Preparation and therapeutic use of 3D cultured fibroblasts

Abstract

The present disclosure provides methods, compositions, and systems for producing and using organoids containing fibroblasts.The organoids can be generated and placed in a specific storage matrix, which provides suitable conditions for transportation and reduces damage to cells in the organoids compared to conventional culture media.The organoids can also be used for the sustained release of fibroblasts for cell therapy and for drug delivery at target sites after administration to individuals in need.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 378,796, filed October 7, 2022, the entire contents of which are incorporated herein by reference.

[0002] Technical Field Embodiments of the present disclosure are in the fields of at least cell biology, molecular biology, immunology, and medicine. [Background technology]

[0003] Beginning with the embryonic stages of human development, fibroblasts play a critical role in all aspects of human physiology, anatomy, and immunology ( 1 ).

[0004] Fibroblasts can be isolated from a variety of tissues. This isolation method requires a high-level laboratory equipped with specific equipment (ultraclean benches, temperature- and humidity-controlled incubators, and various high-purity gases and incubators) for preparation, maintenance, and quality control. Typical medical institutions do not meet these requirements. Therefore, fibroblast products for cell therapy must be transported long distances between laboratories and medical institutions across a specific geographic range (2). Under normal temperature conditions, cells rupture organelles, and nuclear damage ultimately leads to apoptosis. Cell debris can cause severe inflammation after transplantation (3). Currently, there are two types of cell transport: short-distance and long-distance. Short-distance transport is usually limited to 24 hours (4). For transport beyond 24 hours, cells are transported in culture flasks covered with growth medium. They are transported near the body in a 37°C thermostatic suitcase at room temperature, but the amount of cells transported using this method is limited and prone to cell damage. Even if they reach their destination, recovery takes 48 hours. Technically, this method consumes large amounts of expensive culture media, and transporting liquids poses potential public health risks. Transportation methods such as airplanes and high-speed rail are dangerous and inefficient, making them suitable only for transporting small numbers of live cell lines. However, small cell volumes are not suitable for cell therapies, which require up to one billion cells per case. Dry ice transport is widely used to transport large numbers of cells over long distances (5, 6). Cells are frozen in cryovials, placed in solid dry ice, and transported to the destination laboratory or hospital. Cells are then cultured locally for 4–7 days for recovery before use. While this method can transport large numbers of cells at once, it requires the addition of tens of kilograms of dry ice every 12–24 hours. At each stop along the delivery route, local suppliers must be contacted to request dry ice, which is extremely expensive. Dry ice is also difficult to transport on airplanes. Other transportation methods, such as high-speed rail, require complex procedures and certification, inevitably increasing costs.If dry ice is consumed during transport and cannot be replenished, cells may undergo freeze-thaw cycles, which can cause irreversible damage to the cells. Therefore, developing a method for efficiently and safely transporting cells at room temperature is crucial for realizing cell-based therapies. Summary of the Invention

[0005] The embodiments of the present disclosure include compositions, methods and systems for the efficient preparation, culture, maintenance, preservation and / or transport of fibroblasts that are not in a state of a plurality of single cells.The embodiments of the present disclosure include compositions, methods and systems for the efficient preparation, culture, preservation and / or transport of fibroblasts that exist as clusters of, for example, at least 2, 10, 50, 100, 500, 1000, 2000, 5000, 7500, 10000, 25000, 50000, 75000, 100000 cells.In certain embodiments, the clusters of cells (sometimes referred to as organoids) have a specific shape, such as a sphere or a sphere-like shape, a cube or a cubic-like shape, a pyramid or a pyramid-like shape, or a star or a stellate-like shape.In other embodiments, the organoid does not have a specific geometric shape. In certain embodiments, the clusters of fibroblasts comprise 3D fibroblast spheroids.

[0006] The present disclosure encompasses 3D fibroblast organoid, wherein at least about 51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98 or 99% of the cell in organoid is fibroblast.In certain cases, the majority of the cell in 3D fibroblast organoid is fibroblast. 3D fibroblast organoids can contain approximately 50-100%, 50-90%, 50-80%, 50-70%, 50-60%, 60-100%, 60-90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%, 80-90%, or 90-100% fibroblasts, including any value or range derivable therein. 3D fibroblast spheroids can also contain combinations of one or more cell types other than fibroblasts, such as stem cells, epithelial cells, keratinocytes, neural cells, and / or any type of immune cell.

[0007] Any fibroblast organoid (including spheroids), including spheroids, can be used for disease modeling, clinical training, research, and / or therapeutic applications in humans and animals (including all types of mammals). In certain embodiments, the present disclosure relates to the interaction of a first type of cell with a second type of cell and / or a specific factor, and includes modifications to the first and / or second type of cell as a result of the interaction. In certain embodiments, the first type of cell is a fibroblast, and the second type of cell is any type of immune cell (e.g., T cells, NK cells, NKT cells, macrophages, B cells, dendritic cells, or a mixture thereof), any type of stem cell, or a combination thereof. In further specific embodiments, the present disclosure encompasses compositions, methods, and systems in which fibroblasts are modified when exposed to specific cells and / or one or more specific conditions, environments, and / or factors. Specific factors that can modify fibroblasts may include one or more nucleic acids, cytokines, chemokines, growth factors, and / or exosomes before or after the preparation, culture, maintenance, and / or storage of single-cell fibroblasts, or after the generation of 3D fibroblast organoids (including 3D fibroblast spheroids).In addition, in certain embodiments, the present disclosure provides an introduction to use these fibroblast spheroids, or materials derived from fibroblast spheroids, as a sustained release mechanism for fibroblasts and / or fibroblast-derived materials in vivo.In certain embodiments, such materials can be used as a targeting tool for various clinical applications, such as tissue regeneration, immunomodulation, tissue repair, wound healing, or cell migration to specific cell types or tissues.When fibroblast organoids are used as a targeting tool for cell migration to specific cell types or tissues, in certain embodiments, this occurs through activated or modified cell surface markers, nucleic acid modifications, and / or the expression or secretion of one or more chemokines, cytokines, exosomes, and / or growth factors.

[0008] Embodiments of the present disclosure provide in vitro or in vivo methods for producing any type of cellular structure or material derived therefrom, including (1) activated or non-activated fibroblast spheroids, (2) fibroblast spheroid-derived material derived from activated or non-activated spheroid fibroblasts, (3) activated or non-activated immune cells, and / or (4) fibroblast derivatives. Such compositions can be used in any suitable application, including at least the following: generation in vitro or ex vivo followed by introduction in vivo to (1) initiate or maintain long-term immunomodulation, (2) initiate or maintain long-term release of activated or non-activated fibroblasts and / or fibroblast-derived material, or (3) initiate or maintain long-term cell migration and proliferation to a target tissue or organ for repair, regeneration, and / or anti-degeneration. In certain embodiments, the therapeutic composition is the spheroid itself or cells released therefrom; in other or additional embodiments, the therapeutic composition is material derived from cells in the spheroid or released from those cells or released cells. When the therapeutic agent is a cell or a derivative thereof that is released from the spheroid over time, the timing of the release may have a controllable or predictable release rate of the cells or components released from the cells. Such control of time release may result directly or indirectly from the method of preparation of the spheroid. The cells within the spheroid may be allogeneic, autologous, or xenogeneic to the individual receiving the therapeutic agent.

[0009] The fibroblasts may be activated, e.g., have activated or altered cell surface markers, nucleic acid modifications, and / or expression or secretion of one or more chemokines, cytokines, exosomes, and / or growth factors.

[0010] Embodiments of the present disclosure encompass the preparation, culture, maintenance, storage, transport, and / or use of organoid fibroblasts, i.e., fibroblast spheroids and / or fibroblast spheroids containing other cells and / or cellular components, and these structures can be used as long-term release of fibroblasts and / or fibroblast-derived materials in at least clinical applications.

[0011] One clinical application of the structure is a method for storing and / or transporting fibroblasts at a specific desired temperature (e.g., about 0-37°C) using a unique matrix to ensure cell viability and biological activity. The disclosed method differs from any previous method. In one embodiment, the properties of fibroblast spheroid formation are exploited to slow their proliferation and metabolic rates, thereby maintaining fibroblast and / or fibroblast activity and biological function in a normothermic environment for up to about 21 days or longer, for example, in combination with other cell types.

[0012] Another example of the clinical application of the structure is the use of a unique liquid preservation matrix that is not particularly biologically toxic, can be easily applied to single cells and / or formed structures, and meets the requirements for high-density, large-scale cell storage and transportation. In certain embodiments, the cell product does not require post-processing such as separation or purification before use, and does not require the maintenance of specific temperatures or humidity levels throughout the entire storage and transportation process. As a result, the cell product directly meets the requirements of biomedical scientific research, cell therapy for immune and degenerative diseases, cell transplantation for tissue and organ damage, drug screening, and other fields, bringing immeasurable scientific and socio-economic value.

[0013] Embodiments of the present disclosure utilize the ability of fibroblasts to form organoids, such as spheroids, under certain suspension culture conditions, where the fibroblasts spontaneously aggregate to form condensed organoids. In certain embodiments, the fibroblasts in the cell spheroids naturally reduce their proliferation and metabolic rate due to cell contact inhibition, which suppresses oxygen and nutrient consumption.

[0014] The present disclosure provides a unique liquid culture and storage medium or matrix that ensures organoids meet their most basic energy, nutrient maintenance requirements, and / or pH levels suitable for cell survival, while reducing shear stress damage caused by sudden movements during transport. Furthermore, the disclosed method can accommodate long-distance transport requirements under ambient temperature conditions, such as transport for 24 hours or longer, and allows cell products to be stored only in plastic, glass, metal, and / or rubber containers. In certain embodiments, even large-scale transport of high-density cells (e.g., 1 million to 100 billion cells) does not require specialized temperature and gas maintenance equipment, although cryopreservation and / or transport may involve such equipment.

[0015] In certain embodiments, after the cells reach their intended destination (e.g., a university laboratory, a research institute, or any type of medical facility, including a hospital), the matrix can be removed by an appropriate method, such as centrifugation. As shown herein, the recovered cells retain clinically useful viability and complete biological function even after storage at ambient temperature for at least 7 days, and can be used for any type of assay and / or treatment.

