherently cultured in different wells or portions of a tissue culture vessel such as a microplate or multiwell plate (e.g., a plate comprising 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 10,000 wells).

[0174] In embodiments where the culture involves two-dimensional cell culture on the surface of a culture vessel or the like, the culture surface (where cell adhesion is intended) may be coated with one or more substances that promote cell adhesion. Such substances that are useful for enhancing adhesion to a solid support include, for example, type I, type II, and type IV collagen, concanavalin A, chondroitin sulfate, fibronectin, fibronectin-like polymers, gelatin, laminin, poly-D and poly-L-lysine, Matrigel, thrombospondin, osteopontin, poly-D-lysine, human extracellular matrix, Corning® Cell-Tak™ cell and tissue adhesive, Corning PuraMatrix® peptide hydrogel, and / or vitronectin.

[0175] In some embodiments, when the culturing of cells is performed as adherent culture, for example, when the cells are adhered to a solid support, the cells are at 25,000 - 250,000 cells / cm 2 ; 50,000 - 200,000 cells / cm 2 ; 75,000 - 175,000 cells / cm 2 ; or 100,000 - 150,000 cells / cm 2It may be seeded in an amount of. In some embodiments, when the cell culture is carried out as an adherent culture, the cells may be seeded on a solid support under gravity. In other embodiments, the cells may be seeded on a solid support under centrifugation.

[0176] In some embodiments, the seeding and expansion culture of a plurality of cell cultures are preferably at least partially automated and / or high-throughput. In some embodiments, the seeding and culture of a plurality of producer cell cultures are semi-automated. In some embodiments, the seeding and culture of a plurality of producer cell cultures are fully automated.

[0177] The expansion culture may be carried out over different periods. For example, the expansion culture may be carried out over a period of 6 to 96 hours, 12 to 72 hours, 36 to 60 hours, 42 to 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.

[0178] In some embodiments, all or part of the expansion culture is carried out under hypoxic conditions. In some embodiments, the hypoxic conditions are 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% O 2 concentration.

[0179] In some embodiments, all or part of the expansion culture step is carried out under normoxic conditions. In some embodiments, at least the final 6 - 72 hours, final 10 - 48 hours, or final 12 - 36 hours of the culture step is carried out under normoxic conditions. In some embodiments, the normoxic conditions are an O 2 concentration between 20% and 21%.

[0180] For example, any medium suitable for culturing producer cells, including known commercially available cell culture media, may be used. In some embodiments, a basal medium containing a buffer, one or more carbon sources, amino acids, and salts may be used. In some embodiments, to the basal medium, but not limited to, additional buffers, amino acids, antibiotics, proteins, and growth factors and supplements, such as those useful for promoting the growth of a particular cell type or maintaining or altering the differentiated state (e.g., fibroblast growth factor - basic (bFGF), also known as fibroblast growth factor 2 (FGF - 2)), may be added. The basal medium may be, for example, 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 Modified Essential Medium (GMEM), McCoy's 5A medium, Basal Medium Eagle (BME), Medium 199, or any basal medium suitable for the cell type to be cultured, including media containing, consisting of, or comprising variants, combinations, or modifications thereof.

[0181] Additional supplements can be added to the medium to supply trace elements to the cells for optimal growth and expansion. Such supplements include, for example, insulin, transferrin, sodium selenite, Hank's 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 additional amino acids, and combinations thereof. Such amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, 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.

[0182] Optionally, hormones can be further used under cell culture conditions and include, but are not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, β-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, and L-thyronine. β-mercaptoethanol can also be added to the cell culture medium.

[0183] Lipids and lipid carriers can be used to supplement the cell culture medium depending on the cell type. Such lipids and carriers include, but are not limited to, among others, cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic acid-oleic acid-arachidonic acid conjugated to albumin, unconjugated oleic acid and oleic acid conjugated to albumin.

[0184] In certain embodiments, albumin, such as human serum albumin, is present in the medium. Albumin, including human serum albumin, may be, for example, isolated, synthetic, recombinant, and / or modified. The amount of albumin may be adjusted according to the desired culture conditions and / or requirements. In some embodiments, albumin is present in an amount of 0.1 μg / mL to 50 mg / mL, 1 μg / mL to 25 mg / mL, 10 μg / mL to 20 mg / mL, 100 μg / mL to 10 mg / mL, 0.5 mg / mL to 5 mg / mL, 1 mg / mL to 3 mg / mL, or an amount of about 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL or 5 mg / mL.

[0185] In some embodiments, the medium further comprises one or more selected from the group consisting of glutamine; biotin; DL-alpha tocopherol acetate; DL-alpha-tocopherol; vitamin A; catalase; insulin; transferrin; superoxide dismutase; corticosteron; D-galactose; ethanolamine, glutathione; L-carnitine; linoleic acid; progesterone; putrescine; sodium selenite; triodo-I-thyronine; amino acids; sodium pyruvate; lipoic acid; vitamin B12; nucleosides; and ascorbic acid. Also, the medium may be supplemented with one or more carbon sources. The one or more carbon sources may be selected from carbon sources such as glycerol, glucose, galactose, sucrose, fructose, mannose, lactose, or maltose. The medium may contain serum such as fetal bovine serum or bovine fetal serum, or may be a serum-free medium.

