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WO2025080972A1 - Produits d'exosomes dérivés d'organoïdes, procédés de fabrication et procédés d'utilisation - Google Patents

Produits d'exosomes dérivés d'organoïdes, procédés de fabrication et procédés d'utilisation Download PDF

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Publication number
WO2025080972A1
WO2025080972A1 PCT/US2024/050957 US2024050957W WO2025080972A1 WO 2025080972 A1 WO2025080972 A1 WO 2025080972A1 US 2024050957 W US2024050957 W US 2024050957W WO 2025080972 A1 WO2025080972 A1 WO 2025080972A1
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Prior art keywords
exosomes
mto
culture medium
cells
condition
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Timothy D. O'brien
Beth LINDBORG
Amanda VEGOE
Sabarinathan RAMACHANDRAN
Parthasarathy RANGARAJAN
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University of Minnesota Twin Cities
University of Minnesota System
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University of Minnesota Twin Cities
University of Minnesota System
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs

Definitions

  • the exosomes express at least one marker identifying the exosomes as being isolated from culture medium in which a multi-tissue organoid is cultured.
  • this disclosure describes exosomes that express at least one marker identifying the exosomes as being isolated from culture medium in which a multi-tissue organoid is cultured.
  • this disclosure describes a method of treating a subject having, or at risk of having, a condition treatable with exosomes isolated from culture medium in which a multi-tissue-organoid is cultured. Generally, the method includes administering to the subject a pharmaceutical composition that includes an effective amount of exosomes isolated from culture medium in which a multi-tissue-organoid is cultured.
  • the pharmaceutical composition is administered before the subject manifests a symptom or clinical sign of the condition. In one or more embodiments, the pharmaceutical composition is administered after the subject manifests a symptom or clinical sign of the condition.
  • A The biogenesis pathway of an exosome begins with late endosomes, so called multivesicular bodies, containing multiple internal vesicles that merge with the cell membrane and release its internal vesicles into the extracellular matrix.
  • B Simple illustration of an exosome. Exosomes are formed by a bilayer of lipids and contain components such as transmembrane proteins (including MHC molecules, exosomal marker proteins, tetraspanins, etc.), saccharides, cytosolic proteins and nucleic acids.
  • C Detailed isolation schema for the isolation of exosomes. Exosomes were collected and isolated by a series of centrifugation/ultracentrifugation steps. FIG.2. Characterization of exosomes.
  • B Western blots of organoid-derived exosomes at six (sample 1) and eight (sample 2) weeks in culture showing positive bands for exosome markers CD63, flotillin-2, and flotillin-1, and CD9.
  • Organoid-derived exosomes increases the proliferation of C20A4 Chondrocyte Cells.
  • A Gating Strategy for proliferation marker (Ki-67) on C20A4 Chondrocyte Cells (Isotype control). Singlets were gated based on the forward and side scatter parameters (Area vs Height). Chondrocytes were then gated on the singlet population followed by the live cells (L/D - ghost Dye). Ki-67 + population were detected on the live cells based on the isotype control.
  • B Flow cytometry-based detection of Ki-67 + population using the isotype control. TNF ⁇ -treated cells showed decreased proliferation (70.9%) compared to the normal media control (85.5%) after 48 hours in the media control and TNF ⁇ control overlay.
  • FIG.5. Organoid-derived exosomes increases the proliferation of C20A4 Chondrocyte Cells.
  • A Detection of percentage change in Ki-67 + population compared to the media control in C20A4 chondrocytes. Increased proliferation is observed on cells treated with exosomes from Week 5, Week 9, Week 11, and Week 13 of MTO culture media. Detection of % change in Ki- 67 + population compared to the TNF ⁇ control after 48 hours is also represented.
  • B Detection of percent change in Ki-67 + population compared to the media control in TC28a2 chondrocyte cell line. Increase in proliferation is observed on cells treated with exosomes from Week 5, Week 9, Week 11, and Week 16 of MTO culture media.
  • FIG.6 Exosomes from MTO culture medium alters the colony forming ability of the chondrocytes. TC28a2 chondrocyte colonies after treatment with exosomes from different weeks of MTO culture media in the presence and absence of TNF ⁇ .
  • FIG.7 Exosomes from MTO culture medium enhances the colony forming ability of the chondrocytes.
  • A The crystal violet retained by the cells (FIG.6) was washed with 10% acetic acid and the color intensity was measured at 595 nm.
  • Exosomes inhibit the inflammation by inducing expansion of the Treg population and their activation through CD69 expression. Detailed gating strategy to determine the effect of exosomes on regulatory T cell population. Singlets were gated based on the forward and side scatter parameters (Area vs Height).
  • FIG.11 Schematic illustration of exemplary mechanisms of action of MTO exosomes for the treatment of osteoarthritis.
  • Exosomes isolated from MTO culture media increase chondrocyte proliferation.
  • the presence of exosomes increases the level of regulatory T cell (Treg) population and CD4 + CD69 + T cells in human MLR cultures.
  • FIG.12. Therapeutic trial of MTO-derived exosomes in the mouse model of traumatic brain injury.
  • FIG.13 Gating strategy for immunomodulatory molecule (CD9) on MTO exosomes.
  • FIG.15 Expression of immunomodulatory molecules on Week 9 exosomes from Batch- 13 MTO culture medium.
  • A Expression of CD63 compared to unstained control.
  • B Expression of CD81 compared to unstained control.
  • C Expression of CD9 compared to unstained control.
  • FIG.16 Expression of immunomodulatory molecules on Week 10 exosomes from Batch-13 MTO culture medium.
  • A Expression of CD63 compared to unstained control.
  • B Expression of CD81 compared to unstained control.
  • FIG.17 Expression of immunomodulatory molecules on Week 11 exosomes from Batch-13 MTO culture medium.
  • A Expression of CD63 compared to unstained control.
  • B Expression of CD81 compared to unstained control.
  • C Expression of CD9 compared to unstained control.
  • FIG.18 Expression of immunomodulatory molecules on exosomes isolated from different weeks of Batch-13 MTO culture medium.
  • FIG.19 Expression of immunomodulatory molecules on Batch-13 MTO culture exosomes compared to exosomes isolated from parental iPSC cell culture medium.
  • A CD63.
  • B CD81.
  • C CD9.
  • FIG.20 Expression of immunomodulatory molecules on Batch-14 MTO culture exosomes compared to exosomes isolated from parental iPSC cell culture medium.
  • A CD63.
  • B CD81.
  • C CD9.
  • FIG.21 Expression of immunomodulatory molecules on Batch-15 MTO culture exosomes compared to exosomes isolated from parental iPSC cell culture medium.
  • A CD63.
  • B CD81.
  • C CD9.
  • FIG.22 Batch-to-batch characterization if immunological molecules expressed by MTO exosomes compared to iPSC exosomes.