[0016] In various embodiments of the present disclosure, methods for forming fibroblast spheroids include U-shaped / V-shaped ultra-low attachment culture plates, the hanging drop method, applying fibroblasts to hydrophilic regions on a substrate with a primarily hydrophobic surface, and / or forming core-shell structures in pipette tips (e.g., (1) forming a core spheroid in a single pipette tip and culturing it for 48 hours; (2) forming an outer layer of the spheroid with cells in the same pipette tip and culturing it for an additional 48 hours, preferably repeating this process to add additional cell layers and / or cell types to the spheroids; (3) culturing it in the pipette tip for 96 hours, after which the cell spheroids are formed, and then the liquid containing the spheroids in the pipette tip is ejected to recover the cell spheroids, and the liquid matrix is ​​removed by centrifugation or filtration to recover the spheroids). These methods allow for the generation of cell spheroids of different sizes depending on the cell density. In certain embodiments, the size of the spheroids may be in the range of about 50-500 μm, 50-400 μm, 50-300 μm, 50-200 μm, 50-100 μm, 100-500 μm, 100-400 μm, 100-300 μm, 100-200 μm, 200-500 μm, 200-400 μm, 200-300 μm, 300-500 μm, 300-400 μm, or 400-500 μm, and may include any value or range derivable therein. Furthermore, examples of size include about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 μm, and may include any value or range derived therefrom. In one embodiment, fibroblasts are seeded at high density in a container or culture dish using an ultra-low attachment culture plate or a glass culture flask, and allowed to spontaneously form spheroids of different sizes.

[0017] In certain embodiments, the liquid storage matrix can contain one or more biosafe thickening agents (tackifiers) for transport or storage, increasing the viscosity of the medium and enabling stable retention of fibroblast spheroids within the liquid storage matrix. In certain embodiments, the liquid storage matrix contains a basal cell culture medium excluding phenol red, providing essential nutrients and an acid-base balance system. In certain embodiments, the basal medium includes a low-sugar medium, human serum, non-essential amino acids, and / or L-glutamine. Examples of thickening agents for increasing the viscosity of the medium include food-grade additives such as methylcellulose, agar, guar gum, xanthan gum, pectin, collagen, and gelatin, and can be used at concentrations of approximately 0.2-0.5%, 0.2-0.4%, 0.2-0.3%, 0.3-0.5%, 0.3-0.4%, or 0.4-0.5%, or any concentration derived therefrom. Concentration can be 0, about 0.1%, about 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4% or 5%, and can include any range or value.The medium can also contain other materials and conditions for successful culture.In certain embodiments, the thickening agent is non-biologically toxic, metabolized, non-persistent, and safe for cells.In addition, in certain embodiments, fibroblast organoid can survive without damage for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days. Furthermore, in certain embodiments, the organoids may be transported for a period ranging from about 1 to 21 days, 1 to 20 days, 1 to 15 days, 1 to 10 days, 1 to 5 days, 1 to 2 days, 2 to 21 days, 2 to 20 days, 2 to 15 days, 2 to 10 days, 2 to 5 days, 5 to 21 days, 5 to 20 days, 5 to 10 days, 10 to 21 days, 10 to 20 days, 10 to 15 days, 15 to 21 days, 15 to 20 days, or 20 to 21 days, while avoiding shear stress damage during transport.

[0018] Various embodiments of the present disclosure provide for the clinical use of fibroblast spheroids for extended release in any therapeutic application, such as sustained immunomodulatory capacity in autoimmune diseases.

[0019] In various embodiments of the present disclosure, clinical uses of fibroblast spheroids are provided, for example, for the sustained release of one or more fibroblast-derived growth factors.

[0020] In various embodiments of the present disclosure, there is provided the use of fibroblast spheroids for clinical or biological research purposes.

[0021] The compositions and methods of the present disclosure allow for increased viability of fibroblasts in any in vivo application during and after administration, and also allow for increased viability of spheroids to reach damaged tissue areas and continuously grow and repair damaged tissues and organs or reverse organ degeneration.

[0022] In certain embodiments, the present disclosure relates to compositions, methods, and systems in which specific cells are modified by exposure to specific factors secreted from, contained within, or contained within fibroblasts and / or fibroblast spheroids. In certain embodiments, fibroblasts interact with one or more other types of cells (optionally including one or more specific environments and / or factors), resulting in the modification of fibroblasts and / or other types of cells to participate in immune regulation, tissue repair, and reversal of tissue and organ degeneration. In certain embodiments, the other types of cells include at least recruited immune cells and local stem cells.

[0023] In certain embodiments, the disclosed methods include introducing live single-cell suspensions or live 3D fibroblast spheroids, non-activated fibroblasts, and / or fibroblast-derived materials into a target tissue or organ to modify fibroblasts and other cells present in the target tissue or organ and / or surrounding tissues. In certain embodiments, the activity stimulates at least tissue and / or organ repair or reversal of degenerative processes. In some aspects, the fibroblasts and / or one or more factors are exposed to factors produced by modification from one or more cells present at the injury site. In certain embodiments, the one or more factors involved in the exposure may be exogenously provided or endogenous, derived from the environment and / or cells and / or tissue.

[0024] In certain embodiments, the method of the present disclosure is carried out ex vivo, for example, in a culture environment. In certain cases, the method is performed by human hands and does not involve natural or accidental phenomena in the body. The method of the present disclosure is non-natural in certain respects. In certain embodiments, the concentration of cells or cell-derived materials used in the method of exposing one type of cell to another type of cell does not occur in nature or occur accidentally in nature. In certain embodiments, the concentration of one or more factors used in the method of exposing one or more cells does not occur in nature or occur accidentally in nature. Any type of cell modification encompassed by the present disclosure that occurs ex vivo or in vitro does not occur in a similar form in nature in vivo. In this embodiment, cells are isolated from tissue biopsies from donors, characterized, optionally activated, expanded, and reintroduced into organoid structures for the purpose of immune regulation, tissue repair, tissue regeneration, and / or reversal of tissue degeneration.

[0025] Certain embodiments of the present disclosure encompass therapeutic uses of cells, including all types of fibroblasts (including organ fibroblasts), epithelial cells, immune cells, neutrophils, macrophages, keratinocytes, vascular endothelial cells, myofibroblasts, and mixtures thereof. In at least some cases, the fibroblasts or fibroblast-derived materials are modified prior to exposure to the target tissue or organ, which may be by chemical, viral, physical, or epigenetic activation, or by exposure to conditions not normally found in the body. In other cases, immune cells, such as neutrophils, macrophages, tissue-specific fibroblasts, keratinocytes, vascular endothelial cells, myofibroblasts, or their derivatives, are activated or otherwise modified prior to exposure to the fibroblasts.

[0026]

[0006] Embodiments of the present disclosure provide a means for utilizing fibroblasts as allogeneic, autologous (or xenogeneic or syngeneic) therapeutic cells by modifying culture conditions. In one embodiment of the present disclosure, fibroblasts are extracted from a less immunogenic source (e.g., placental fibroblasts, omental tissue-derived fibroblasts, umbilical cord blood-derived fibroblasts, etc.) or other source that can produce cells that can be used for therapy or clinical research.

[0027] In one embodiment of the present disclosure, fibroblasts are cultured in vitro as spheroids to maintain their viability and proliferation. The present disclosure provides for modifying known culture techniques to reduce fibroblast recognition by the recipient's immune system. In one embodiment, the fibroblast spheroids are cultured in xeno-free culture conditions, e.g., in a xeno-free medium. For example, the medium may not contain fetal bovine serum. In certain embodiments, fetal bovine serum may be replaced with one or more factors that promote reduced immunogenicity of the fibroblast spheroids, such as human platelet-rich plasma, platelet lysate, umbilical cord blood serum, autologous serum, and / or one or more defined cytokines, such as one or a combination of fibroblast growth factor, epidermal growth factor, leukemia inhibitory factor, insulin-like growth factor, angiopoietin, and vascular endothelial growth factor.

[0028] In one embodiment of the present disclosure, an effective amount of fibroblast spheroids prepared by the methods encompassed by the present disclosure is administered to treat or prevent one or more diseases. In certain embodiments, the administration of fibroblast spheroids and / or fibroblast spheroid-derived materials improves immune response, wound repair, cell differentiation, and / or tissue remodeling in a recipient individual.

[0029] Embodiments of the present disclosure provide methods for administering universal donor-derived fibroblast spheroids (wherein the fibroblasts may be genetically modified, xenogeneic, allogeneic, or autologous) with one or more factors, individually or in combination, to stimulate and maintain a desired immune response, tissue repair, and / or tissue regeneration. In certain embodiments, methods are provided for administering universal donor fibroblast spheroids and / or fibroblast spheroid-derived material with one or more chemokines and / or cytokines and / or growth factors. In one embodiment, universal donor fibroblast spheroids, derived from fibroblast spheroids treated under conditions that reduce immunogenicity, are utilized to stimulate fibroblast growth factors necessary for tissue repair, tissue regeneration, and / or reversal of organ degeneration.

[0030] Embodiments of the present disclosure provide methods for reducing the immunogenicity of specific types of fibroblast spheroids. The fibroblast spheroids may be derived from various tissues or organs, including, but not limited to, skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, foreskin, etc., and may be obtained by biopsy or autopsy, as appropriate. In some aspects, the cells include fibroblast spheroids of fetal origin, neonatal origin, adult origin, or a combination thereof.

[0031] The fibroblast spheroids used in any of the methods of the present disclosure may, in certain embodiments, be exposed to particular media components.