[0186] After expansion culture of the producer cells, the medium used for the expansion cell culture is preferably removed and replaced with a medium for vesicle formation; after culturing the producer cells in the medium for vesicle formation, a conditioned medium is thereby produced. Preferably, the conditioned medium contains extracellular vesicles.

[0187] In some embodiments where the expanded producer cells are to be used for the analysis, development, testing, and / or optimization of the vesiculation medium, a plurality of different vesiculation medium preparations may be tested in parallel (against a plurality of producer 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 10,000, at least 50,000, or at least 100,000 different vesiculation medium preparations may be tested in parallel or sequentially.

[0188] A plurality of vesiculation medium preparations can be made, for example, by mixing or combining two or more different types of media together. In some embodiments, one or more of the media for mixing or combining are known in the art and / or are 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 media are mixed or combined together to create a particular vesiculation medium preparation. The media for mixing can be any medium suitable for the cell type to be cultured, including, for example, 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 Minimum Essential Medium (GMEM), McCoy's 5A medium, Basal Medium Eagle (BME), Medium 199, or variants, combinations, or modifications thereof, or media containing, consisting of, or including them.

[0189] The medium for mixing may contain one or more supplements or additives. 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 adrenomedullin, angiopoietin, autocrine motility factor, bone morphogenetic protein (BMP), 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 somatotropin (FBS), glial cell line-derived neurotrophic factor (GDNF), neurturin, 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).It may be selected from 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), renalinase (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).

[0190] In some embodiments, one or more growth factors may each independently be present in an amount of 0.001 μg / mL to 1000 μg / mL, an amount of 0.01 μg / mL to 100 μg / mL, an amount of 0.1 μg / mL to 10 μg / mL, an amount of 0.05 μg / mL to 5 μg / mL, an amount of 0.5 μg / mL to 2.5 μg / mL, or an amount of about 0.5 μg / mL, about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL or about 5 μg / mL.

[0191] Other supplements or additives may include, for example, a carbon source (such as glycerol, glucose, galactose, sucrose, fructose, mannose, lactose, or maltose), albumin, biotin, DL alpha tocopherol acetate, DL alpha - tocopherol, vitamin A, catalase, superoxide dismutase, corticosterone, D - galactose, ethanolamine, glutathione, L - carnitine, putrescine, sodium selenite, triiodo - L - thyronine, sodium pyruvate, lipoic acid, vitamin B12, nucleoside, β - mercaptoethanol, insulin, transferrin, sodium selenium, Hank's balanced salt solution, Earle's salt solution, antioxidant supplement, MCDB - 201, phosphate buffered saline (PBS), N - 2 - hydroxyethylpiperazine - N’ - ethanesulfonic acid (HEPES), nicotinamide, ascorbic acid and / or ascorbic acid - 2 - phosphate, and additional amino acids, and combinations thereof. Such amino acids include, but are not limited to, L - alanine, L - arginine, L - aspartic acid, L - asparagine, L - cysteine, L - cystine, 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 can be added, and hormones include, but are not limited to, D - aldosterone, diethylstilbestrol (DES), dexamethasone, β - estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, and L - thyronine.

[0192] Lipids and lipid carriers can also be used as additives or supplements. Such lipids and carriers include, but are not limited to, among others, cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic acid-oleic acid-arachidonic acid conjugated to albumin, unconjugated oleic acid, and oleic acid conjugated to albumin.

[0193] In some embodiments, a panel of vesicle formation medium preparations is produced by mixing or combining two or more different types of media from an initial selection of different media. In some embodiments, the different media of the initial selection include 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 media.

[0194] In some embodiments, the initial selection of different media is performed based on the suitability of a particular medium for a particular type of extracellular vesicle-secreting cell, culture conditions, desired results, and other considerations. In some embodiments, the initial selection of different media is performed by screening different media for desired characteristics, for example, culturing extracellular vesicle-secreting cells in the medium to obtain a conditioned medium, and analyzing one or more characteristics of the extracellular vesicles in the recovered conditioned medium, the recovered cells, or both.

[0195] In some embodiments, a panel of vesicle formation medium preparations is produced by mixing or combining two or more different types of media without performing any initial selection or screening of different media.

[0196] Alternatively, the plurality of vesicle formation medium preparations can be made by adding one or more supplements, additives, etc., for example, in different combinations and / or at different concentrations, to an existing medium (or a mixture of different types of media, as described above).

[0197] In some embodiments, the mixing or combination of different media; and / or the addition of one or more additives or supplements for preparing the vesicle formation medium preparation can be facilitated or strategized using one or more statistical methods. In some embodiments, a design of experiments (DoE) approach is used. In some embodiments, the mixing or combination of different media; and / or the addition of one or more additives or supplements for preparing the vesicle formation medium preparation may be facilitated or strategized using software (e.g., Design-Expert software by StatEase).

[0198] In some embodiments, the supplement or additive may be one or more growth factors. In some embodiments, the one or more growth factors are adrenomedullin, angiopoietin, autocrine motility factor, bone morphogenetic protein (BMP), 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), bovine fetal somatotropin (Somatotrophin) (FBS), glial cell line-derived neurotrophic factor (GDNF), neurturin, 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),It may be selected from 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), lenalase (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).