  • A MTO derived exosomes from three separate batches and baseline IPSC derived exosomes were profiled and compared for the expression of CD9, CD63 and CD81 by flowcytometry. Percentage expression of immunomodulatory molecules on exosomes isolated from different batches of MTO culture medium.
  • B Mean fluorescent intensity (MFI) of Immunomodulatory molecules on exosomes isolated from different batches of MTO culture medium.
  • FIG.23 Batch-to-batch characterization if immunological molecules expressed by MTO exosomes compared to iPSC exosomes.
  • A MTO derived exosomes from three separate batches and baseline IPSC derived exosomes were profiled and compared for the expression of CD9, CD63 and CD81 by flowcytometry. Percentage expression of immunomodulatory molecules on exosomes isolated from different batches of MTO culture medium.
  • B Mean fluorescent
  • Organoid-derived exosomes increase the proliferation of chondrocytes. Effect of exosomes on the proliferation of chondrocytes – Ki-67 based assay representing Ki67 expression in mitotic cells. TC28a2 chondrocyte cells were treated with exosomes of week 10 MTO culture medium. Proliferation marker, Ki-67 level was detected on control and exosome treated cells through immunohistochemistry. In the magnified image, the cells marked by the arrow are found to be in the active stage of cell cycle showing positive signal for Ki67. FIG.24. Organoid-derived exosomes increase the proliferation of chondrocytes.
  • Ki67 level was detected on Tc28a2 chondrocytes treated with TNF ⁇ (50 ng/million cells) and exosomes from Batches 13 to 17 on week 10 of the MTO culture medium. The cells were also treated with control iPSC exosomes and the level of expression of Ki67 was detected.
  • the merged images of DAPI and Ki67 show the reversal of the TNF ⁇ -mediated decrease in proliferation on TC28a2 chondrocytes upon treatment with week 10 exosomes compared to the iPSC exosomes.
  • FIG.25 The merged images of DAPI and Ki67 show the reversal of the TNF ⁇ -mediated decrease in proliferation on TC28a2 chondrocytes upon treatment with week 10 exosomes compared to the iPSC exosomes.
  • Organoid-derived exosomes increase the proliferation of chondrocytes. Characterization of chondrocyte proliferation using exosomes from 10-week MTOs.
  • A Effect of Week 10 exosomes from batches 13 to 17 on the proliferation of chondrocytes – Ki-67+ Cell Percentage.
  • B Percentage increase in Ki-67 + Cells on MTO exosome treatment compared to iPSC exosome treated chondrocytes. Frequency increase in the Ki67+ proliferating chondrocytes in MTO exosome treatment compared to the IPSC exosome treatment.
  • FIG.26 Organoid-derived exosomes increase the proliferation of chondrocytes.
  • Exosome treatment revert the TNF ⁇ mediated inhibition in Ki67+ cell percentage to near normal (control) levels in C20a4 (left) and TC28a2 (right) chondrocyte cell lines.
  • FIG.27 Exosomes inhibit the inflammation by inducing the regulatory T cell population, Treg and Tr1.
  • A Detailed gating strategy to determine the effect of exosomes on regulatory T cell population (Treg and Tr1) and their activation status (CD69 expression). Singlets were gated based on the forward and side scatter parameters (Area vs. Height). Lymphocytes were then gated on the singlet population followed by the live cells (L/D - ghost Dye).
  • CD4 + T cell population were detected on the live cells which were then gated to identify CD25-FOXP3 double positive Treg cells and CD49b-LAG3 double positive Tr1 cells.
  • Activation marker, CD69 expression was measured in regulatory T cell (Treg and Tr1) populations.
  • MTO Multi-Tissue Organoid
  • MTO Exosomes from week 10 to 12 from eight separate batches were tested at a dose of 100,000 exosome particles/million cells on a mixed lymphocyte reaction (MLR) culture in the presence of peripheral blood mononuclear cells (PBMCs) and their effect on the frequency (%) of Treg cells was determined.
  • MLR mixed lymphocyte reaction
  • PBMCs peripheral blood mononuclear cells
  • MTO exosomes from multiple batched show a significant increase in the frequency of immunomodulatory Treg cells compared to untreated media control. Potency of MTO exosomes to increase the frequency of Treg cells is consistent across the tested batches (Batch 26 to Batch 32).
  • FIG.30 Graphic representation of Dynamic Weight Bearing data shown in Tables 10-12, showing percent of animals in each group that score excellent or good at each time point.
  • FIG.31 Graphical representation of data shown in Tables 10-12, indicating the percent of rats in each group that achieved an excellent score at any time point in the study.
  • DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS This disclosure describes, in one aspect, exosomes generated by multi-tissue organoids. In another aspect, this disclosure describes methods of preparing exosomes generated from a multi-tissue organoid.
  • the multi-tissue organoids can be generated by culturing iPSCs in culture medium (e.g., a chemically defined maintenance cell culture medium) that includes a minimum of at least 0.01% hyaluronic acid such as, for example, at least 0.01% hyaluronic acid, at least 0.02% hyaluronic acid, at least 0.05% hyaluronic acid, at least 0.1% hyaluronic acid, at least 0.2% hyaluronic acid, at least 0.3% hyaluronic acid, at least 0.4% hyaluronic acid, at least 0.5% hyaluronic acid, at least 0.6% hyaluronic acid, at least 0.7% hyaluronic acid, at least 0.8% hyaluronic acid, at least 0.9% hyaluronic acid, at least 1.0% hyaluronic acid, at least 2.0% hyaluronic acid, at least 3.0% hyaluronic acid, at least
  • the multi-tissue organoids can be generated by culturing iPSCs in culture medium (e.g., a chemically defined maintenance cell culture medium) that includes a maximum of no more than 10% hyaluronic acid such as, for example, no more than 5% hyaluronic acid, no more than 4% hyaluronic acid, no more than 3% hyaluronic acid, no more than 2% hyaluronic acid, no more than 1% hyaluronic acid, no more than 0.5% hyaluronic acid, no more than 0.4% hyaluronic acid, no more than 0.3% hyaluronic acid, no more than 0.2% hyaluronic acid, or no more than 0.1% hyaluronic acid.
  • culture medium e.g., a chemically defined maintenance cell culture medium
  • hyaluronic acid such as, for example, no more than 5% hyaluronic acid, no more than 4% hyaluronic acid, no
  • the multi-tissue organoids can be generated by culturing iPSCs in culture medium (e.g., a chemically defined maintenance cell culture medium) that includes hyaluronic acid at a concentration that falls within a range having endpoints defined by any minimum amount of hyaluronic acid described above and any maximum amount of hyaluronic acid described above that is greater than the selected minimum amount.
  • culture medium e.g., a chemically defined maintenance cell culture medium
  • hyaluronic acid at a concentration that falls within a range having endpoints defined by any minimum amount of hyaluronic acid described above and any maximum amount of hyaluronic acid described above that is greater than the selected minimum amount.