[0032] In embodiments of the present disclosure, a method for producing fibroblast organoids is provided, the method comprising producing a plurality of fibroblast organoids and suspending the organoids in a medium containing low-glucose medium, about 0-20% human serum, about 0-5% non-essential amino acids and / or about 0-5% L-glutamine, and optionally one or more viscosity enhancing agents. In certain embodiments, the amount of human serum can be about 0-20%, 0-15%, 0-10%, 0-5%, 1-20%, 1-15%, 1-10%, 1-5%, 5-20%, 5-15%, 5-10%, 10-20%, 10-15%, or 15-20%, including any value or range derivable therein. The amount of serum can be about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20%. The amount of non-essential amino acids can be about 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 1-5%, 1-4%, 1-3%, 1-2%, 2-5%, 2-4%, 2-3%, 3-5%, 3-4%, or 4-5%, including any value or range derivable therein. The amount of non-essential amino acids can be about 0, 1, 2, 3, 4, or 5%. Non-essential amino acids include glycine, L-alanine, L-asparagine monohydrate, L-aspartic acid, L-glutamic acid, L-proline, L-serine, L-histidine, isoleucine, L-lysine hydrochloride, L-serine, L-tryptophan, and / or L-valine. The amount of L-glutamine can be about 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 1-5%, 1-4%, 1-3%, 1-2%, 2-5%, 2-4%, 2-3%, 3-5%, 3-4%, or 4-5%, and can include about 0, 1, 2, 3, 4, or 5%. In certain embodiments, the amount of thickener is about 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 1-5%, 1-4%, 1-3%, 1-2%, 2-5%, 2-4%, 2-3%, 3-5%, 3-4%, or 4-5%, including about 0, 1, 2, 3, 4, or 5%. Examples of thickeners include methylcellulose, agar, guar gum, xanthan gum, pectin, collagen, and / or gelatin.

[0033] In certain embodiments, the size of the resulting organoids may be about 50-500 μm, 50-400 μm, 50-300 μm, 50-200 μm, 50-100 μm, 100-500 μm, 100-400 μm, 100-300 μm, 100-200 μm, 200-500 μm, 200-400 μm, 200-300 μm, 300-500 μm, 300-400 μm, or 400-500 μm, including about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 μm. In certain embodiments, organoids are produced by the following methods: (a) using U-shaped / V-shaped ultra-low attachment culture plates; (b) the hanging drop method; (c) applying cells to hydrophilic regions on a substrate with a predominantly hydrophobic surface; (d) forming core-shell structures within pipette tips (e.g., (1) creating a core spheroid within a single pipette tip and culturing for 48 hours; (2) forming a second layer of cells on the spheroid within the same pipette tip and culturing for an additional 48 hours, and repeating this process as needed to add additional cell layers or cell types; (3) after 96 hours of culturing within the pipette tip, ejecting and collecting the liquid containing the spheroids from the pipette tip, and then removing the liquid matrix by centrifugation or filtration to collect the spheroids). In certain embodiments, the number of cells within each organoid is about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10Fibroblast organoids can be produced by culturing them for about 24-48 hours in an incubator at about 37°C, 5% carbon dioxide, and 80% or greater humidity. In certain cases, organoids can be cultured for about 24-48, 24-44, 24-40, 24-36, 24-30, 24-28, 28-48, 28-44, 28-40, 28-36, 28-30, 30-48, 30-44, 30-40, 30-36, 36-48, 36-44, 36-40, 40-48, 40-44, or 44-48 hours, including any value or range between about 24 and 48 hours. The temperature can be about 20-37°C, 20-35°C, 20-30°C, 20-27°C, 20-25°C, 20-22°C, 22-37°C, 22-35°C, 22-33°C, 22-30°C, 22-27°C, 22-25°C, 25-37°C, 25-35°C, 25-33°C, 25-30°C, 25-27°C, 27-37°C, 27-35°C, 27-33°C, 27-30°C, 30-37°C, 30-35°C, 30-33°C, 33-37°C, 33-35°C, or 35-37°C.

[0034] In certain embodiments, fibroblast organoids can be cultured in a carbon dioxide environment of about 3, 4, 5, 6, 7, or 8%, including ranges of about 3-8%, 3-7%, 3-6%, 3-5%, 3-4%, 4-8%, 4-7%, 4-6%, 4-5%, 5-8%, 5-7%, 5-6%, 6-8%, 6-7%, or 7-8%. Humidity can be about 65, 70, 75, 80, 85, or 90%, including about 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%, 70-75%, 75-90%, 75-85%, 75-80%, 80-90%, 80-85%, or 85-90%. The process of organoid preparation can include adjusting one or more parameters to control the size of the organoid obtained.In certain embodiments, the parameters that are changed include: fibroblast concentration, the type and / or amount of medium, the type and / or amount of sugar in medium, the type and / or amount of serum, the type and / or amount of non-essential amino acid, the amount of L-glutamine, the amount of carbon dioxide in the incubator that contains cells, the temperature of incubator, the time of producing spheroid, or combinations thereof.

[0035] In some embodiments, organoids are placed in containers made of plastic, metal, glass, rubber, etc. In some embodiments, organoids can be stored after production, and storage can occur at room temperature. In some cases, organoids can be stored at about 2-37°C. Organoids can also be cryogenically frozen.

[0036] In some embodiments, organoids are transported within, for example, approximately 0-50 days. Transport periods include 0-50 days, 0-15 days, 0-10 days, 0-5 days, 1-20 days, 1-15 days, 1-10 days, 1-5 days, 5-20 days, 5-15 days, 5-10 days, 10-20 days, 10-15 days, 15-20 days, 20-25 days, or 25-30 days, including any intermediate values ​​or ranges therein. Organoids can be transported at room temperature, e.g., approximately 2-37°C. After transport, organoids are removed from the culture medium by centrifugation, gravity, filtration, microfluidic cassettes, mechanical interaction, chemical interaction, or a combination thereof. After transport, at least some cells are dissociated from the organoids. Cells can be dissociated from the organoids by exposure to an effective amount of one or more proteases, such as trypsin. The amount of trypsin is about 0.1 to 0.3%, and may be about 0.25%.

[0037] In various embodiments, the organoids are used within 0-21 days of any of the methods of manufacture encompassed herein. In some embodiments, the organoids are subjected to one or more research assays or used for therapy and are used within 0-20 days, 0-15 days, 0-10 days, 0-5 days, 1-20 days, 1-15 days, 1-10 days, 1-5 days, 5-20 days, 5-15 days, 5-10 days, 10-20 days, 10-15 days, or 15-20 days of any of the methods of manufacture encompassed herein. In some embodiments, the organoids are subjected to research assays or used for therapy within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days of any of the methods of manufacture encompassed herein. The range of days used from any of the methods of manufacture described herein. Organoid can be used in one or more research assays as an alternative to animal models, for example, in preclinical drug testing and drug development.In some embodiments, the research assay is to use organoid to study organ function.In certain embodiments, the research assay is to use organoid to meet the requirements of drug screening.In certain embodiments, any method encompassed herein can further comprise the step of exposing organoid to one or more chemicals or drugs.

[0038] In certain embodiments, any of the methods encompassed herein may further comprise administering a therapeutically effective amount of organoids to an individual in need thereof. In certain embodiments, the individual has an autoimmune, chronic, degenerative, genetic, infectious, or other disorder or disease, or is in need of immunomodulation, tissue repair, tissue regeneration, organ repair, and / or organ regeneration. The autoimmune disorder and / or disease may be multiple sclerosis, eczema, and / or psoriasis. The infectious disorder and / or disease may be viral, bacterial, fungal, and / or protozoal. The genetic disorder and / or disease may be cancer. The organoids may be administered locally or systemically. Administration may be by injection, infusion, spray, and / or inclusion in a 3D matrix. In certain embodiments, the organoids are administered to the individual one or more times. In certain examples, when multiple doses are administered, the interval between doses may be 1 to 12 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, or 1 to 5 years or more, including any intermediate values ​​or ranges therebetween.

[0039] In various embodiments, the fibroblasts in organoid can be separated from organoid after administration, and can be separated as sustained release.The release period can be any period, for example, from about 1 hour to about 15 days.In certain embodiments, fibroblasts migrate to specific tissue or organ, particularly to the area that needs tissue repair or the area that needs to cause immune response.Specific organs include heart, liver, lung, stomach, spleen, gallbladder, kidney, brain, bladder, and / or intestine.Specific tissues include connective tissue, epithelial tissue, muscle tissue, and / or nerve tissue.

[0040] In various embodiments, the fibroblasts in the organoid are activated, and can be activated by one or more chemicals, RNA, microRNA, RNAi, DNA, viral nucleic acid, and / or exosome.In some embodiments, the fibroblasts contain one or more therapeutic agents that are released from the fibroblasts.Any method can further comprise modifying the fibroblasts to contain one or more therapeutic agents.Examples of therapeutic agents include proteins, nucleic acids, exosomes, growth factors, microRNA, RNAi, mRNA, or combinations thereof.

[0041] Embodiments of the present disclosure include compositions comprising organoids produced by any of the methods described herein. The organoids may be contained in a liquid storage matrix. The composition may contain organoids at room temperature. The organoids may be frozen. The composition may contain organoids in a container such as plastic, glass, metal, or rubber. In certain embodiments, the composition contains organoids in a liquid storage matrix containing low-glucose medium, about 0-20% human serum, about 0-5% non-essential amino acids and / or about 0-5% L-glutamine, and optionally one or more viscosity enhancing agents.

[0042] The foregoing has outlined broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter, and these form the subject of the claims of the invention. Those skilled in the art will appreciate that the conception and specific embodiments disclosed herein may readily serve as a basis for designing or modifying other structures for carrying out the same purposes of the present invention. Those skilled in the art will also realize that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features believed characteristic of the present invention, both as to its organization and method of operation, together with other objects and advantages, will be better understood in connection with the following description and the accompanying drawings. It is to be expressly understood, however, that these drawings are for purposes of illustration and understanding only and are not intended to limit the scope of the invention. [Brief explanation of the drawings]

[0043] [Figure 1] Mouse fibroblasts can be formed into spheroids by the hanging drop method.

[0044] [Figure 2] Mouse fibroblasts can maintain 100% viability after one week of storage at room temperature.

[0045] [Figure 3] Mouse fibroblasts can maintain normal fibroblast morphology, proliferation, and migration ability after storage at room temperature for one week.

[0046] [Figure 4] Human skin fibroblasts (for example, human dermal fibroblasts, e.g., HDFs) can maintain 100% viability even when stored at room temperature for one week.

[0047] [Figure 5]Human dermal fibroblasts in spheroids maintained 95% viability after 2 weeks of storage at room temperature. Viability of HDF spheroids was measured using AO / PI. No significant difference was observed between HDFs stored at 4°C for 2 weeks and the unstored control group.

[0048] [Figure 6] Human dermal fibroblasts within spheroids can maintain 89% viability after 4 weeks of storage at 4°C, as detected by AO / PI staining. The storage medium can be 1X DPBS, 10% FBS, and 10% DMEM / F12.