[0199] In some embodiments, one or more growth factors may each independently be present in an amount of 0.001 μg / mL to 1000 μg / mL, an amount of 0.01 μg / mL to 100 μg / mL, an amount of 0.1 μg / mL to 10 μg / mL, an amount of 0.05 μg / mL to 5 μg / mL, an amount of 0.5 μg / mL to 2.5 μg / mL, or an amount of about 0.5 μg / mL, about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL or about 5 μg / mL.

[0200] Other supplements or additives may include, for example, a carbon source (such as glycerol, glucose, galactose, sucrose, fructose, mannose, lactose, or maltose), albumin, biotin, DL alpha tocopherol acetate, DL alpha - tocopherol, vitamin A, catalase, superoxide dismutase, corticosterone, D - galactose, ethanolamine, glutathione, L - carnitine, putrescine, sodium selenite, triiodo - L - thyronine, sodium pyruvate, lipoic acid, vitamin B12, nucleoside, β - mercaptoethanol, insulin, transferrin, sodium selenium, Hank's balanced salt solution, Earle's salt solution, antioxidant supplement, MCDB - 201, phosphate buffered saline (PBS), N - 2 - hydroxyethylpiperazine - N’ - ethanesulfonic acid (HEPES), nicotinamide, ascorbic acid and / or ascorbic acid - 2 - phosphate, and additional amino acids, and combinations thereof. Such amino acids include, but are not limited to, L - alanine, L - arginine, L - aspartic acid, L - asparagine, L - cysteine, L - cystine, 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 can be added, and hormones include, but are not limited to, D - aldosterone, diethylstilbestrol (DES), dexamethasone, β - estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, and L - thyronine.

[0201] Lipids and lipid carriers can also be used as additives or supplements. Such lipids and carriers include, but are not limited to, among others, cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic acid-oleic acid-arachidonic acid conjugated to albumin, unconjugated oleic acid, and oleic acid conjugated to albumin.

[0202] Vesicle formation culture may be carried out over various periods. For example, the culture may be carried out over a period of 6 to 96 hours, 12 to 72 hours, 36 to 60 hours, 42 to 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.

[0203] In some embodiments, all or part of the vesicle formation culture is carried out under hypoxic conditions. In some embodiments, the final 6 to 72 hours, the final 10 to 48 hours, or the final 12 to 36 hours of the culture is carried out under hypoxic conditions. In some embodiments, the hypoxic conditions are at an O 2 concentration of 0% to 15%, 0% to 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%.

[0204] In some embodiments, all or part of the vesicle formation culture step is performed under normoxic conditions. In some embodiments, at least the final 6 - 72 hours, the final 10 - 48 hours, or the final 12 - 36 hours of the culture step is performed under normoxic conditions. In some embodiments, the normoxic conditions are an O 2 concentration between 20% and 21%.

[0205] In some embodiments, removal of the expansion medium from the expanded cell culture; washing of the cell culture; formulation of the vesicle formation medium preparation; addition of the vesicle formation medium preparation to the cell culture; culturing of the cell culture (expansion and / or vesicle formation culture); and / or collection of the conditioned medium is at least partially automated (e.g., semi-automated or fully automated), and / or high-throughput. For example, an automated liquid handler, such as a Biomek® automated liquid handler manufactured by Beckman Coulter, may be used.

[0206] During and / or after culturing of the producer cells in the vesicle formation medium (the vesicle formation stage where the conditioned medium containing extracellular vesicles is produced), one or more characteristics of the cultured cells (e.g., including total number of cells, 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) may be tested.

[0207] In some embodiments, the cultured producer cells are counted (e.g., by measuring cell density) and / or analyzed by using a cell viability assay. In some embodiments, during and / or after culturing of the producer 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 of those embodiments, the DNA-labeling dye or nuclear staining dye is a fluorescent dye.

[0208] In some embodiments, during and / or after culturing producer cells in a vesicle formation medium, the producer cells are stained with acridine orange and propidium iodide and counted using a cell counter. In some embodiments, the counting of live and / or dead cells is at least partially automated (e.g., semi-automated or fully automated) and / or high-throughput. For example, a Cellaca Cell Counter manufactured by Nexcelom may be used.

[0209] Additionally, or alternatively, one or more properties of the extracellular vesicles in the conditioned medium (which is being made by the vesicle formation culture) are analyzed using one or more assays (including, for example, particle number; particle concentration; particle size distribution; protein concentration; concentration of protein profile; RNA profile; potency; marker expression; host cell protein assessment; quantification and / or characterization of residual DNA; appearance; pH; osmolality, etc.) to determine one or more properties of the extracellular vesicles.

[0210] 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 may be a high-throughput immunoassay. In some embodiments, the immunoassay may be, for example, enzyme-linked immunosorbent assay (ELISA), competitive binding assay, immunometric assay, radioimmunoassay (RIA), fluorescence immunoassay (FIA), chemiluminescent immunoassay (CLIA), counting immunoassay (CIA), or flow cytometry or FACS.