  • Exosomes are nano-spherical membrane structures formed by a bilayer of lipids, as illustrated in FIG.1A. They are membrane bound vesicles derived from endosomes that act as intercellular messengers during physiological and pathological conditions. Exosomes are typically defined by their size and surface markers. Exosomes range in a size from about 30 nm to about 200 nm. Exosomes typically express one or more surface markers which may include CD9, CD63, CD81, and other tetraspanins.
  • exosomes can possess various transmembrane components such as proteins, lipids, and saccharides, as well as various cytosolic proteins and nucleic acids.
  • the cargo contained within an exosome (or a population of exosomes) can include a variety of proteins, polypeptides, glycoproteins, lipids, and/or various types of RNA (FIG.1B).
  • the particular transmembrane components and/or cytosolic components of an exosome is determined, at least in part, by the cell and/or tissue from which the exosome is derived.
  • the biogenesis pathway of an exosome begins with late endosomes, so called multivesicular bodies, containing multiple internal vesicles that merge with the cell membrane and release its internal vesicles into the extracellular matrix (FIG.1A).
  • multivesicular bodies containing multiple internal vesicles that merge with the cell membrane and release its internal vesicles into the extracellular matrix (FIG.1A).
  • This biogenesis of exosomes via multivesicular bodies differentiates exosomes from other extracellular vesicles such as apoptotic bodies and microvesicles.
  • Extracellular vesicles vary in their size, origin within the donor cell and function. Microvesicles vary greatly in size (100 nm to 1000 nm), with their cargo predominantly consisting of cytoplasmic content, such as mRNA, miRNA and proteins, as they are directly released from the cell membrane.
  • Apoptotic bodies the largest of the extracellular vesicles (800– 5000 nm), are released during the execution phase of the apoptotic process, potentially carrying organelles, receptors, and similar cytoplasmic content.
  • the cargo of exosomes typically includes DNA, messenger RNA (mRNA), microRNA (miRNA), proteins, and growth factors.
  • mRNA messenger RNA
  • miRNA microRNA
  • Exosomes are present in most bodily fluids including serum, uterine and peritoneal fluid, urine, and breast milk.
  • the lipid bilayer of exosomes protects the cargo from degradation and allows for distant communication throughout the body. Exosome cargo is known to regulate biological processes.
  • NTA instruments are equipped with one or more lasers and an optical microscope connected to a digital camera.
  • NTA enables one to characterize particles in solution ranging from about 10 nm to about 2000. Particles are visualized by the light they scatter upon laser illumination, and their Brownian motion is monitored.
  • NTA software enables sizing of single particles by tracking their mean squared displacement and thereby calculating their theoretical hydrodynamic diameter. Based on knowing the analyzed sample volume, NTA also allows for an estimation of particle concentration. Exosomes isolated from multi-tissue organoid (MTO) culture medium were subjected to NTA characterization.
  • MTO multi-tissue organoid
  • compositions and methods herein can involve identifying exosomes by screening for any suitable exosome marker.
  • exosome markers include, but are not limited to, CD63, flotillin-2, flotillin-1, CD9, and CD81.
  • exosomes typically show low/no immunogenicity and can be easier to handle, store, and/or ship compared to cellular therapies. Exosomes also reduce or eliminate certain issues associated with cell transplantation such as, for example, immune rejection, low cell survival/engraftment, off-target engraftment and tumorigenicity.
  • exosomes can be included in a pharmaceutical composition for administering to a subject.
  • pharmaceutical compositions that includes exosomes derived from multi-tissue organoids. This disclosure also describes methods of treating a subject having, or at risk of having, a condition treatable with exosomes derived from multi-tissue organoids.
  • a composition can be administered via known routes including, for example, oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.).
  • a pharmaceutical composition can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol).
  • a composition also can be administered via a sustained or delayed release.
  • a pharmaceutical composition that includes MTO-generated exosomes may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture.
  • the composition may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle.
  • the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like.
  • the formulation may further include one or more additives including, but not limited to, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like.
  • a formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing the MTO-derived exosomes into association with a carrier that constitutes one or more accessory ingredients.
  • a formulation may be prepared by uniformly and/or intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.
  • the amount of MTO-derived exosomes administered can vary depending on various factors including, but not limited to, the weight, physical condition, and/or age of the subject, and/or the route of administration.
  • the absolute number of MTO-derived exosomes included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight, and physical condition of the subject, and/or the method of administration. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of MTO-derived exosomes effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.
  • the method can include administering sufficient MTO- derived exosomes to provide a dose of, for example, from about 500,000 exosomes to about 100 trillion exosomes to the subject, although in one or more embodiments the methods may be performed by administering MTO-derived exosomes in a dose outside this range.
  • the method can include administering sufficient MTO- derived exosomes to provide a minimum dose of, for example, at least 500,000 exosomes, at least one million exosomes, at least 10 million exosomes, at least 20 million exosomes, at least 50 million exosomes, at least 100 million exosomes, at least 500 million exosomes, at least one billion exosomes, at least five billion exosomes, at least 10 billion exosomes, at least 50 billion exosomes, or at least 100 billion exosomes.
  • the method can include administering sufficient MTO- derived exosomes to provide a maximum dose of, for example, no more than 100 trillion exosomes, no more than 50 trillion exosomes, no more than 10 trillion exosomes, no more than one trillion exosomes, no more than 500 billion exosomes, no more than 100 billion exosomes, no more than 50 billion exosomes, no more than 10 billion exosomes, or no more than 1 billion exosomes.
  • Exosomes are said to be present in amounts “no more than” a reference amount when exosomes are not absent but are present in an amount up to the reference amount.
  • the method can include administering sufficient MTO- derived exosomes to provide a dose characterized by a range having endpoints defined by any a minimum dose identified above and any maximum dose identified above that is greater than the selected minimum dose.
  • the method can include administering sufficient MTO-derived exosomes to provide a dose of, for example, from one billion exosomes to one trillion exosomes, from one million exosomes to 10 trillion exosomes, from 100 million exosomes to 500 billion exosomes, from 50 billion exosomes to 10 trillion exosomes, etc.
  • the method can include administering sufficient MTO-derived exosomes to provide a dose equal to any minimum dose or any maximum dose listed above.
  • the method can include administering sufficient MTO-derived exosomes to provide a dose of one million exosomes, one billion exosomes, 10 billion exosomes, 50 billion exosomes, 100 billion exosomes, one trillion exosomes, etc.
  • a single dose may be administered all at once, continuously for a prescribed period of time, or in multiple discrete administrations. When multiple administrations are used, the amount of each administration may be the same or different.
  • a dose of 100 billion per day may be administered as a single administration of 100 billion exosomes, continuously over 24 hours, as two or more equal administrations (e.g., two administrations of 50 billion exosomes), or as two or more unequal administrations (e.g., a first administration of 75 billion exosomes followed by a second administration of 25 billion exosomes).
  • the interval between administrations may be the same or different.
  • MTO-generated exosomes may be administered, for example, from a single dose to multiple doses per week, although in one or more embodiments the method can involve a course of treatment that includes administering doses of the MTO- generated exosomes at a frequency outside this range.