[0049] [Figure 7] Human dermal fibroblasts within spheroids can be stored at 4°C for 2 and 4 weeks, then replated for 5 days and maintain their migratory and proliferative capacity upon readhesion to cell culture dishes.

[0050] [Figure 8] Human dermal fibroblasts can express both mesenchymal and HDF-specific biomarkers even after 4 weeks of storage at 4°C. Cryosections obtained after 4 weeks show a dense layer at the periphery of the spheroids and a low-density region in the center. Staining of the cryosections showed that HDF spheroids highly expressed fibroblast-specific markers (actin, S00A4, and vimentin) and also partially expressed mesenchymal markers (CD44 and CD90). DETAILED DESCRIPTION OF THE INVENTION

[0051] I. Definition Examples Following long-standing patent law practice, the words "a" and "an" herein, when used in conjunction with the word "comprising," including the claims, mean "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be practiced in combination with any other method or composition described herein.

[0052] As used herein, the terms "about" or "approximately" refer to a variation of up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, when "about" or "approximately" precedes a numerical value, it refers to a range of ±15%, 10%, 5%, or 1% of that value. With respect to biological systems and processes, the term can refer to a range within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold. Unless otherwise specified, "about" refers to an acceptable range of error for the particular value.

[0053] As used herein, "activated immune cells" refers to immune cells that have been treated with one or more stimuli capable of inducing one or more changes in metabolism, immune, epigenetic, secretion of growth factors, expression of surface markers, and production and secretion of microvesicles.

[0054] As used herein, "administered" or "administering" refers to any method of providing a composition to an individual so that the composition exerts its intended effect on the patient. For example, one method of administration is an indirect mechanism using a medical device such as a catheter, applicator gun, syringe, gel matrix, or 3D matrix containing one or more cell types, cell-derived products, and / or growth factors and / or antibiotics. Another example is a direct mechanism of administration, such as administration to a local tissue, oral ingestion, transdermal patch, topical application, inhalation, or suppository.

[0055] As used herein, "allogeneic" means tissue or cells from another individual that are immunologically incompatible or compatible in their natural environment, and are from one or more individuals of the same species.

[0056] As used herein, "autologous" refers to tissues or cells taken from or transplanted into the body of the same individual (e.g., autologous blood transfusion, autologous bone marrow transplant, etc.).

[0057] As used herein, "agent" refers to a nucleic acid, cytokine, chemokine, transcription factor, epigenetic factor, growth factor, hormone, or combinations thereof (including whole cell lysates).

[0058] As used herein, "xenogeneic" refers to tissue or cells obtained from a species different from that of the patient.

[0059] A "cell culture" is an artificial system constructed outside the body that contains viable cells that may be quiescent, senescent, or actively dividing. In cell culture, cells are grown and maintained at a temperature of typically 37°C and in an environment containing varying concentrations of oxygen and carbon dioxide. Culture conditions vary widely depending on the cell type, and changes in conditions for a particular cell type can result in the development of different phenotypes. The factors most commonly altered in a culture system are the medium and the oxygen concentration in the culture medium. The medium can be varied in terms of the concentrations of nutrients, growth factors, and other components. Growth factors are often derived from animal blood, such as calf serum.

[0060] Throughout this specification, unless the context clearly indicates otherwise, the words "comprise," "comprises," and "comprising" shall be understood to mean the inclusion of the recited step, element, or group of steps or elements, but not the exclusion of other steps, elements, or group of steps or elements. "Consisting of" means including and limited to only the elements that follow the phrase. That is, the phrase "consisting of" indicates that the recited elements are required, and that no other elements are present. "Consisting essentially of" means including the elements recited after the phrase, and including only other elements that do not interfere with or contribute to the disclosed activity or function. Thus, "consisting essentially of" indicates that the recited elements are required, but that the presence of other elements is optional so long as they do not affect the function.

[0061] As used herein, the term "individual" refers to a human or animal, whether or not the individual is institutionalized in a medical institution or receiving outpatient treatment. The individual may also receive one or more medical compositions via the internet. An individual includes humans or non-human animals of any age, including adults, children, and infants. The terms "subject" and "individual" are used interchangeably and refer to any living organism or animal subject to a method or material, including mammals such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), farm animals (e.g., cows, sheep, goats, pigs, turkeys, chickens), household pets (e.g., dogs, cats, rodents), horses, and transgenic non-human animals.

[0062] As used herein, the terms "an embodiment," "one embodiment," "particular embodiment," "related embodiment," "further embodiment," "additional embodiment," and combinations thereof mean that a particular feature, structure, or characteristic described in an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of these terms in various places in the specification do not necessarily all refer to the same embodiment. Furthermore, these features, structures, or characteristics may be combined in one or more embodiments, where appropriate.

[0063] As used herein, the terms "reduce," "suppress," "mitigate," "prevent," "reduce," "prevent," and grammatical variations thereof (e.g., "lower," "smaller," etc.) refer to a clinically meaningful reduction in the amount and / or severity of a symptom in a treated subject compared to the occurrence of that symptom in an untreated subject. In certain embodiments, the amount and / or severity of a symptom in a treated subject is reduced by at least 10%, 25%, 50%, 75%, and / or 90% compared to an untreated subject.

[0064] As used herein, "transplantation" refers to the process of taking living tissue or cells and transplanting them into another part of the body or into the body of another individual.

[0065] "Treatment," "treat," or "treating" refers to a method of alleviating the effects of a disease or condition. Treatment can also refer to a method of alleviating the disease or condition itself, and is not limited to methods that target only its symptoms. Treatment can refer to any reduction from pre-treatment levels, including complete elimination of the disease, condition, or its symptoms. Thus, in the methods disclosed herein, "treatment" refers to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity or progression of an established disease, including a reduction in the severity of at least one symptom. For example, a method of reducing the immunogenicity of cells is considered "treatment" if there is a significant reduction in immunogenicity compared to pre-treatment levels in the same subject or a control subject. Thus, this reduction is understood and contemplated herein to mean a 10% to 100% reduction compared to native or control levels, or any level therebetween. "Treatment" does not necessarily mean a cure for a disease or condition, but rather an improvement in its prognosis. In certain embodiments, "treatment" refers to reducing the severity or extent of at least one symptom and / or delaying the onset of at least one symptom.

[0066] " organoid " in this paper refers to the self-organized three-dimensional tissue culture, and is derived from the cell that has self-renewal ability and differentiation ability.In some cases, it refers to the organoid that has a specific shape such as spheroid or a shape similar thereto.In some cases, the cell that has self-renewal ability and differentiation ability is fibroblast.

[0067] As used herein, "fibroblast spheroid-derived materials" refers to exosomes, cells released from spheroids, lysates, conditioned media, and / or apoptotic bodies.

[0068] As used herein, the term "tackifier" refers to a biologically safe substance that can increase the viscosity of a culture medium, and in certain embodiments, functions to preserve fibroblast spheroids in a liquid matrix.

[0069] II. General Examples Embodiments of the present disclosure relate to methods and compositions for preparing 3D cellular structures (such as organoids), enabling the structures to be used as sustained-release agents or for improved handling, such as transportation. Embodiments may combine one or more of the organoid preparation methods, preparative compositions, transportation methods, and / or methods of use. Organoids are manufactured to initiate or maintain desired activity in a target tissue and / or organ, culture vessel, research facility, clinical facility, or a combination thereof. Such activity may be for research and / or therapeutic purposes. Such activity may include improved survival and / or biological activity after transportation, as well as the ability to sustain the sustained release of specific factors (cells themselves and / or factors produced by the cells). Examples of research applications include drug testing, organ development analysis, alternatives to animal testing, tissue regeneration, biological assay development, organoid formation testing, pharmacological testing, and biological response testing.

[0070] In certain embodiments, organoids are manufactured to (1) initiate or maintain the sustained release of immunomodulatory agents, (2) initiate or maintain the sustained release of activated or inactivated fibroblasts and / or fibroblast-derived materials, and (3) initiate or maintain migration and proliferation into target tissues or organs for the purpose of repair, regeneration, and / or reversal of involution. The cellular structure of organoids, rather than a collection of single cells, increases cell contact inhibition, and fibroblasts naturally have reduced proliferation and metabolic rates, minimizing oxygen and nutrient consumption.

[0071] A. Example of organoid production method In various embodiments, the methods and compositions of the present disclosure provide specific means for preserving and transporting fibroblasts in the form of organoids, ensuring their viability and biological activity efficiently, for example, under ambient temperature conditions. In certain embodiments, fibroblast spheroids may be naturally generated. They may be suspended in a liquid culture medium that ensures basic energy needs, nutrient maintenance, and an appropriate pH, reducing shear stress damage that occurs during transportation, for example. The production of organoids can meet long-distance transportation requirements and allows for high-density and large-scale cell product packing under ambient temperature conditions or in plastic, glass, metal, and / or rubber containers. In some cases, the liquid matrix can be removed by centrifugation or other means after arriving at the destination. Organoids may be stored before removing the medium. In certain embodiments, the harvested cells exhibit clinically beneficial viability and intact biological function within 0-21 days, 0-15 days, 0-10 days, 0-5 days, 1-21 days, 1-15 days, 1-10 days, 1-5 days, 5-21 days, 5-15 days, 5-10 days, 10-21 days, 10-15 days, or 15-21 days. Thus, the cells may be used within 0-21 days, 0-15 days, 0-10 days, 0-5 days, 1-21 days, 1-15 days, 1-10 days, 1-5 days, 5-21 days, 5-15 days, 5-10 days, 10-21 days, 10-15 days, or 15-21 days after production or arrival at their destination. The cells may be used immediately after manufacture or arrival, or any number of days after 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more. Destinations may include clinical use, clinical trial use, and / or research and development use.