[0211] In some embodiments, extracellular vesicles in the conditioned medium are captured using a reagent (such as a protein) that has an affinity for the extracellular vesicles. In some embodiments, the reagent is the Tim4 protein (or a variant or derivative thereof) that has an affinity for phosphatidylserine presented on the surface of the extracellular vesicles. In some embodiments, the captured extracellular vesicles can then be detected using a different reagent that binds to or detects the extracellular vesicles, such as a reagent that binds to or detects any one or more markers presented on the extracellular vesicles. Such markers can be any marker presented on the extracellular vesicles. Such markers can be, for example, selected from tetraspanins (such as CD9, CD63, and CD81), ceramide, MHC class I, MHC class II, integrin, adhesion molecules, phosphatidylserine, sphingomyelin, cholesterol, cytoskeletal proteins (such as actin, gelsolin, myosin, tubulin), enzymes (such as catalase, GAPDH, nitric oxide synthase, LT synthase), nucleic acids (such as RNA, miRNA), heat shock proteins (such as HSC70, HSP60, HSP70, HSPA5, CCT2, and HSP90), exosome biogenesis proteins (ALIX, Tsg101), LT, prostaglandin, and S100 proteins.Other exemplary extracellular vesicle markers that can be analyzed and / or detected include, for example, CD3, flotillin (e.g., flotillin-1, flotillin-2), TSG101 (tumor susceptibility 101), CD4, CD19, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CD1c, CD25, CD49e, ROR1 (neurotrophic tyrosine kinase, receptor-related 1), CD209, SSEA-4 (stage-specific embryonic antigen-4), HLA-ABCG, CD40, 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, annexin (e.g., annexin 2, annexin 5), programmed cell death 6 interacting protein (Alix), fibronectin 1, galectin 3 binding protein, α-2-macroglobulin, hemoglobin subunit β, gelsolin, β-actin, β-2-microglobulin, stomatin, moesin, peroxiredoxin 2, RAP1B (member of the RAS oncogene family), filamin A, integrin, selectin, syntenin (e.g., syntenin-1), syndecan binding protein (SDCBP), 14-3-3 protein, IMMT (mitochondrial protein), Disco interacting protein 2 homolog B (DIP2B), members of the four transmembrane protein family TSPAN6 and TSPAN3, arrestin domain-containing protein 1 (ARRDC1), immunoglobulin superfamily member 8 (IGSF8), CD82, TfR2, LAMP1 / 2, heparan sulfate proteoglycan, EMMPRIN, ADAM10, NT5E, complement binding proteins (e.g., CD55 and CD59) glycophorin A, AChE-E, amyloid beta A4 / APP, ESCRT-I / II / III, one or more of caveolin.

[0212] In some embodiments, the reagent that binds to or detects extracellular vesicles is an antibody. In some embodiments, the reagent, such as an antibody, binds to or detects CD9, CD63, or CD81. In some embodiments, the reagent is an antibody that binds to CD63.

[0213] In some embodiments, the seeding and expansion culture of multiple cell cultures are preferably at least partially automated and / or high-throughput. In some embodiments, the seeding and culture of multiple cell cultures are semi-automated. In some embodiments, the seeding and culture of multiple cell cultures are fully automated.

[0214] In some embodiments, the analysis of the conditioned medium to estimate or determine the number and / or type of extracellular vesicles therein is performed by using high-throughput ELISA, capturing extracellular vesicles using Tim4 protein (or its variant or derivative), and detecting the captured extracellular vesicles using an anti-CD63 antibody.

[0215] In some embodiments, the extracellular vesicles in the conditioned medium are characterized at the single extracellular vesicle level. In some embodiments, single extracellular vesicles are analyzed for the presence of one or more markers and / or their size. In some embodiments, single extracellular vesicles are analyzed using microscopy techniques. In some embodiments, the microscopy technique is super-resolution microscopy. In some embodiments, the super-resolution microscopy technique is direct stochastic optical reconstruction microscopy (dSTORM).

[0216] In some embodiments, extracellular vesicles are analyzed at the single extracellular vesicle level for the presence of one or more markers presented on the extracellular vesicles. Such markers may be any markers presented on the extracellular vesicles. Such markers may be selected, for example, from tetraspanins (e.g., CD9, CD63, and CD81), ceramide, MHC class I, MHC class II, integrin, 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, flotillin (e.g., flotillin-1, flotillin-2), TSG101 (tumor susceptibility 101), CD4, CD19, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CD1c, CD25, CD49e, ROR1 (neurotrophic tyrosine kinase, receptor-related 1), CD209, SSEA-4 (stage-specific embryonic antigen-4), HLA-ABCG, CD40, 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, annexin (e.g., annexin 2, annexin 5), programmed cell death 6 interacting protein (Alix), fibronectin 1, galectin 3 binding protein, α-2-macroglobulin, hemoglobin subunit β, gelsolin, β-actin, β-2-microglobulin, stomatin, moesin, peroxiredoxin 2, RAP1B (member of the RAS oncogene family), filamin A, integrin, selectin, syntenin (e.g., syntenin-1), syndecan binding protein (SDCBP), 14-3-3 protein, IMMT (mitochondrial protein), Disco interacting protein 2 homolog B (DIP2B), members of the four-pass transmembrane protein family TSPAN6 and TSPAN3, arrestin domain-containing protein 1 (ARRDC1), immunoglobulin superfamily member 8 (IGSF8), CD82, TfR2, LAMP1 / 2, heparan sulfate proteoglycan, EMMPRIN, ADAM10, NT5E, complement binding proteins (e.g., CD55 and CD59) glycophorin A, AChE-E, amyloid beta A4 / APP, ESCRT-I / II / III, one or more of caveolin.