  • a course of treatment involves administering multiple doses within a certain period, the amount of each dose may be the same or different.
  • a course of treatment can include a loading dose initial dose, followed by a maintenance dose that is lower than the loading dose.
  • the interval between doses may be the same or be different.
  • MTO-generated exosomes may be administered from a once per week to a single once-off dose, although in one or more embodiments the methods may be performed by administering MTO-derived exosomes at a frequency outside of this range.
  • MTO-generated exosomes may be administered at a minimum frequency of at least once per week, at least once per month, at least once per year, at least once every two years, at least once every three years, at least once five years, at least once every 10 years, or as a single once-off dose.
  • MTO-generated exosomes may be administered at a maximum frequency of no more than once every five years, no more than one every three years, no more than one every two years, no more than once per year, no more than one per month, or no more than once per week.
  • MTO-generated exosomes may be administered at a frequency characterized by a range having endpoints defined by any a minimum frequency identified above and any maximum frequency identified above that is more frequent than the selected minimum frequency.
  • MTO-generated exosomes may be administered at a frequency of from a once-off does to once per week, from once every three years to once per week, from once every five years to once per month, etc.
  • MTO-generated exosomes may be administered at a frequency equal to any minimum frequency or any maximum frequency listed above.
  • MTO-generated exosomes may be administered as a single once-off dose or at a frequency of once every three years, once every five years, etc.
  • a course of treatment can have a duration of a single dose to the remaining life of the subject.
  • the course of treatment with MTO-generated exosomes can have a minimum duration of a once-off dose, at least six months, at least one year, at least three years, at least five years, or until complete recovery.
  • the course of treatment with MTO-generated exosomes can have a maximum duration of the remaining life of the subject, no more than 10 years, no more than five years, no more than three years, no more than one year, no more than six months, or no more than three months.
  • the duration of a course of treatment can be characterized by a range having endpoints defined by any a minimum duration identified above and any maximum duration identified above that is greater than the selected minimum duration.
  • the duration of a course of treatment can be from a single once-off dose to the remaining life of the subject, from six months to five years, from three months to three years, etc.
  • the term “sign” or “clinical sign” refers to an objective physical finding relating to a particular condition capable of being found by one other than the patient.
  • at risk refers to a subject that may or may not actually possess the described risk.
  • a subject “at risk” of having a condition is a subject possessing one or more risk factors associated with the condition such as, for example, genetic predisposition, ancestry, age, sex, geographical location, lifestyle, or medical history. Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
  • a composition can be administered before, during, or after the subject first exhibits a symptom or clinical sign of the condition. Treatment initiated before the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the likelihood that the subject experiences clinical evidence of the condition compared to a subject to which the composition is not administered, decreasing the severity of symptoms and/or clinical signs of the condition, and/or completely resolving the condition.
  • total knee or hip arthroplasty total knee or hip replacement
  • Traditional non-surgical treatments for osteoarthritis can improve symptoms but cannot restore articular cartilage, regenerate cartilage, or modify degenerative processes.
  • surgical arthroplasty results in long-term functional improvement and improves quality of life, such treatments are only suitable for the end stage of the diseases, and instability and infection are the most common limitations, necessitating further revision surgery.
  • MTO-generated exosomes can be applied earlier in the course of osteoarthritis than surgical treatments, or even prophylactically to subjects at risk for developing osteoarthritis.
  • MTO-generated exosome therapy can reduce the likelihood or severity of osteoarthritis, slow or reverse the progression of disease, regenerate damaged cartilage, reduce inflammation and pain, and improve the subject’s quality of life.
  • chondrocytes were cultured in the presence or absence of TNF ⁇ with exosomes purified from the culture supernatant from organoid cultures at different stages of organoid development. Following 48 hours of culture with exosomes, chondrocyte proliferation was analyzed by enumerating the numbers of Ki-67 + chondrocytes. Ki- 67 is a nonhistone nuclear protein that is a convenient and reproducible biomarker for cell proliferation.
  • Human chondrocyte cell lines (e.g., TC28a2 and C20A4) are widely used as a model cell line for studying normal and pathological cartilage repair mechanisms related to chondrocyte biology and physiology.
  • Chondrocyte cell lines TC28a2 and C20A4 were cultured in the presence and absence of exosomes isolated from multi-tissue organoid (MTO) culture medium at various time points during organoid development. Exosomes isolated from different weeks of the batch 4 MTO culture medium was used. The exosomes were dosed based on their protein cargo measurement. For the proliferation assay, exosomes with an overall cargo content of 50 ⁇ g of protein per million cells were used.
  • MTO multi-tissue organoid
  • TNF ⁇ an inhibitor of chondrocyte proliferation
  • a normal complete media 1% FBS
  • TNF ⁇ mesenchymal stem cells
  • MSCs mesenchymal stem cells
  • chondrocytes were treated with exosomes isolated from different time points of MTO development in the presence and absence of TNF ⁇ . After 48 hours of the treatment period, the cells were collected and stained for the expression of Ki-67 using a Ki-67- PE staining kit.
  • An isotype control (IgG1) was also performed for each sample. Isotype controls are primary antibodies that lack specificity to the target but match the class and type of the primary antibody used in the application.
  • FIG.5A and FIG.5B The effect of MTO-derived exosomes on the change in proliferation of chondrocytes cell lines C20A4 and TC28a2 compared to the TNF ⁇ control cells are presented in FIG.5A and FIG.5B.
  • Exosomes derived from MTO culture media at Week 5, Week 9, Week 11, Week 13, and Week 15 of MTO development reversed the TNF ⁇ -mediated inhibition of proliferation on C20A4 and TC28a2 chondrocytes (FIG.5A and FIG.5B).
  • Exosomes from Week 13 (Exo4) reversed the TNF ⁇ -mediated inhibition of proliferation to the maximum level (15.4%) on C20A4 cells (FIG.5A).
  • exosomes from Week 9 were most effective reversing the TNF ⁇ -mediated inhibition of chondrocyte proliferation (FIG.5B).
  • the exosome-mediated increases in proliferation reversed the TNF ⁇ -mediated inhibition to near normal level.
  • exosomes from Week 16 showed the lowest level of reversal of TNF ⁇ -mediated inhibition: C20A4 (2.9%, FIG.5A) and TC28a2 (1.5%, FIG.5B).
  • Exosomes secreted from earlier weeks of MTO may contain cargo that might induce more proliferation compared to exosomes from the later weeks.
  • Exosomes from MTO culture medium alters the colony forming ability of the chondrocytes
  • the colony formation assay is an in vitro cell survival and proliferation assay based on the ability of a single cell to grow into a colony of at least 50 cells.
  • the assay essentially tests every cell in the population for its ability to retain their reproductive integrity over a prolonged period of time. This is an important feature as it reveals phenotypic effects that require time and possibly several cell divisions to develop. When analyzing therapeutic use of exosomes this is particularly important. Through this assay the long-term effect of an exosome on proliferation can be determined. TC28a2 chondrocytes were used for this assay.