[0072] In the initial or subsequent steps of the method, fibroblasts are engineered to form organoids (including spheroids) under appropriate suspension culture conditions. For fibroblast spheroid formation, U / V-bottom ultra-low attachment culture plates or the hanging drop method can be used depending on the conditions. For example, organoids of different sizes can be generated depending on the fibroblast density in the medium. In some cases, manipulating the surface tension on the substrate can promote organoid formation. In certain cases, placing individual cells or cell clusters on a surface that is hydrophobic except for the hydrophilic regions can promote organoid generation. In some cases, spheroids can be generated by forming core-shell structures within a pipette tip (e.g., (1) generating a core spheroid within a single pipette tip and culturing it for 48 hours; (2) forming a second layer on the spheroid with cells within the same pipette tip and culturing it for 48 hours, repeating the process by adding additional layers of cells or cell types as needed; (3) culturing it within the pipette tip for 96 hours, then expelling the liquid containing the spheroids from the tip and recovering the spheroids by removing the liquid matrix via centrifugation or filtration). Alternatively, fibroblasts can be seeded directly into ultra-low attachment culture plates or glass culture flasks at high density within the vessel or dish, allowing clumps of different sizes to form naturally with slow agitation.

[0073] In certain embodiments, the size of the spheroids can range from 50 micrometers to 500 micrometers, although smaller and larger sizes are also possible. In certain embodiments, the size of the spheroids can be about 50-500, 50-400, 50-300, 50-200, 50-100, 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, 300-400, or 400-500 micrometers, including any value or range derivable therein. The spheroid size can be about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 micrometers. In certain cases, the size of the organoid is generally controlled by the culture time and culture medium (and other culture conditions as needed).

[0074] In certain embodiments, organoid comprises specific range of cell number.In certain cases, the cell number in organoid is generally controlled by culture time and culture medium (and other conditions as needed).In certain embodiments, organoid can comprise about 103, 104, 105, 106, 107, 108, 109 or 1010 cells per organoid, and can comprise any value or range derived therefrom.

[0075] In one embodiment, fibroblast spheroids are generated by culturing at approximately 37°C for approximately 24-48 hours. The incubator may be set to approximately 5% carbon dioxide and 80% or higher humidity. Stem cell masses can also form naturally at 20-37°C, but other conditions may be used in some cases.

[0076] In certain embodiments, fibroblast spheroids may be cultured in an incubator under appropriate conditions for 24-48, 24-44, 24-40, 24-36, 24-30, 24-28, 28-48, 28-44, 28-40, 28-36, 28-30, 30-48, 30-44, 30-40, 30-36, 36-48, 36-44, 36-40, 40-48, 40-44, or 44-48 hours. In certain embodiments, the incubator may be set to a specific temperature of approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37°C. Temperature ranges may include about 20-37, 20-35, 20-30, 20-27, 20-25, 20-22, 22-37, 22-35, 22-33, 22-30, 22-27, 22-25, 25-37, 25-35, 25-33, 25-30, 25-27, 27-37, 27-35, 27-33, 27-30, 30-37, 30-35, 30-33, 33-37, 33-35, or 35-37°C.

[0077] In certain embodiments, the incubator uses specific gas concentrations, such as carbon dioxide, oxygen, and / or nitrogen. Carbon dioxide can be 3, 4, 5, 6, 7, or 8%. Carbon dioxide concentration ranges can include about 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8%. In certain embodiments, humidity can be about 65, 70, 75, 80, 85, or 90%, and can include about 65-90, 65-85, 65-80, 65-75, 65-70, 70-90, 70-85, 70-80, 70-75, 75-90, 75-85, 75-80, 80-90, 80-85, or 85-90%. The oxygen concentration in the incubator can be set in the range of 0.5 to 25%, and the nitrogen concentration can be set in the range of 1 to 75%.

[0078] Humidity ranges of about 65-90, 65-85, 65-80, 65-75, 65-70, 70-90, 70-85, 70-80, 70-75, 75-90, 75-85, 75-80, 80-90, 80-85, or 85-90% may be used. Other incubator conditions and culture periods may exist or be required.

[0079] In various embodiments, the size of the generated spheroids can be at least partially controlled. The desired size can be specified for manufacturing purposes, transportation purposes, and / or research or therapeutic applications. In certain embodiments, the multiple organoids used are approximately the same size. In other embodiments, first and second multiple organoids of different sizes can be combined before transportation and / or research or therapeutic use, each generated under different culture conditions (e.g., culture duration). The size of the organoids can be determined by adjusting the concentration of fibroblasts used to initiate spheroid formation. Concentration adjustment can be achieved by dilution, selection of medium type, sugars, serum, non-essential amino acids, L-glutamine, carbon dioxide concentration, temperature, culture time, etc.

[0080] In some embodiments, the size of the organoids can be adjusted according to clinical needs (see below). For example, when tissue repair, regeneration, and / or reversal of involution are required, organoids can be manufactured with a size adjusted according to the tissue site. In some embodiments, the organoids function as a sustained release vehicle for delivering fibroblasts themselves or factors derived therefrom as therapeutic components. The sustained release period can be until improvement in the site is detected visually or by assay, until healing is confirmed, or until immune regulation, fibroblast activation or inactivation, or migration and proliferation to the target tissue or organ can be initiated or maintained.

[0081] In certain embodiments, organoids are stored under appropriate conditions after production. In certain cases, the temperature of the storage environment is about 2°C to 37°C. In certain embodiments, the storage environment may include temperatures ranging from 2°C to 37°C, 2°C to 35°C, 2°C to 30°C, 2°C to 25°C, 2°C to 15°C, 2°C to 10°C, 2°C to 5°C, 5°C to 37°C, 5°C to 35°C, 5°C to 30°C, 5°C to 25°C, 5°C to 20°C, 5°C to 15°C, 5°C to 10°C, 10°C to 37°C, 10°C to 35°C, 10°C to 30°C, 10°C to 25°C, 10°C to 20°C, 20°C to 37°C, 20°C to 35°C, 20°C to 30°C, 20°C to 25°C, 25°C to 37°C, 25°C to 35°C, 25°C to 30°C, 30°C to 37°C, 30°C to 35°C, or 35°C to 37°C.

[0082] B. Embodiments of Transport and / or Therapeutic Uses of Organoids After organoids are generated, they can be preserved or transported to a desired destination. Organoids can be suspended in a liquid basal medium containing one or more viscosity-enhancing agents to form a liquid preservation matrix. The liquid portion of the liquid preservation matrix can contain any basic cell culture medium compatible with fibroblasts. The medium can include basic nutrients and an acid-base balance system. Components can include low-glucose medium (e.g., DMEM low-glucose medium and / or other media containing 1 mM to 20 mM (e.g., 5 mM) glucose), approximately 0 to 20% human serum, approximately 0 to 5% non-essential amino acids, and approximately 0 to 5% L-glutamine. The viscosity-enhancing agent can be a food-additive-grade viscosity-enhancing agent such as methylcellulose, and can be used at a specific concentration ranging from 0 to 5%. The basal medium can also contain other substances and / or conditions that promote culture, such as growth factors, sugars, and amino acids.

[0083] In embodiments in which the liquid storage matrix for storage and / or transport comprises serum, particularly human serum, the amount of serum can be 0-20%, 0-15%, 0-10%, 0-5%, 1-20%, 1-15%, 1-10%, 1-5%, 5-20%, 5-15%, 5-10%, 10-20%, 10-15%, or 15-20%, including any intermediate values ​​or ranges thereof. The amount of serum can be any value between 0 and 20%, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20%.

[0084] In embodiments in which the storage and / or transport medium contains non-essential amino acids, the amount may be 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 1-5%, 1-4%, 1-3%, 1-2%, 2-5%, 2-4%, 2-3%, 3-5%, 3-4%, or 4-5%, including intermediate values ​​or ranges thereof. The amount of non-essential amino acids may be 0, 1, 2, 3, 4, or 5%. Non-essential amino acids may include glycine, L-alanine, L-asparagine monohydrate, L-aspartic acid, L-glutamic acid, L-proline, L-serine, L-histidine, isoleucine, L-lysine hydrochloride, L-tryptophan, and / or L-valine.

[0085] In embodiments where the liquid storage matrix for storage and / or transport comprises about 0-5% L-glutamine, the amount can be 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 1-5%, 1-4%, 1-3%, 1-2%, 2-5%, 2-4%, 2-3%, 3-5%, 3-4%, or 4-5%. The amount of L-glutamine can be 0, 1, 2, 3, 4, or 5%.

[0086] In embodiments in which the liquid storage matrix for storage and / or transport includes one or more thickening agents, the amount of thickening agent(s) can be 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 1-5%, 1-4%, 1-3%, 1-2%, 2-5%, 2-4%, 2-3%, 3-5%, 3-4%, or 4-5%. When more than one thickening agent is included, the amount can range from 0-5%, e.g., 0, 1, 2, 3, 4, or 5%, including intermediate amounts and ranges therein. When multiple thickening agents are used, their combined amount may or may not be within the above ranges.

[0087] In some embodiments, fibroblast spheroids are generated and then placed in a suitable container. The number of spheroids per container can vary as needed, but in certain cases ranges from 1 to 10 million cells per mL of storage liquid matrix. In certain embodiments, the fibroblast spheroids are packaged and stored in a sealed container, including a sterile container. In certain embodiments, the container is a plastic container made of a chemically inert material, such as polyethylene, polypropylene, or melamine. The container can also be made of other materials that are not harmful to the cells, such as glass, coated metal, or other polymers, such as polystyrene, branched polymer hydrogels, and cycloolefin polymers.

[0088] Embodiments of the present disclosure allow spheroids to be transported under ambient conditions, which may be before or after storage. In certain embodiments, spheroids are transported at a temperature range of 2-35°C. Spheroids may be transported at temperatures ranging from 2-35°C, 2-30°C, 2-25°C, 2-20°C, 2-15°C, 2-10°C, 2-5°C, 5-35°C, 5-30°C, 5-25°C, 5-20°C, 5-15°C, 5-10°C, 10-30°C, 10-25°C, 10-20°C, 10-15°C, 15-35°C, 15-30°C, 15-25°C, 15-20°C, 20-35°C, 20-30°C, 20-25°C, 25-35°C, 25-30°C, or 30-35°C, including any intermediate values ​​or ranges thereof. Spheroids can be shipped at any temperature between 2 and 35°C, for example, at each integer temperature between 2 and 35°C (2, 3, 4, ..., 34, 35°C). In certain embodiments, the shipping temperature does not exceed about 38°C and does not fall below 0°C, although in other embodiments, the spheroids are shipped at a low temperature.