[0217] In some embodiments, extracellular vesicles are analyzed for the presence of one or more of CD9, CD63, and CD81 at the single extracellular vesicle level. In some embodiments, single extracellular vesicles are analyzed for the presence of CD9, CD63, and CD81.

[0218] In some embodiments, prior to extracellular vesicles being analyzed at the single extracellular vesicle level, the extracellular vesicles are captured from the conditioned medium before being labeled with one or more antibodies that bind to a marker of interest. In some embodiments, the extracellular vesicles are captured on a solid phase, such as a chip or a cartridge, before being labeled with one or more antibodies that bind to a marker of interest. In some embodiments, the antibodies are labeled with fluorophores.

[0219] The conditioned medium can, in some embodiments, undergo one or more additional processing steps. For example, the conditioned medium and / or the extracellular vesicles in the conditioned medium can be subjected to removal, recovery, concentration, enrichment, isolation, purification, refrigeration, freezing, cryopreservation, lyophilization, sterilization, etc. The conditioned medium can also be pre-clarified or clarified by one or more centrifugation and / or filtration techniques. The extracellular vesicles can be enriched, purified, or further concentrated, for example, by centrifugation, ultracentrifugation, filtration, ultrafiltration, gravity, sonication, density gradient ultracentrifugation, tangential flow filtration, size exclusion chromatography, ion exchange chromatography, affinity capture, polymer-based precipitation, or organic solvent precipitation.

[0220] Experiment Non-limiting embodiments of the present invention are illustrated in the following examples. Although efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, concentrations, percent changes, etc.), some experimental error and deviation is to be expected. It is to be understood that these examples are provided solely by way of the drawings and are not intended to limit the scope of the invention, which is regarded as consisting of various embodiments of the invention. Not all of the steps described below in each example are required, nor does the order of the steps in each example need to be as presented.

Example

[0221] Example 1 Development of a high-throughput medium for extracellular vesicle production from iPSC-derived cells iPSC-derived cells were used as a model for extracellular vesicle production in a high-throughput assay to test multiple preparations of vesicle-forming media. This high-throughput semi-automated assay enabled the generation and simultaneous testing of multiple different preparations of vesicle-forming media while reducing the experimental period. A flowchart of the various steps of an exemplary assay performed is shown in FIG. 1.

[0222] To prepare multiple different preparations of vesicle-forming media for analysis, the medium mixing was strategized using a design of experiments (DoE) approach with Design-Expert software (StatEase). Six different varieties of media (including commercially available media) were used as starting media for mixing. An automated liquid handler (Biomek i7 automated liquid handler manufactured by Beckman Coulter) was used to mix these six different varieties of media to create 31 different mixtures (M1 - M31) of vesicle-forming media. Next, the resulting panel of 31 medium mixtures was tested as follows.

[0223] After the expansion culture of iPSC-derived cells in a multi-well plate, the expansion medium was removed from the cell culture wells and replaced with either a benchmark control medium ("benchmark") or one of the vesicle-forming medium mixtures (M1 - M31). Replicate wells were used for the control and vesicle-forming medium mixtures. Next, the cells were cultured to produce conditioned medium containing extracellular vesicles. All of the removal of the expansion medium, replacement with the vesicle-forming medium, vesicle-forming culture, and removal of the conditioned medium were performed in a semi-automated manner (using a Biomek i7 automated liquid handler manufactured by Beckman Coulter).

[0224] After collecting the conditioned medium, the cultured cells were evaluated in a semi-automated manner for cell density and cell viability. That is, live and dead cells were stained with AOPI (acridine orange and propidium iodide) and counted using an automated Cellaca Cell Counter manufactured by Nexcelom. The results are shown in Figure 2, which represents the live cell density after culturing with the control and M1 - M31 vesicle formation medium preparations. As can be seen from Figure 2, this procedure enabled efficient determination of the effects of different vesicle formation medium preparations on cell viability and identification of the vesicle formation medium preparations that promoted cell growth.

[0225] In addition, the collected conditioned medium was then analyzed using different vesicle formation media to determine extracellular vesicle production. Extracellular vesicle production was determined using a high-throughput Tim4-based 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). The captured extracellular vesicles were detected using an anti-CD63 antibody. The results are shown in Figures 3A and 3B. Figure 3A represents the data on the total extracellular vesicle production, and Figure 3B represents the extracellular vesicle production per cell. The total number of extracellular vesicles was estimated based on the amount of CD63 on the surface of the extracellular vesicles. As can be seen from Figures 3A and 3B, this procedure enabled efficient determination of the effects of different vesicle formation medium preparations on extracellular vesicle production and identification of the vesicle formation medium preparations that promoted extracellular vesicle production. Figures 3A and 3B also show that preparation M17 produced more extracellular vesicles than the benchmark control medium and more than the other vesicle formation medium preparations tested.