  • the cells were treated with exosomes (50 ⁇ g/million cells) collected from different weeks of the batch3 MTO culture supernatants.
  • the colonies were compared with the media control and TNF ⁇ -treated control wells to determine the effect of exosomes on the colony forming ability of the TC28a2 chondrocytes. Since the TC28a2 chondrocytes grow as a dispersed colony (FIG.6), quantification of the crystal violet retained by the cells on each condition was measured. The results of the quantification were depicted in FIG. 7A. Based on the optical density measured at 595 nm, percentage change in proliferation was determined keeping the values of the control wells (media alone and TNF ⁇ ) as 100%.
  • the change in proliferation percentage in the exosome-treated wells compared to the respective controls were determined and the data is presented in FIG.7B.
  • Treatment with exosomes show a significant increase in the proliferation as there was an increase in chondrocyte percentage upon treatment with exosomes compared to the TNF ⁇ control wells.
  • Exosomes from early stage of the MTO culture media show a very high increase in proliferation with as high as 48.0%.
  • the decrease in chondrocyte percentage compared to media control in FIG.7B could be due to over proliferation and necrosis of the cells.
  • the Ki-67 + proliferation assay and colony formation assay show that exosomes isolated from MTO culture media collected at up to Week 15 of multi-tissue organoid development reversed TNF ⁇ -mediated decreases in chondrocyte proliferation.
  • Exosomes inhibit inflammation by inducing the Treg population and CD69 expression Regulatory T (Treg) cells are a specialized subset of immunosuppressive CD4 T cells that express the X-chromosome-encoded, lineage-specific transcription factor FOXP3.
  • Treg cells maintain peripheral tolerance by providing essential suppression of autoreactive CD4 T cells that have escaped negative selection in the thymus.
  • Treg cells act as negative regulators of inflammation in various biological contexts, including infection, metabolic disease, tissue repair, and cancer.
  • Treg cells respond to inflammation by sharply increasing their suppressive function.
  • Activated Treg cells upregulate immunosuppressive molecules and tissue homing receptors and undergo polycomb-mediated repression of Foxp3-bound genes, which may inhibit pro-inflammatory functions.
  • Gating strategies for defining the Treg population are depicted in FIG.8 and FIG.27. In short, area versus height on forward and side scattered population was used to gate for the singlet population. The lymphocytes were then selected, and the live population was gated based on the Live-Dead ghost dye. CD4 + T cells were gated from the live population and the Tregs were defined by the expression of both CD25 and FOXP3 (FIG.8; FIG.27).
  • exosomes from the MTO culture medium induce the proliferation on CD25 + FOXP3 + regulatory T cells (FIG.9; FIG.28A).
  • Human PBMC responder sample (Sample Code: Human 23) that was used in the study stimulated with mismatched human PBMC sample (Sample code: STC3) show a marked increase in the level of Treg population in the presence of exosomes.
  • the CD4 + CD25 + FOXP3 + Treg cells are actively engaged in mitigating autoimmunity and aberrant or excessive immune responses.
  • the transcription factor Forkhead box P3 (denoted “FOXP3” in humans, and “Foxp3” in mice) is a well-characterized marker of Treg cells, and its expression typically is considered a requisite for Treg cell differentiation and function.
  • FOXP3 deficiency leads to the scurfy phenotype in mice and to immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome in humans.
  • Treg-cell function is impaired in several autoimmune and inflammatory diseases, including colitis, rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus.
  • MTO exosomes might provide a practical approach to the treatment of autoimmune and inflammatory diseases.
  • CD69 C-type lectin receptor CD69 (encoded in NK gene cluster), an early activation antigen of lymphocytes, also was examined.
  • CD69 is involved in regulating immune responses in murine models of asthma, arthritis, colitis, myocarditis, pathogen clearance, and tumors.
  • CD69 activation induces TGF- ⁇ expression and suppresses the production of pro- inflammatory cytokines IL-17 and IFN- ⁇ .
  • Treating human PBMCs with exosomes isolated from the MTO culture medium increased expression of Treg (FIG.28A) and Tr1 (FIG.28B) regulatory T cells. The level of expression was consistent across multiple batches of MTO exosome treatment compared to untreated media control PBMCs.
  • the activation marker, CD69 also increased in Treg and Tr1 cells upon MTO exosome treatment compared to the untreated media control (FIG.10, FIG. 29A). Activation of CD69 leads to ERK phosphorylation and thus stabilizes TGF- ⁇ on the cell surface of lymphocytes.
  • CD4 + CD69 + CD25 ⁇ T cells inhibit development of graft-versus host disease.
  • CD69 + T cells are able to induce indoleamine 2,3-dioxygenase (IDO) in tumor-associated macrophages and hence downregulate inflammatory immune responses. Therefore CD4 + CD69 + CD25 ⁇ T cells have been introduced as novel regulatory cell type whose effector functions depend mainly on TGF- ⁇ . Exosomal membranes are enriched in endosome-specific tetraspanins (CD9 and CD81).
  • CD9 and CD81 regulate systemic inflammation, including functions that are related to modulating CD19 and MHC class II molecules in B cells, with CD4 in T cells, and with integrins in B cells and T cells.
  • the many involvements of CD81 stem from its ability to stabilize and facilitate the organization of its specific partner molecules such as CD9 in response to stimuli.
  • exosomes from IPSC-derived medium and MTO-derived medium were incubated with antibodies against CD9 and CD81. The level of expression of CD9 and CD81 was determined via flow cytometry and the results were presented (FIG.29B).
  • Exosomes isolated from week 12 MTO culture medium is found to be showing high levels of CD9 and CD81 compared to the exosomes from regular IPSC culture medium.
  • High levels of CD9 and CD81 with the anti- inflammatory potential being a part of the MTO exosomal cargo could contribute to the anti- inflammatory effects of MTO-derived exosomes.
  • Expression profile of immunomodulatory molecules on early, mid, and late weeks of MTO exosomes FIGS.13-18 provide data showing increased expression percentage and mean fluorescent intensity (MFI) of immune suppressive markers CD9, CD81, and CD63 in exosomes collected from Week 8 to Week 11 from Batch 13 of the MTO culture medium compared to the exosomes collected from iPSC culture medium (control).
  • MFI mean fluorescent intensity
  • FIG.3A shows the gating strategy for assessing CD9 expression on MTO exosomes.
  • FIGS.19-22 show expression profiles of immunosuppressive markers CD9, CD63, and CD82 in different batches of MTOs. The profiling suggests that exosomes isolated from MTO culture medium show an increased expression of the markers compared to the exosomes isolated from control iPSC culture medium across various batches.
  • the nucleus of the cells were labelled with DAPI (Red) and Ki-67 level marked by the expression ranging from green (low) to orange (High) spectrum of colors based on the intensity of Ki-67 expression, with blue to purple being no expression of Ki-67 (FIG.23).