[0089] In certain embodiments, the cell containers are not exposed to intense energy or light waves, such as intense light sources found in airports, X-rays, or other intense light sources, at least during part or all of their transport. In certain cases, the cell containers and / or incubators in which they are being prepared are not exposed to light at least during part or all of their transport.

[0090] Generated organoids can be transported and / or used within approximately 0-50 days of production. In some cases, organoids are transported and / or used on the day of production, or between 1-50 days (1, 2, 3, ..., 50 days), including any intermediate values ​​or ranges. In some embodiments, organoids can be transported and / or used within 1 to 50 days, 1 to 40 days, 1 to 30 days, 1 to 25 days, 1 to 20 days, 1 to 10 days, 1 to 5 days, 5 to 50 days, 5 to 40 days, 5 to 30 days, 5 to 25 days, 5 to 20 days, 5 to 10 days, 10 to 50 days, 10 to 40 days, 10 to 30 days, 10 to 25 days, 10 to 20 days, 20 to 50 days, 20 to 40 days, 20 to 30 days, 20 to 25 days, 25 to 50 days, 25 to 40 days, 25 to 30 days, 30 to 50 days, 30 to 40 days, or 40 to 50 days.

[0091] In certain embodiments, fibroblast spheroids are manipulated to dissociate at least some cells from the organoid, thereby allowing the dissociated fibroblasts to regain their proliferative capacity or exhibit a higher proliferation rate than when they are present within the organoid. In certain embodiments, the organoids are exposed to one or more proteases, mechanical disruption (e.g., pipetting up and down), and / or sonication. In certain cases, the amount of trypsin is about 0.1-0.3%, e.g., about 0.25%. In certain cases, the spheroids are exposed to about 0.25% trypsin and placed in a 37°C incubator to restore proliferation of the dissociated fibroblasts. Additionally, fibroblasts can be reseeded directly into the container used for shipping and cultured at room temperature.

[0092] In certain embodiments, a plurality of fibroblasts can be released from the spheroid. The release rate can vary depending on the size, contents, activation method, and / or administration environment of the spheroid. In certain embodiments, a plurality of fibroblasts can be released from the spheroid and migrate to a desired site, such as a specific organ or tissue. The ability of fibroblasts to migrate to a desired site can be natural or artificially conferred. In certain embodiments, the released fibroblasts can be activated by activating or altering cell surface markers, nucleic acid modifications, or expressing or secreting one or more chemokines, cytokines, exosomes, or growth factors, and then migrate to the desired site. In certain embodiments, a plurality of fibroblasts can be released from the spheroid, migrate to the desired site, and adherently proliferate from the spheroid. In certain embodiments, once the spheroid is attached to a desired surface, the cells can migrate and proliferate from the spheroid to the site. Furthermore, in some embodiments, the fibroblast spheroids can be separated from the storage liquid matrix and applied directly to the implantation site for tissue regeneration or immunotherapy.

[0093] In some embodiments, the size of organoid can be changed to meet various clinical needs by adjusting cell concentration and / or adjusting the growth period of organoid.For example, when tissue needs repair, regeneration and / or atrophy reversal, the size of organoid may be specific depending on the tissue site.The organoid of a certain size may be desired to be able to be injected through a syringe needle.In another embodiment, the organoid of a certain size may be desired to be injected into a specific site according to the size of vein / artery.

[0094] In some embodiments, organoids are used as therapeutic substance delivery vehicles, and the therapeutic substance delivered is fibroblasts themselves and / or one or more fibroblast-derived substances. In these cases, the organoids function as a sustained-release mechanism for releasing the therapeutic substance over a desired period of time. The desired period can be the period until improvement at the site is observed visually or by assay. The desired period can also be the period until complete healing at the site is observed. The desired period can also be the period required for the initiation or maintenance of immunomodulation. It can also be set as a maintenance period after administration of activated or non-activated fibroblasts and / or fibroblast-derived substances. Furthermore, the desired sustained-release period can also include the period for the initiation or maintenance of migration and / or proliferation to the target tissue or organ, for example, for repair, regeneration, or reversal of atrophy.

[0095] In certain embodiments, the size of fibroblast spheroid can be adjusted to adjust the scope and duration of the in vivo sustained release function of fibroblast and / or fibroblast-derived material from organoid.This adjustment includes producing larger size organoid for longer sustained release, or producing smaller size organoid when short-term release is sufficient.For example, for organ atrophy reversal, larger organoid may be produced than for local tissue repair.

[0096] In the present disclosure, organoid fibroblasts can be used as a means of " slow release " of fibroblasts and / or their secretions.These therapeutic agents can be used for immunomodulation, chronic disease treatment, tissue repair, tissue regeneration, specific tissue migration targeting, and then proliferation and / or differentiation.

[0097] In certain embodiments, organoid-derived fibroblasts and / or fibroblast-derived materials can be released from the spheroids over time to induce immunomodulatory and other therapeutic biological responses. For immunomodulation, organoids can be used to treat autoimmune diseases such as multiple sclerosis (MS) and skin diseases involving immune disorders such as eczema and psoriasis. Cancers of all types can also be treated, as cancer cells exploit immunomodulation to evade the immune response. Organoids can also be used as biologically sustained release agents in the thymus and spleen.

[0098] In certain embodiments, fibroblast organoids are administered to individuals in need by appropriate administration methods.Administration methods can be local or systemic, and can include injection, infusion, spraying, and / or inclusion in 3D matrix.In certain embodiments, administration is performed once or multiple times.When multiple administrations are performed, the interval between administrations can be 1 to 12 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, or 1 to 5 years or more.Any of these values ​​or ranges can be included.

[0099] In certain embodiments, spheroids are administered to or onto damaged tissue, after which the spheroids migrate and / or proliferate to the desired site and regenerate the damaged tissue by differentiation and / or stem cell recruitment.

[0100] In some embodiments, organoid fibroblasts are activated to express one or more cell surface molecules, so that they can migrate and proliferate to specific tissues or organs, and contribute to tissue repair and / or immune response induction at that site.Fibroblasts can be activated by one or more chemicals (including small molecule compounds), RNA, microRNA, RNAi, DNA, viral nucleic acid, and / or exosome.Specific examples include 6SM, p53 peptide, I09 / RG7388, NUT / nutlin-3a, IQK, 41P, 2FS, TX6 / TX64014, estradiol, 4-hydroxytamoxifen, bazedoxifene, AZD9496, testosterone, bicalutamide, PT2399, ZTD, VH298, T28, 0WC, 76Z, JQ1, or combinations thereof.

[0101] III. Specific examples of research objectives In some embodiments, the organoid produced can be used for all kinds of research purposes.The organoid for research use is particularly suitable for research, because it has the ability of self-organization, and is very similar to real human organ, and in some cases is histologically indistinguishable from it, being a 3D culture system.Organoid can be used to obtain information about the mechanisms underlying human development and organ regeneration, highlighting its value in basic biology research, in addition to the application of pharmaceutical testing and molecular medicine.

[0102] Organoids can be commercially available for research use.The organoids produced can be transported to research institutions for any purpose by the methods and compositions encompassed herein.In certain embodiments, organoids are used as an alternative to animal models in preclinical drug testing.The application of these organoids is to explore organ function and explore drug development approaches.

[0103] The organoids of the present disclosure can be used to study infectious diseases (viral, bacterial, fungal, protozoal, etc.), genetic diseases, cancer, etc.

[0104] Organoids for research use can be derived from fibroblasts, either genetically engineered or derived from patient biopsies. Therefore, organoid production (including the type of fibroblasts, their modification / activation, and organoid size) can be tailored to the specific needs of researchers or research institutions (private or public). For example, organoids can be produced to characterize biological processes unique to the human body that cannot be modeled in other animals.

[0105] IV. Kits of the Present Disclosure Any of the compositions described herein may be incorporated into a kit. This kit may include, but is not limited to, fibroblasts, tissues containing fibroblasts, organoids containing fibroblasts, culture reagents, one or more thickening agents, or a combination thereof, each contained in a suitable container. The kit may also include an organ-on-a-chip system.

[0106] The components of the kit may be packaged in a suitable solution, if desired. In certain embodiments, the components of the kit may be in aqueous media or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, particularly as appropriate, for dispensing. Where multiple components are included in the kit, second, third, or other additional containers may be included for separately placing the additional components. However, various combinations of components may be included within a container, such as a vial. The kits of the present disclosure will also generally include a means for containing the components in a sealed state for sale. Such containers may include injection-molded or blow-molded plastic containers or the like, which accommodate the desired containers.

[0107] Regardless of the number or type of containers, the kits of the present disclosure may also include or be packaged with instruments to aid in the injection / administration and / or placement of the organoids into an animal, which may be a syringe, pipette, tweezers, or other medically approved delivery device. [Example]

[0108] I. Example 1 - Generation of spheroids from mouse fibroblasts Mouse skin fibroblasts maintain high viability after storage at room temperature for 7 days.

[0109] A mouse fibroblast cell line was isolated from mouse skin fibroblasts. For validation experiments, cells were cultured under a standard cell culture environment (e.g., 37°C, 5% carbon dioxide, 90% humidity) until they reached 80% confluence. An example of the experimental procedure is as follows. 1. Isolate cells, remove media and wash twice with 1x PBS. 2. Dissociate cells with 0.25% trypsin for 3 minutes. 3. Harvest the cells by suspending them in an appropriate medium (e.g., medium containing low glucose, human serum, non-essential amino acids and / or L-glutamine). 4. Cell concentration increased to 8 x 10 5 Adjust the concentration to cells / mL and prepare stem cell spheroids using the hanging drop method (25 µL per drop). 5. Place the culture plate in a normal cell culture environment for 48 hours. 6. Harvest the stem cell pellet from the hanging drop. 7. Centrifuge the stem cell pellet at 1000 rpm for 3 minutes and remove the supernatant. 8. Add storage matrix to resuspend the cells and adjust the cell density in the liquid matrix to 5 million cells / mL. 9. Add the cells to a 15 mL plastic centrifuge tube, seal with parafilm, and store in the dark at room temperature (e.g., approximately 20°C) for days 0 to 7. 10. On day 7, place the tube in the incubator for 2 hours, then centrifuge the cells, remove the supernatant, and wash twice with 1x PBS. 11. Dissociate cells with 0.25% trypsin for 3 minutes. 12. Resuspend cells in stem cell medium (e.g., low-glucose DMEM containing 10% human serum, 1% non-essential amino acids, and / or 5% L-glutamine). 13. Cells can be tested, applied, or continuously cultured by returning them to normal culture conditions.