[0226] To determine the accuracy of the above-described automated liquid handling steps, extracellular vesicle production using an automated liquid handling process was compared with the corresponding steps performed by manual handling. The above-described Tim4-based extracellular vesicle ELISA assay was used for comparison. The results are shown in Figure 4. The CD63 level in the benchmark medium was set to 100%, and the x-axis and y-axis indicate the relative EV production levels. As shown in Figure 4, extracellular vesicle production using an automated liquid handler was closely correlated with extracellular vesicle production using manual handling, and the accuracy and robustness of the high-throughput semi-automated extracellular vesicle production process were confirmed.

[0227] Next, extracellular vesicles in the conditioned medium were characterized at the single extracellular vesicle level using a super-resolution microscope (direct stochastic optical reconstruction microscopy (dSTORM)). That is, for each sample, 10 μL of the conditioned medium was used as the input sample. Extracellular vesicles in the input sample were captured on a chip using an EV Profiler kit (manufactured by ONi) and labeled with CD63, CD81, and CD9 fluorophore-conjugated antibodies included in the kit. Next, individual fluorophores were localized using a Nanoimager S (manufactured by ONi). Next, the acquired images were processed and analyzed using CODI software (https: / / oni.bio / applications / ) and R.

[0228] As shown in Figures 5A, 5B, and 5C, the total number of extracellular vesicles determined from single extracellular vesicle (dSTORM) analysis (Figure 5A) and nanoparticle tracking analysis (NTA) (Figure 5B) was closely correlated with the CD63 expression level determined using an EV ELISA assay (Figure 5C).

[0229] Figure 6 shows the results of a human cardiomyocyte viability assay conducted to evaluate EV function. EVs purified from the conditioned medium and mock control medium using ultracentrifugation were used as input samples. The effect of the EVs was tested in a cardiomyocyte viability assay as described in the pamphlet of International Publication No. WO 2022 / 106890 A1. EVs produced by the candidate media for vesicle formation were functional in the staurosporine CM2 viability assay.

[0230] Figure 7 shows the results of a HUVEC scratch wound healing assay conducted to evaluate EV function. EVs purified from the conditioned medium and mock control medium using ultracentrifugation were used as input samples. A scratch wound healing assay developed by Essen BioSciences as IncuCyte® was used according to the manufacturer's instructions. The results indicate that EVs produced by the candidate media for vesicle formation were functional in the wound healing assay.

[0231] The above experiments show that when iPSC-derived cells were used as a model, a medium for vesicle formation that promotes cell proliferation and stimulates extracellular vesicle secretion compared to the benchmark control medium could be identified through this high-throughput approach. These experiments further show that the unexpected effects of the medium on the cellular microenvironment affect not only cell expansion and cell quality but also extracellular vesicle yield, quality, and purity.

[0232] Example 2 High-Throughput Medium Development for Extracellular Vesicle Production from Primary MSCs Primary mesenchymal stem cells (MSCs) were used as a model for extracellular vesicle production in a high-throughput assay to test multiple preparations of media for vesicle formation. This high-throughput semi-automated assay enabled the generation and simultaneous testing of multiple different preparations of media for vesicle formation while reducing the experimental period. A flowchart of the various steps of an exemplary assay performed is shown in FIG. 1.

[0233] To prepare multiple different vesicle formation medium preparations for analysis, the medium mixing was strategized using the design of experiments (DoE) approach with Design-Expert software (StatEase). Six different varieties of media (including commercially available media) were used as starting media for mixing. An automated liquid handler (Biomek i7 automated liquid handler manufactured by Beckman Coulter) was used to mix these six different varieties of media to create 46 different mixtures (M1 - M46) of vesicle formation media. Next, the panel of 46 resulting medium mixtures was tested as follows.

[0234] After the expansion culture of MSCs in a multi-well plate, the expansion medium was removed from the cell culture wells and replaced with either a benchmark control medium (“Benchmark 1 and Benchmark 2”), or one of the vesicle formation medium mixtures (M1 - M46). Replicate wells were used for some of the control and vesicle formation medium mixtures. Next, the cells were cultured to create a conditioned medium containing extracellular vesicles. All of the removal of the expansion medium, replacement with the vesicle formation medium, vesicle formation culture, and removal of the conditioned medium were performed in a semi-automated manner (using the Biomek i7 automated liquid handler manufactured by Beckman Coulter).

[0235] After collecting the conditioned medium, the cultured cells were evaluated for cell density in a semi-automated manner. That is, live cells were stained with PrestoBlue™ cell viability reagent (ThermoFisher Scientific), and the fluorescence intensity was measured by a plate reader. The results are shown in Figure 8, which represents the live cell density after culture with the control and M1 - M46 vesicle formation medium preparations (see the right vertical axis). As can be seen from Figure 8, this procedure was able to efficiently determine the effect of different vesicle formation medium preparations on cell viability and identify a vesicle formation medium preparation that promotes cell growth.