  • the cells marked by the arrow are found to be in the active stage of cell cycle also showing positive signal for Ki-67.
  • the Ki-67 level was detected on Tc28a2 chondrocytes treated with TNF ⁇ (50 ng/million cells) and exosomes from Batches 13 to 17 on week 10 of the MTO culture medium.
  • the cells were also treated with control iPSC exosomes and the level of expression of Ki-67 was detected.
  • the merged images of DAPI and Ki-67 show the reversal of the TNF ⁇ -mediated decrease in proliferation on TC28a2 chondrocytes upon treatment with Week 10 exosomes from batches 13 – 17 compared to the iPSC exosomes (FIG.24).
  • the percentage proliferative Ki-67 positive cells on control and exosome treated chondrocytes were calculated and results are presented in FIG.25.
  • FIG.25B shows the percentage increase in Ki-67 + cells on MTO derived exosome treatment compared to iPSC exosome treated chondrocytes.
  • a flow-based assay was also performed using C20A4 and TC28a2 chondrocytes that were treated with exosomes isolated from different weeks of MTO in the presence or absence of TNF ⁇ . After 48 hours of the treatment period, the cells were collected and stained for the expression of Ki-67. Isotype controls are used as negative controls to help differentiate non-specific background signal from specific antibody signal. The detailed gating strategy (FIG.4B) was followed to determine the Ki-67 + cell percentage on different treatment conditions. Cells treated with TNF ⁇ (10 ng/ml) shows a significant decrease in the proliferation of chondrocyte cell lines (FIG.4B).
  • exosomes from week 9 were found to exhibit maximum increase in proliferation (12.5%) after TNF ⁇ -mediated inhibition. This increase in proliferation brought the TNF ⁇ -mediated inhibition to near normal level.
  • Exosomes from week 16 shows the lowest level of increase in proliferation from the TNF ⁇ treated C20A4 (2.9%) and TC28a2 (1.5%) chondrocytes (FIG.5).
  • Exosomes secreted from earlier weeks (13 weeks or less) of MTO might contain the necessary cargo to induce more proliferation compared to exosomes from later weeks of MTO culture medium.
  • exosomes reversed the TNF ⁇ inhibition in proliferation of TC28a2 (right panel) and C20A4 (left panel) similar to the proliferation observed in the media controls (FIG 26).
  • the overall possible mechanism of action of MTO exosomes for the treatment of osteoarthritis is depicted in FIG.11. From the Ki-67 + proliferation assay and colony formation assay, it is evident that exosomes isolated from MTO culture media at up to Week 15 of MTO development reverses the TNF ⁇ -mediated decrease in chondrocyte proliferation. Apart from the role of inhibiting the proliferation, TNF ⁇ also induces inflammation that can lead to osteochondral defects.
  • TNF ⁇ also may interfere with the healing process of chondral and osteochondral defects that occurs naturally or in low inflammatory environment after a cartilage repair procedure.
  • a significant correlation between inflammation and disease severity has been observed in human and animal models of osteoarthritis.
  • This disclosure provides data showing that MTO exosomes contain high levels of anti-inflammatory molecules such as CD9 and CD81 in their cargo compared to exosomes from regular iPSC culture medium. Exosomes purified from MTO- development inhibits TNF ⁇ -induced inflammation in human peripheral blood mononuclear cells. Regulatory T cell (Treg) function is impaired in several autoimmune and inflammatory diseases, including osteoarthritis.
  • Groups 1-4 received weekly intra- articular injections into the right femoro-tibial joint beginning on day 0, consisting of saline (Group 1), 2.20 ⁇ 10 11 exosomes in saline (Group 2), TCA + Tramadol (Group 3), and FGF-18 (Group 4).
  • rats were assessed by dynamic weight bearing analysis. In normal rats the weight bearing should be approximately equal between left and right hind limbs which is indicated by a score of 0.50 (as in the Na ⁇ ve rats in Group 5). Scores for each rat in all five groups are shown in FIG.30. Scores were categorized as Excellent if above 0.45 and Good if above 0.40.
  • the data indicates a therapeutic effect for MTO-derived exosomes for the treatment of osteoarthritis. While described above in the context of an exemplary embodiment in which osteoarthritis is the condition treated using MTO-generated exosomes, the compositions and methods described herein can involve treating other conditions.
  • Exemplary alternative conditions treatable using MTO-generated exosomes include, but are not limited to, cartilage injury, autoimmune diseases (e.g., immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, etc.), inflammatory diseases (e.g., colitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, immune-mediated inflammatory disease, etc.), neurodegenerative disorders (e.g., Alzheimer’s disease, Parkinson’s disease, traumatic brain injury, etc.), stroke, and conditions involving mitochondrial dysfunction (e.g., aging, diabetes, metabolic disorders, obesity, etc.).
  • autoimmune diseases e.g., immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, etc.
  • inflammatory diseases e.g., colitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, immune-mediated inflammatory disease, etc.
  • Table 3 lists additional proteins that are markers characteristic of chondrocytes and indicate that the source of the exosomes is cells in the chondrocytic lineage.
  • Table 4 lists examples of proteins present in the exosomes that have been shown to have chondrogenic and/or chondrotrophic properties that are relevant to potential cartilage regenerative effects of the MTO-derived exosomes.
  • Table 5 lists several surface proteins present in the MTO-derived exosomes that are relevant to the identity of exosomes. Some of these surface markers also have important immunomodulatory effects (noted below), which may be relevant to anti-inflammatory actions of the MTO-derived exosomes.
  • Table 6 lists several proteins present in the MTO-derived exosomes that are immunomodulatory and/or anti- inflammatory.
  • Table 7 lists examples of proteins present in MTO-derived exosomes that are of mitochondrial origin and which may assist in mitochondrial function.
  • Table 8 lists examples of proteins present in MTO-derived exosomes that may stimulate cell growth and tissue regeneration, and may have anti-aging activities.
  • Table 3. Cartilage Extracellular Matrix Components Detected in MTO-Derived Exosomes and Type 12 collagen COL12A1 Cartilage ECM component
  • the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
  • the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention. EXAMPLES The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
  • iPSC line referred to as 1024 (ATCC-BYS0110, American Type Culture Collection, Manassas, VA) was expanded in culture on vitronectin (VTN-N, Thermo Fisher Scientific, Inc., Waltham, MA) in Essential 8 Medium (E8, Thermo Fisher Scientific, Inc., Waltham MA) for two or three passages. iPSCs were harvested using sodium citrate buffer and briefly centrifuged for five minutes at room temperature, 250 ⁇ g.
  • Multi-tissue organoid (MTO) induction was initiated by resuspending expanded iPSCs ( ⁇ 20 million cells) in 40 ⁇ l of buffered hyaluronic acid (HA). Cells in HA were then transferred to a G-REX 100 bioreactor (Wilson Wolf Manufacturing Corp., New Brighton, MN) containing E8 media and incubated at 37 °C in 5% CO 2 . E8 culture medium containing 1% antibiotic-antimycotic (Thermo Fisher Scientific, Inc., Waltham, MA) was changed every 3-4 days over the entirety of multi-tissue organoid (MTO) culture.