[0110] This method resulted in mouse skin fibroblasts with a 100% survival rate and normal cell morphology, proliferation, and migration abilities (Figures 1-8).

[0111] II. Example 2 - Generation of spheroids from human fibroblasts Human fibroblasts showed high viability even after pelleting and storage at room temperature for 7 days.

[0112] A human fibroblast cell line was isolated from fibroblasts derived from an individual. Skin fibroblasts were used in the validation experiment. The cells were cultured under a standard cell culture environment (37°C, 5% carbon dioxide, 90% humidity) until they reached 80% confluence. An example of the experimental procedure is as follows: 1. Isolate the cells, remove the medium, and wash twice with 1x PBS. 2. Dissociate cells with 0.25% trypsin for 3 minutes. 3. Harvest the cells by suspending them in an appropriate medium (e.g., medium containing low glucose, human serum, non-essential amino acids and / or L-glutamine). 4. Cell concentration increased to 8 x 10 5 Adjust the cell / mL and prepare stem cell spheroids at 25 µL / drop using the hanging drop method. 5. Place the culture plate in a normal cell culture environment for 48 hours. 6. Harvest the stem cell pellet from the hanging drop. 7. Centrifuge the stem cell pellet at 1000 rpm for 3 minutes and remove the supernatant. 8. Add storage matrix to resuspend cells and adjust cell concentration to 5 million cells / mL in liquid matrix. 9. Add the cells to a 15 mL plastic centrifuge tube, seal with parafilm, and store in the dark at room temperature (e.g., about 20° C.) for days 0 to 7. 10. On day 7, place the tube in the incubator for 2 hours, centrifuge the cells, remove the supernatant, and wash twice with 1x PBS. 11. Dissociate cells with 0.25% trypsin for 3 minutes. 12. Suspend the cells in stem cell medium (e.g., low-glucose DMEM containing 10% human serum, 1% non-essential amino acids, and 5% L-glutamine). 13. Cells can be tested, applied, or continued in culture by returning to normal culture conditions.

[0113] Using this method, human dermal fibroblasts achieved 100% viability and maintained normal cell morphology, proliferation, and migration ability (Figure 4).

[0114] III. Example 3 - Generation of spheroids from human fibroblasts Human dermal fibroblast (HDF) spheroids survive well and are capable of colony formation even when stored at 4°C for up to 4 weeks.

[0115] A human fibroblast cell line was isolated from fibroblasts of an individual. Skin fibroblasts were used in the validation experiment. The cells were cultured under a standard cell culture environment (e.g., 37°C, 5% carbon dioxide, 90% humidity) until they reached 80% confluence. An example of the experimental procedure is as follows. 1. Isolate the cells, remove the medium, and wash twice with 1x PBS. 2. Dissociate cells with 0.25% trypsin for 3 minutes. 3. Harvest the cells by suspending them in an appropriate medium (e.g., medium containing low glucose, human serum, non-essential amino acids and / or L-glutamine). 4. Cell concentration increased to 8 x 10 5 Adjust the cell / mL and prepare stem cell spheroids at 25 µL / drop using the hanging drop method. 5. Place the culture plate in a normal cell culture environment for 48 hours. 6. Harvest the stem cell pellet from the hanging drop. 7. Centrifuge the stem cell pellet at 1000 rpm for 3 minutes and remove the supernatant. 8. Add storage matrix to resuspend cells and adjust cell concentration to 5 million cells / mL in liquid matrix. 9. Add the cells to a 15 mL plastic centrifuge tube, seal with parafilm, and store in the dark at room temperature (e.g., about 20° C.) for days 0 to 14. 10. On day 14, place the tube in the incubator for 2 hours, centrifuge the cells, remove the supernatant, and wash twice with 1x PBS. 11. Dissociate cells with 0.25% trypsin for 3 minutes. 12. Suspend the cells in stem cell medium (e.g., low-glucose DMEM containing 10% human serum, 1% non-essential amino acids, and 5% L-glutamine). 13. Cells can be tested, applied, or continued in culture by returning to normal culture conditions.

[0116] Using this method, human dermal fibroblasts achieved a viability of 89-95% and retained normal cell morphology, proliferation, and migration ability (Figures 5-7).

[0117] IV. Example 4 - Validation of human fibroblast-derived spheroids HDF spheroids survived well and were capable of colony formation even after storage at 4°C for up to 4 weeks. HDFs within the spheroids retained the dermal fibroblast phenotype (Figure 8).

[0118] Frozen sections can be used to verify that the HDFs in the spheroids retain the dermal fibroblast phenotype. 10 μm spheroid sections can be stained with anti-CD90 antibody to detect mesenchymal stem cells and vimentin to detect HDFs. Nuclei can be stained with DAPI nuclear stain. 1% fetal serum in 1x DPBS can be used as a blocking buffer, incubation buffer, and mounting medium. An example experimental procedure is as follows. 1. Air-dry the frozen tissue sections briefly at room temperature (RT). 2. Circle the section with a hydrophobic pen. 3. Rehydrate sections in staining dish with 1x DPBS for 10 minutes twice. 4. Add blocking buffer and block for 1 hour at RT in a humid chamber. 5. Remove the blocking buffer and add the incubation buffer containing the primary antibody. 6. Incubate with primary antibody overnight at 4°C in a humid chamber (Important: Some antibodies require incubation at RT; see the antibody datasheet). 7. Wash in staining dish 3 times for 10 minutes with 1x DPBS at RT. 8. Transfer the slides to a humid chamber and add incubation buffer containing the secondary antibody diluted to the manufacturer's recommended concentration. 9. Incubate the slides for 1 hour at RT. 10. Wash 3 times with 1x DPBS for 10 minutes at RT. 11. Optional: Add DAPI solution and stain for 10 minutes in 1x DPBS at RT. 12. Wash 3 times with 1x DPBS for 10 minutes at RT. 13. Remove the hydrophobic ink from around the tissue section. 14. Mount the slides and observe under a microscope. (References) 1.Tschumperlin, DJ, Fibroblasts and the ground they walk on.Physiology(Bethesda), 2013.28(6):p.380-90. 2.Lee, JH, YSHan, and S.H.Lee, Long-Duration Three-Dimensional Spheroid Culture Promotes Angiogenic Activities of Adipose-Derived Mesenchymal Stem Cells.BiomolTher(Seoul), 2016.24(3):p.260-7. 3.Shimura,M.,etal.,Room temperature-induced apoptosis of Jurkat cells sensitive to both caspase-1 and caspase-3 inhibitors.Cancer Lett,1998.132(1-2):p.7-16. 4.Veronesi,E.,et al.,Transportation conditions for prompt use of ex vivo expanded and freshly harvested clinical-grade bone marrow mesenchymal stromal / stem cells for bone regeneration.Tissue Eng Part C Methods,2014.20(3):p.239-51. 5.Yong,K.W.,et al.,Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications:Current Methods and Challenges.Biopreserv Biobank,2015.13(4):p.231-9. 6.Jang,T.H.,et al.,Cryopreservation and its clinical applications.Integr Med Res,2017.6(1):p.12-18.

[0119] While the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the present invention. Moreover, the scope of the present application is not limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods, or steps described in the specification. As will be readily apparent to those skilled in the art from this disclosure, any now-existing or hereafter developed process, machine, manufacture, composition of matter, means, methods, or steps that perform substantially the same function or achieve substantially the same results as the corresponding examples described herein can be utilized in accordance with the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Claims

1. 1. A method for producing fibroblast organoids, comprising: producing a plurality of fibroblast organoids; suspending the fibroblast organoids in a preservation matrix comprising a low-glucose base medium, 0-20% human serum, 0-5% non-essential amino acids, and / or 0-5% L-glutamine, and optionally at least one adhesive; A method comprising:

2. 10. The method of claim 1, wherein the preservation matrix comprises about 0-20%, 0-15%, 0-10%, 0-5%, 1-20%, 1-15%, 1-10%, 1-5%, 5-20%, 5-15%, 5-10%, 10-20%, 10-15%, or 15-20% human serum.

3. 3. The method of claim 1 or 2, wherein the storage matrix comprises about 0, 1, 2, 3, 4, or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% human serum.

4. 4. The method of any one of claims 1 to 3, wherein the storage matrix comprises about 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 1-5%, 1-4%, 1-3%, 1-2%, 2-5%, 2-4%, 2-3%, 3-5%, 3-4%, or 4-5% of non-essential amino acids.

5. The method of any one of claims 1 to 4, wherein the storage matrix comprises about 0, 1, 2, 3, 4, or 5% non-essential amino acids.

6. 6. The method of any one of claims 1 to 5, wherein the storage matrix comprises about 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 1-5%, 1-4%, 1-3%, 1-2%, 2-5%, 2-4%, 2-3%, 3-5%, 3-4%, or 4-5% L-glutamine.

7. 7. The method of any one of claims 1 to 6, wherein the storage matrix comprises about 0, 1, 2, 3, 4, or 5% L-glutamine.

8. 8. The method of any one of claims 1 to 7, wherein the storage matrix comprises about 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 1-5%, 1-4%, 1-3%, 1-2%, 2-5%, 2-4%, 2-3%, 3-5%, 3-4%, or 4-5% of at least one adhesive.

9. The method of any one of claims 1 to 8, wherein the storage matrix comprises about 0, 1, 2, 3, 4, or 5% of at least one adhesive.

10. The method of any one of claims 1 to 9, wherein the at least one adhesive comprises methylcellulose, agar, guar gum, xanthan gum, pectin, collagen, and / or gelatin.

11. The method of any one of claims 1 to 10, wherein the fibroblast organoids are about 50-500 μm, 50-400 μm, 50-300 μm, 50-200 μm, 50-100 μm, 100-500 μm, 100-400 μm, 100-300 μm, 100-200 μm, 200-500 μm, 200-400 μm, 200-300 μm, 300-500 μm, 300-400 μm, or 400-500 μm in size.

12. 12. The method of any one of claims 1 to 11, wherein the fibroblast organoids are about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 μm in size.