[0236] In addition, the collected conditioned medium was then analyzed using different vesicle formation media to determine extracellular vesicle production. Extracellular vesicle production was determined using a high-throughput Tim4-based extracellular vesicle ELISA (EV ELISA) assay, where Tim4 was used to capture extracellular vesicles in the conditioned medium, and the captured extracellular vesicles were detected using anti-CD63, anti-CD9, and anti-CD81 antibodies. The results are shown in FIGS. 8 and 9, which represent data on the total extracellular vesicle production and extracellular vesicle production per fixed number of cells, respectively. The total number of extracellular vesicles was estimated based on the amounts of CD63, CD9, and CD81 on the surface of the extracellular vesicles. As can be seen from FIGS. 8 and 9, this procedure enabled efficient determination of the effects of different vesicle formation medium preparations on extracellular vesicle production and identification of a vesicle formation medium preparation that promotes extracellular vesicle production. FIGS. 8 and 9 also show that several preparations containing preparation M3 produced more extracellular vesicles than the benchmark control medium and more than other vesicle formation medium preparations tested.

[0237] Next, extracellular vesicles in the conditioned medium were characterized at the single extracellular vesicle level using a super-resolution microscope (direct stochastic optical reconstruction microscopy (dSTORM)). Specifically, for each sample, 4.5 μL of purified EVs were used as the input sample. Extracellular vesicles in the input sample were captured on a chip using an EV Profiler kit (manufactured by ONi), then labeled with CD63, CD81, and CD9 antibodies and analyzed. Next, individual fluorophores were localized using a Nanoimager S (manufactured by ONi). The acquired images were then processed and analyzed using CODI software (https: / / oni.bio / applications / ) and R. The results are shown in FIG. 10.

[0238] Next, primary MSC cell culture and vesicle formation were performed using T-150 flasks. The collected extracellular vesicles were analyzed using an EV ELISA assay.

[0239] Figures 11A and 11B show the expression levels of CD63, CD9, and CD81 determined using an extracellular vesicle ELISA assay for vesicles produced in a specific vesicle formation medium by a manual method in a flask (Figure 11A) and by using an automated liquid handler in a multi-well plate (Figure 11B). As can be seen by comparing the results in Figures 10, 11A, and 11B, the total number of extracellular vesicles determined from single extracellular vesicle (dSTORM) analysis was closely correlated with the expression levels of CD63, CD9, and CD81 determined using the extracellular vesicle ELISA assay.

[0240] The HUVEC seeding assay was performed to evaluate EV function. EVs purified from the conditioned medium and mock medium using ultracentrifugation were used as input samples. As shown in Figure 12, EVs purified from the conditioned medium stimulated the growth of HUVEC cells.

[0241] Therefore, the above experiments indicate that, using MSCs as a model, a vesicle formation medium that promotes cell proliferation and stimulates extracellular vesicle secretion compared to a benchmark control medium could be identified through this high-throughput approach. These experiments further show that the unexpected effects of the medium on the cellular microenvironment affect not only cell expansion and cell quality but also extracellular vesicle yield, quality, and purity.

Claims

**Claim 1** A high-throughput method for analyzing, developing, and / or optimizing a medium for extracellular vesicle production, comprising: (a) culturing cells in a first medium, wherein cell division occurs during said culturing, and said culturing is performed in plurality; (b) after step (a), removing said first medium from said plurality of cell cultures, adding different candidate vesicle-forming media to different cell cultures among said plurality of cell cultures, and further culturing said plurality of cell cultures to produce a conditioned medium containing extracellular vesicles; (c) recovering from said plurality of cell cultures, either or both of said conditioned medium and said cells after said culturing of step (b); (d) analyzing at least one property of either or both of said extracellular vesicles in said recovered conditioned medium and said recovered cells; wherein each of steps (a) to (c) is semi-automated using automated liquid handling. **Claim 2** The method according to claim 1, wherein at least one of steps (a) to (c) is fully automated. **Claim 3** The method according to claim 1, wherein each of steps (a) to (c) is semi-automated or fully automated. **Claim 4** The method according to any one of claims 1 to 3, wherein said cells are iPSC-derived cells and / or primary stem cells. **Claim 5** The method according to any one of claims 1 to 4, wherein said culturing is two-dimensional cell culture. **Claim 6** The method according to claim 5, wherein said two-dimensional cell culture comprises culturing said cells on the surface of a culture vessel. **Claim 7** The method according to claim 6, wherein the surface of said culture vessel is coated with a substance for promoting cell adhesion. **Claim 8** The method according to claim 7, wherein said substance for promoting cell adhesion is vitronectin or fibronectin. **Claim 9** The method according to any one of claims 1 to 8, wherein said plurality of cell cultures are cultured in one or more multi-well plates or micro-well plates. **Claim 10** The method according to any one of claims 1 to 9, wherein said different candidate vesicle-forming media in step (b) are obtained by mixing or combining two or more media and / or adding one or more additives or supplements to one or more media to produce a panel of different candidate vesicle-forming media. **Claim 11** ​ The method of claim 10, wherein the panel of candidate media for vesicle formation is produced using an automated liquid handler. **Claim 12** The method of claim 11, wherein the panel of candidate media for vesicle formation is produced by mixing or combining two or more different media from an initial selection of at least five different media. **Claim 13** The method of claim 11, wherein the panel of candidate media for vesicle formation is produced by mixing or combining two or more media from an initial selection of at least 10 different media. **Claim 14** The method of claim 11, wherein the panel of candidate media for vesicle formation is produced by mixing or combining two or more media from an initial selection of at least 20 different media. **Claim 15** The method of claim 11, wherein the panel of candidate media for vesicle formation is produced by mixing or combining two or more media from an initial selection of at least 50 different media. **Claim 16** The method according to any one of claims 11 to 15, wherein the panel of candidate media for vesicle formation comprises at least 10 candidate media for vesicle formation. **Claim 17** The method of claim 16, wherein the panel of candidate media for vesicle formation comprises at least 20 candidate media for vesicle formation. **Claim 18** The method of claim 16, wherein the panel of candidate media for vesicle formation comprises at least 30 candidate media for vesicle formation. **Claim 19** The method of claim 16, wherein the panel of candidate media for vesicle formation comprises at least 50 candidate media for vesicle formation. **Claim 20** The method of claim 16, wherein the panel of candidate media for vesicle formation comprises at least 100 candidate media for vesicle formation. **Claim 21** The method according to any one of claims 1 to 20, wherein the cells comprise progenitor cells. **Claim 22** The method according to any one of claims 1 to 21, wherein the cells are pre-refrigerated or cryopreserved. **Claim 23** The method according to any one of claims 1 to 22, wherein the at least one characteristic of the recovered cells from step (c) to be analyzed is selected from the group consisting of the cell number, cell viability, cell density, cell morphology, cell identity, cell karyotype, cell transcriptome, hypertrophy, cell integrity, cell adhesion, cell physiology, and / or ATP content. **Claim 24** The method according to claim 23, wherein the at least one characteristic is selected from the group consisting of cell number and cell viability.