  • MTO multi-tissue organoid
  • Exosome samples were diluted 50 times in 0.02- ⁇ m-filtered PBS to obtain a concentration within the range of 108 ⁇ 109 particles/ml.
  • the exosome analyses were performed with the NTA software (version 3.1 Build 3.1.54, Malvern Panalytical, Ltd., Malvern, UK) using 60 seconds of video captures per sample (in triplicate). Camera level was set to 14 and detection threshold was set to 3.
  • Western Blot Analysis Purified exosomes were characterized by semi-quantitative Western blot analysis by measuring the presence of CD63 and flotillin-1 content in the exosomes using specific antibodies.
  • SDS-sample buffer was added to the exosome sample and the sample was incubated for five minutes at 95 °C and separated on 4-12% precast acrylamide gels (Invitrogen, Carlsbad, CA). After transfer to PVDF membranes (Millipore, Bedford, MA) and blocked overnight (5% milk and 0.05% Tween-20 in PBS), primary antibody was added for one hour, followed by PBS washing and the application of secondary HRP-conjugated antibody. Chemiluminescence detection of bands was performed with ECL Plus reagent (GE Healthcare, Buckinghamshire, UK). Analysis of Exosomes by Flow Cytometry Purified exosomes were stained with a known exosome marker CD63.
  • Exosome pellets were resuspended in anti-CD63-PerCP in PBS and incubated at 4°C for 30 minutes. After the incubation period, exosomes were washed with PBS (10 ml) to remove the excess antibody. The solution was transferred to a centrifuge (AMICON-Ultra 15 Centrifugal Filter Units, Merck KGAA, Darmstadt, Germany) and spun at 3200 ⁇ g for 30 minutes. The collected exosomes were resuspended in 200 ⁇ l of PBS and the samples were used for flow cytometric analysis for the expression of CD63.
  • AMICON-Ultra 15 Centrifugal Filter Units Merck KGAA, Darmstadt, Germany
  • the stained exosomes were washed twice with 3 ml of 1 ⁇ BD Perm/Wash buffer, and then resuspended in 300 ⁇ l of the stain buffer prior to flow cytometric analysis.
  • a minimum of 200,000 events were acquired on three-laser flow cytometer (FACSCANTO II equipped with FACSDIVA 6.1.3, BD Biosciences, Franklin Lakes, NJ).
  • Relative percentages and mean fluorescence intensity (MFI) of each of the populations were determined using the FlowJo 10.1. software (TreeStar). Chondrocyte Cell lines The following cell lines were used for the study.
  • T/C-28a2 cell line (MilliporeSigma, Burlington, MA) was established by transfecting primary cultures (day 5) of costal cartilage from a 15-year-old female with a retroviral vector expressing simian virus SV40 large T antigen.
  • C- 20/A4 cell line (MilliporeSigma, Burlington, MA) was established by transfecting primary cultures of rib chondrocytes from a 5-year-old male with vectors expressing origin-defective simian virus SV40 large T antigen.
  • Ki-67 based chondrocyte Proliferation Assay Chondrocytes were treated with exosomes isolated from different weeks of multi tissue organoid (MTO) in the presence and absence of TNF ⁇ , an inhibitor of chondrocyte proliferation. After 48 hours of the treatment period, the cells were collected and stained for the expression of Ki-67 using a Ki-67-PE staining kit (BD Biosciences, Franklin Lakes, NJ). In short, cells were trypsinized after treatment, then added to a 12 ⁇ 75 mm test tube, and first stained with a Live- Dead Ghost Dye for 20 minutes at 4 °C in the dark. The cells were then washed twice with PBS.
  • MTO multi tissue organoid
  • the permeabilization procedure was performed according to the manufacturer’s protocol.250 ⁇ l of CYTOFIX/CYTOPERM (BD Biosciences, Franklin Lakes, NJ) solution was added per tube and vortexed well. After incubating for 20 minutes at 4°C, the cells were washed twice in 1 ⁇ BD PERM/WASH buffer (BD Biosciences, Franklin Lakes, NJ), 3 ml per tube. The permeabilized cells were thoroughly resuspended in 100 ⁇ l of 1 ⁇ PERM/WASH buffer (BD Biosciences, Franklin Lakes, NJ) before the Ki-67 intracellular staining. A titrated anti-Ki-67 fluorochrome- conjugated antibody was added and incubated at 4°C for 30 minutes in the dark.
  • a test tube without the isotype antibody was used as a negative control.
  • the stained cells were washed twice with 3 ml of 1 ⁇ PERM/WASH buffer (BD Biosciences, Franklin Lakes, NJ), and then resuspended in 300 ⁇ l of the stain buffer prior to flow cytometric analysis using a FACSCANTO system (BD Biosciences, Franklin Lakes, NJ).
  • Colony Formation Assay TC28a2 chondrocytes were used for this assay.
  • the cells were treated with exosomes (50 ⁇ g/million cells) collected from different weeks of the batch 3 multi-tissue organoid (MTO) culture supernatants.
  • MTO multi-tissue organoid
  • CFSE-Based Chondrocyte Proliferation Assay Chondrocytes were labeled with 2.5 ⁇ M carboxyfluorescein succinimidyl ester (CFSE, Invitrogen, Cat# C34554, Carlsbad, CA) and were cultured with exosomes isolated from the MTO culture medium. Following four days of culture with exosomes, proliferation of the chondrocytes was analyzed by flow cytometry by enumerating the numbers of CFSElo chondrocytes and by measuring the levels of CFSE.
  • CFSE carboxyfluorescein succinimidyl ester
  • MLR Mixed Lymphocyte Reaction
  • Activation markers and proliferation percentage were analyzed on CD4 + T cells, CD8 + T cells, and CD20 + B cells.
  • the effect of exosomes on regulatory T cell population (Treg) was studied.
  • a minimum of 200,000 events were acquired on three-laser CANTO II (BD Biosciences, Franklin Lakes, NJ) with FACSDIVA 6.1.3 (BD Biosciences, Franklin Lakes, NJ).
  • Relative percentages and mean fluorescence intensity (MFI) of each of the populations were determined using the FlowJo 10.1. software (BD Biosciences, Franklin Lakes, NJ).
  • RNA sequencing library was prepared using the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA). After library preparation, the constructed library was sequenced using Illumina HiSeq2000/2500, and the reading length was 1 ⁇ 50 bp. Curated reads were analyzed using the RNA analysis pipeline as described earlier (O’Grady et al., BMC Biology 20:72, 2022).