13. The method according to any one of claims 1 to 12, wherein the fibroblast organoids are produced by one of the following methods: (a) Placing fibroblasts in U- or V-bottom ultra-low attachment culture plates; (b) Hanging drop method; (c) disposing fibroblasts on hydrophilic regions of a substrate comprising a hydrophobic surface; (d) A method for forming core-shell structures within pipette tips.

14. The fibroblast organoids are cultured at approximately 10 per organoid. ３ , 10 ４ , 10 ５ , 10 ６ , 10 ７ , 10 ８ , 10 ９ , or 10 １０ The method of any one of claims 1 to 13, comprising fibroblasts.

15. The method according to any one of claims 1 to 14, wherein the fibroblast organoid is produced by culturing for about 24 to 48 hours in an incubator having a temperature of about 37°C, 5% carbon dioxide, and a humidity of more than 80%.

16. 16. The method of any one of claims 1 to 15, wherein the culturing is for about 24 to 48 hours, 24 to 44 hours, 24 to 40 hours, 24 to 36 hours, 24 to 30 hours, 24 to 28 hours, 28 to 48 hours, 28 to 44 hours, 28 to 40 hours, 28 to 36 hours, 28 to 30 hours, 30 to 48 hours, 30 to 44 hours, 30 to 40 hours, 30 to 36 hours, 36 to 48 hours, 36 to 44 hours, 36 to 40 hours, 40 to 48 hours, 40 to 44 hours, or 44 to 48 hours.

17. 17. The method of any one of claims 1 to 16, wherein the culturing is within the range of about 24 to 48 hours, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

18. 18. The method of any one of claims 1 to 17, wherein the culturing is carried out at a temperature of about 20-37°C, 20-35°C, 20-30°C, 20-27°C, 20-25°C, 20-22°C, 22-37°C, 22-35°C, 22-33°C, 22-30°C, 22-27°C, 22-25°C, 25-37°C, 25-35°C, 25-33°C, 25-30°C, 25-27°C, 27-37°C, 27-35°C, 27-33°C, 27-30°C, 30-37°C, 30-35°C, 30-33°C, 33-37°C, 33-35°C, or 35-37°C.

19. 19. The method of any one of claims 1 to 18, wherein the culturing is carried out at a temperature within the range of about 20 to 37°C, including 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37°C.

20. 20. The method of any one of claims 1 to 19, wherein the culturing is carried out under about 3-8%, 3-7%, 3-6%, 3-5%, 3-4%, 4-8%, 4-7%, 4-6%, 4-5%, 5-8%, 5-7%, 5-6%, 6-8%, 6-7%, or 7-8% carbon dioxide.

21. 21. The method of any one of claims 1 to 20, wherein the culturing is carried out under about 3, 4, 5, 6, 7, or 8% carbon dioxide.

22. 22. The method of any one of claims 1 to 21, wherein the culturing is carried out at a humidity of about 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%, 70-75%, 75-90%, 75-85%, 75-80%, 80-90%, 80-85%, or 85-90%.

23. 23. The method of any one of claims 1 to 22, wherein the humidity is about 65, 70, 75, 80, 85, or 90%.

24. 24. The method of any one of claims 1 to 23, wherein the manufacturing process comprises modifying one or more parameters to control the size of the resulting organoids.

25. 25. The method of claim 24, wherein the one or more parameters comprise fibroblast concentration, type and / or amount of base medium, type and / or amount of sugar in the base medium, type and / or amount of serum in the liquid preservation matrix, type of non-essential amino acids, amount of L-glutamine, amount of carbon dioxide in the incubator, temperature in the incubator, duration of fibroblast spheroid production, or a combination thereof.

26. The method of any one of claims 1 to 25, comprising placing the organoid in a container.

27. 27. The method of claim 26, wherein the container is plastic, glass, metal, and / or rubber.

28. The method according to any one of claims 1 to 27, wherein the fibroblast organoids are preserved.

29. 29. The method of claim 28, wherein the fibroblast organoids are stored at room temperature.

30. 29. The method of claim 28, wherein the fibroblast organoids are stored at about 0-37°C.

31. The method according to any one of claims 1 to 30, wherein the fibroblast organoids are cryopreserved.

32. The method according to any one of claims 1 to 31, wherein the fibroblast organoid is transported.

33. 33. The method of claim 32, wherein the fibroblast organoids are transported within about 0 to 50 days.

34. 34. The method of claim 32 or 33, wherein the transport occurs for a period of 0 to 50, 0 to 15, 0 to 10, 0 to 5, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, 15 to 20, 20 to 25, or 25 to 30 days.

35. The method of any one of claims 32 to 34, wherein the transport is performed at ambient temperature.

36. The method of any one of claims 32 to 34, wherein the transport is performed at about 0 to 37°C.

37. 37. The method of any one of claims 32 to 36, further comprising removing the fibroblast organoids from the storage matrix after the transport.

38. 38. The method of claim 37, wherein removal from the storage matrix is ​​accomplished by centrifugation, gravity, filtration, a microfluidic cassette, mechanical manipulation, and / or chemical manipulation.

39. The method of any one of claims 32 to 38, further comprising dissociating at least a portion of the fibroblasts from the fibroblast organoids after the transport.

40. 40. The method of claim 39, wherein said dissociation is effected by exposure to an effective amount of at least one protease.

41. 41. The method of claim 40, wherein the at least one protease comprises about 0.1-0.3% trypsin.

42. 42. The method of claim 41, wherein the at least one protease is about 0.25%.

43. The method according to any one of claims 1 to 42, wherein the fibroblast organoid is used within 0 to 21 days from the date of production by the method of claim 1.

44. 44. The method of any one of claims 1 to 43, wherein the fibroblast organoids are used in an assay or for therapeutic use within 0 to 20, 0 to 15, 0 to 10, 0 to 5, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 days from the date of production by the method of claim 1.

45. 45. The method of any one of claims 1 to 44, wherein the use is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days within 0 to 20 days.

46. 46. ​​The method of claim 44 or 45, wherein the assay is used as an alternative to an animal model.

47. 47. The method of any one of claims 44 to 46, wherein the assay is used in preclinical drug testing or drug discovery.

48. 48. The method of any one of claims 44 to 47, wherein the assay uses fibroblast organoids to study organ function.

49. The method of any one of claims 44 to 48, wherein the assay is used to fulfill drug screening requirements.

50. 50. The method of any one of claims 1 to 49, further comprising exposing said fibroblast organoids to one or more chemicals.

51. 51. The method of any one of claims 1 to 50, further comprising exposing said fibroblast organoids to one or more agents.

52. 52. The method of any one of claims 1 to 51, further comprising administering a therapeutically effective amount of said fibroblast organoid to an individual in need thereof.

53. 53. The method of claim 52, wherein the individual has an autoimmune, chronic, degenerative, genetic, infectious disease / disorder or is in need of immunomodulation, tissue repair, tissue regeneration, organ repair, or organ regeneration.

54. 54. The method of claim 53, wherein the autoimmune disease / disorder is multiple sclerosis, eczema, and / or psoriasis.

55. 55. The method of claim 53 or 54, wherein the infectious disease / disorder is viral, bacterial, fungal, and / or protozoal.

56. 56. The method of any one of claims 53 to 55, wherein the genetic disease / disorder is cancer.

57. The method of any one of claims 52 to 56, wherein the fibroblast organoid is administered locally or systemically.

58. 58. The method of any one of claims 52 to 57, wherein the fibroblast organoids are administered by injection, infusion, spraying, or in a 3D matrix.

59. 59. The method of any one of claims 52 to 58, wherein the fibroblast organoids are administered one or more times.

60. 60. The method of claim 59, wherein when multiple doses are administered, the interval between doses is 1 to 12 hours, 1 to 7 days, 1 to 4 weeks, or 1 to 12 months, or 1 to 5 years or more.

61. 61. The method of any one of claims 52 to 60, wherein the fibroblasts dissociate from the fibroblast organoids after administration.

62. 62. The method of claim 61, wherein the release is sustained release.

63. 63. The method of claim 62, wherein the sustained release occurs over a period of about 1 hour to 15 days.

64. 64. The method of any one of claims 61 to 63, wherein the fibroblasts migrate to a specific tissue or organ.

65. 65. The method of claim 64, wherein the tissue or organ is in need of repair or induction of an immune response.

66. 66. The method of claim 64 or 65, wherein the tissue is connective tissue, epithelial tissue, muscle tissue, and / or nerve tissue.

67. 67. The method of any one of claims 64 to 66, wherein the organ is the heart, liver, lung, stomach, spleen, gallbladder, kidney, brain, bladder, and / or intestine.

68. 68. The method of any one of claims 52 to 67, wherein the fibroblasts are non-activated.

69. 68. The method of any one of claims 52 to 67, wherein the fibroblasts are activated.

70. 70. The method of claim 69, wherein the activation is achieved by one or more chemical agents, RNA, microRNA, RNAi, DNA, viral nucleic acid, and / or exosomes.

71. 71. The method of any one of claims 52 to 70, wherein the fibroblasts contain and release one or more therapeutic agents.

72. 72. The method of claim 71, further comprising modifying the fibroblasts to contain one or more therapeutic agents.

73. 73. The method of claim 71 or 72, wherein the therapeutic agent comprises a protein, a nucleic acid, an exosome, a growth factor, a microRNA, an RNAi, and / or an mRNA.

74. 74. The method of any one of claims 71 to 73, wherein the therapeutic agent comprises RNA, microRNA, RNAi, DNA, viral nucleic acid, and / or exosomes.

75. A composition comprising an organoid produced by the method of any one of claims 1 to 31.

76. 76. The composition of claim 75, wherein the organoid is contained in a liquid preservation matrix.

77. 77. The composition of claim 75 or 76, wherein the organoid is at room temperature.

78. 78. The composition of any one of claims 75 to 77, wherein the organoid is frozen.

79. The composition of any one of claims 75 to 78, wherein the organoid is in a container.

80. 80. The composition of claim 79, wherein the container is plastic, glass, metal, and / or rubber.

81. 81. The composition of any one of claims 75 to 80, wherein the preservation matrix comprises a low glucose-based medium, about 0-20% human serum, about 0-5% non-essential amino acids, and / or about 0-5% L-glutamine, and optionally further comprises at least one adhesive agent.

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