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

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

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

28. The at least one characteristic of the extracellular vesicles to be 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 concentration of the protein profile, the RNA profile, the potency, or the marker expression, according to any one of claims 1 to 22.

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

30. 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 presented on the extracellular vesicles. The method according to claim 29.

31. The method according to claim 30, wherein the marker is tetraspanin.

32. The method according to claim 31, wherein the tetraspanin is selected from the group consisting of CD9, CD63, and CD81.

33. The method according to claim 31, wherein the tetraspanin is CD63.

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

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

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

37. The method according to claim 36, wherein the ELISA is a Tim4-capture ELISA for detecting CD63 expression.

38. The method according to any one of claims 23 to 37, wherein the measurement of the at least one characteristic is at least partially automated.

39. The method according to claim 28, wherein at least one characteristic of the extracellular vesicles to be analyzed is analyzed at the single extracellular vesicle level.

40. The method according to claim 39, wherein at least one characteristic being analyzed at the single extracellular vesicle level is marker expression.

41. The analysis is performed using a super-resolution microscope. The method according to claim 39 or 40.

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

43. The method according to any one of claims 40 to 42, wherein the marker expression to be analyzed includes analysis of the expression of at least one tetraspanin.

44. The method according to claim 43, wherein the tetraspanin is selected from the group consisting of CD9, CD63, and CD81.

45. The method according to any one of claims 39, 41, or 42, wherein the size of individual extracellular vesicles is analyzed.

46. The method according to any one of claims 42 to 45, wherein an extracellular vesicle subpopulation is analyzed using the analysis.

47. The culturing includes culturing the cells on the surface of a culture vessel that is a multi-well plate or a microwell plate. The different vesicle formation medium candidates in step (b) are obtained by mixing together or combining two or more media from an initial selection of at least two different media to produce a panel of different vesicle formation medium candidates. The method includes, in step (d), analyzing the cell number and cell viability using an automated cell counter. The method further includes, in step (d), measuring the total number of the 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 by a high-throughput immunoassay. The method further includes, in step (d), analyzing marker expression and / or the vesicle size of individual extracellular vesicles by a super-resolution microscope. The method according to claim 1.

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

49. The method according to claim 48, wherein the ELISA is a Tim4-capture ELISA that detects CD63 expression.

50. The method according to any one of claims 47 to 49, wherein the analysis comprises analyzing one or more of CD9, CD63, and CD81 expression by the super-resolution microscope.

51. The method according to claim 50, wherein the analysis comprises analyzing CD9, CD63, and CD81 expression by the super-resolution microscope.

52. The method according to claim 50 or 51, further comprising analyzing the size of individual extracellular vesicles by the super-resolution microscope.

53. The method according to any one of claims 1 to 52, further comprising selecting a vesicle formation medium from among the vesicle formation medium candidates based on the results of the analysis in step (d).

54. The method according to claim 53, wherein the selected medium provides an improvement in one or more of cell proliferation, cell viability, total number of extracellular vesicles, number of extracellular vesicles per cell, marker expression on extracellular vesicles, and extracellular vesicle size as compared to a control benchmark medium or a non-mixed medium.

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

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

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

58. The method according to any one of claims 47 to 57, wherein the panel of vesicle formation medium candidates is produced by mixing or combining three or more different media.

59. The method according to any one of claims 47 to 57, wherein the panel of vesicle formation medium candidates is produced by mixing or combining four or more different media.

60. The method according to any one of claims 47 to 59, wherein the combined mixing or combination of the two or more media is derived from an initial selection of at least five different media.

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

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

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