  • RNA-Seq coverage reads were aligned and mapped to version GRCh38 of the human genome with STAR aligner V. 2.5.2b (Dobin et al., Bioinformatics 29(1):15-21, 2013) and visualized with J-Circos (An et al., Bioinformatics 31(9):1463-1465, 2015). For quantification, reads were first mapped to the set of NCBI RefSeq rRNA sequences using STAR aligner V. 2.5.2b (Dobin et al., Bioinformatics 29(1):15-21, 2013).
  • Unmapped reads were then mapped to the GRCh38.90 human transcriptome from Ensembl (cDNA + noncoding RNA) using Salmon v.0.8.2 (Patro et al., Nat Biotechnol 32(5):462-464, 2014). Finally, remaining reads were once again mapped to the GRCh38.90 human transcriptome using Salmon v.0.8.2. Genes with an abundance level of at least 1 transcript per million (TPM) averaged across the replicates were considered to be reliably detected. To investigate differential gene expression, mRNA and lncRNA read counts were summed to the gene level using tximport and compared using DESeq2 (Love et al., Genome Biology 15:550, 2014).
  • TC28a2 cells were initially seeded in glass bottom dishes (ibidi GmbH, Gräfelfing, Germany) at a density of 1 ⁇ 10 5 cells per well, grown for 24 hours in complete medium. The cells were treated with exosomes (50 ⁇ g/million cells) collected from week 10 of batch13-batch17 MTO culture supernatants. Control cells were treated with iPSC culture medium exosomes. After 24 hours of exosome treatment in the presence of TNF ⁇ (10 ng/ml), the cells were stained to look at Ki-67 expression and finally analyzed by means of confocal microscopy.
  • the protein-protein interactions were investigated under confocal microscope (TCS SP8, Leica Camera, Hesse, Germany), and the cellular samples were processed with a standard immunofluorescence protocol: fixation with formaldehyde 3.7% for 10 minutes, permeation with 0.1% Triton X-100 in phosphate buffered saline (PBS) for 10 minutes, block with bovine serum albumin (BSA) 2% for two hours.
  • Ki-67 protein was directly investigated through a mouse IgG anti-Ki-67, conjugated with PE dye (Ki-67 Monoclonal Antibody, PE, eBioscience). Expression pattern of Immunomodulatory Molecules on MTO Exosomes Exosomes from the MTO show elevated levels of immune suppressive markers.
  • MTO exosomes were incubated with antibodies against CD9, CD63, and CD81, known exosomal markers with immunoinhibitory potential.
  • the level of expression of CD9 and CD81 was determined via flow cytometry and the results are presented (FIG.13-22).
  • Exosomes isolated from different weeks of Batch-13, Batch-14, and Batch-15 MTO culture medium show high levels of CD9, CD63, and CD81 compared to the exosomes from regular iPSC culture medium.
  • a detailed gating strategy and expression profile of the immunoinhibitory markers such as CD9, CD63, and CD81 from different weeks of MTO exosomes are provided in FIGs.13-22.
  • Groups 1-4 received weekly intra-articular injections into the right femoro-tibial joint beginning on day 0, consisting of saline (Group 1), 2.2 ⁇ 10 11 exosomes in saline (Group 2), TCA (triamcinolone acetonide) + Tramadol (Group 3), and FGF-18 (Group 4). On Days 7, 38, and 72 rats were assessed by dynamic weight bearing analysis.
  • Exosome treatment also showed a far superior therapeutic response than treatment with FGF-18 (FIG.30), which has been shown to be highly effective in some models of osteoarthritis.
  • FIG.31 shows the percent of animals in each group that achieved an excellent Dynamic Weight Bearing score at any time point in the study, demonstrating a superior therapeutic effect versus saline controls. Overall, the data indicates a therapeutic effect for MTO-derived exosomes for the treatment of osteoarthritis.
  • Table 10 Day 7 Treatment Group Group 1 Group 2 Group 3 Group 4 Group 5 Vehicle Exosomes TCA+Tram FGF18 Na ⁇ ve Table 11.
  • TBI mice Two TBI mice (1M, 1F) received IV tail vein saline injections on Days 7, 14, and 21 (sham treated TBI controls), and 4 TBI mice (2M, 2F) received 10 10 MTO-derived exosomes in saline IV in the tail vein on Days 7, 14, and 21 (exosome treated).
  • Barnes Maze memory assessments were conducted on Days 26, 27, and 28. Mice were sacrificed on Day 28 and brains were fixed and processed for histopathological assessments. Data shown in FIG.12.

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Abstract

Un procédé de préparation d'exosomes comprend généralement la fourniture d'un organoïde multi-tissu, la culture de l'organoïde multi-tissu dans un milieu de culture, la collecte d'au moins une partie du milieu de culture, et l'isolement d'exosomes à partir du milieu de culture. Les exosomes expriment au moins un marqueur identifiant les exosomes comme étant isolé d'un milieu de culture dans lequel un organoïde multi-tissu est cultivé. Les exosomes peuvent être utilisés dans des procédés pour traiter un sujet présentant, ou risquant de présenter, une pathologie traitable avec des exosomes isolés du milieu de culture dans lequel un organoïde multi-tissu est cultivé. Généralement, le procédé comprend l'administration au sujet d'une composition pharmaceutique qui comprend une quantité efficace d'exosomes isolés à partir d'un milieu de culture dans lequel un organoïde multi-tissu est cultivé.
PCT/US2024/050957 2023-10-11 2024-10-11 Produits d'exosomes dérivés d'organoïdes, procédés de fabrication et procédés d'utilisation Pending WO2025080972A1 (fr)

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WO2021096929A1 (fr) * 2019-11-11 2021-05-20 Anand Rene Exosomes humains dérivés de neurones pour la maladie d'alzheimer et les co-morbidités de ceux-ci
US20210220456A1 (en) * 2020-01-17 2021-07-22 Regents Of The University Of Minnesota Tumor cell-derived exosomes and method of treating colorectal cancer
US20220249569A1 (en) * 2013-05-08 2022-08-11 Prokidney Organoids comprising isolated renal cells and uses thereof
US20230251245A1 (en) * 2021-12-17 2023-08-10 The University Of Chicago Methods of Using Multi-Tissue Organoids
CN117384834A (zh) * 2023-10-19 2024-01-12 中国人民解放军空军军医大学 一种软骨类器官外泌体的制备方法及其应用

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US20220249569A1 (en) * 2013-05-08 2022-08-11 Prokidney Organoids comprising isolated renal cells and uses thereof
WO2021096929A1 (fr) * 2019-11-11 2021-05-20 Anand Rene Exosomes humains dérivés de neurones pour la maladie d'alzheimer et les co-morbidités de ceux-ci
US20210220456A1 (en) * 2020-01-17 2021-07-22 Regents Of The University Of Minnesota Tumor cell-derived exosomes and method of treating colorectal cancer
US20230251245A1 (en) * 2021-12-17 2023-08-10 The University Of Chicago Methods of Using Multi-Tissue Organoids
CN117384834A (zh) * 2023-10-19 2024-01-12 中国人民解放军空军军医大学 一种软骨类器官外泌体的制备方法及其应用

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