Novel method for automatically generating cell spheroids with a core-shell structure

Abstract

A method is provided for preparing spheroids in a pipette tip from one or more cells. The spheroids may consist of a single cell type or multiple cell types. Methods are also provided for utilizing the spheroids in fully or partially automated assays, high-throughput screening, and clinical applications.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 514,865, filed on 21 July 2023, which is incorporated herein by reference in its entirety.

[0002] The aspects of this disclosure relate at least to the fields of cell biology, molecular biology, immunology, and medicine. [Background technology]

[0003] Cell spheroids (also known as three-dimensional cell cultures) provide a more physiologically accurate model compared to conventional two-dimensional cell cultures. Cell spheroids offer several advantages over two-dimensional cultures. Firstly, they can more accurately reproduce tissue structure and cell behavior, making them suitable for disease mechanism studies, drug screening, and tissue engineering applications. Secondly, cell spheroids are known to exhibit higher predictive value in evaluating drug efficacy and toxicity compared to two-dimensional cultures. Furthermore, cell spheroids enable the analysis of cell signaling, migration, invasion, and other cellular processes that are better represented in a three-dimensional context.

[0004] There are several methods for forming cell spheroids, which can be classified into four groups based on their formation mechanisms: 1) gravity method, 2) micropattern method, 3) microfluidic method, and 4) electrospun method. The gravity method is further divided into two types: 1) hanging drop method and 2) microwell method. The hanging drop method is a common method for forming cell spheroids in three-dimensional culture. In this method, cells are suspended in droplets of culture medium, and the droplets are placed upside down on the lid of a tissue culture dish. Gravity causes the cells to aggregate, forming spheroids within the droplets. The microwell method is used to produce cell spheroids in a controlled and reproducible manner. In this method, cells are confined to a specific shape (usually spherical or cylindrical) using small microwells or microfabricated molds. The cells are seeded within these microwells and self-assemble to form spheroids. The microwell method allows for precise control of spheroid size, shape, and density, making it useful for a variety of applications, including tissue engineering, drug screening, and cell behavior studies. These two techniques provide a simple and versatile approach to generating uniform and clearly defined cell spheroids for scientific research and biomedical applications.

[0005] Micropatterning is a method for creating cell spheroids with distinct shapes and spatial structures. This method utilizes a micropatterned substrate on which specific adherent or non-adherent regions are formed on the culture surface using techniques such as microcontact printing or photolithography. Cells are seeded onto these patterns, proliferate, and transform into the desired spheroid shape while altering intercellular contact and organization. This method allows for precise control of cell positioning, intercellular interactions, and tissue-like structures. Micropatterning is useful in studying cell behavior, analyzing tissue development, and creating complex multicellular structures that more accurately mimic in vivo conditions.

[0006] Microfluidics is an innovative approach to creating cell spheroids with precisely controlled size, shape, and composition. This method utilizes microscale channels and chambers to manipulate fluids and cells in a highly controlled manner. Cells are introduced into these microfluidic devices, grow through controlled flow and spatial arrangement, and assemble into spheroids. This method offers advantages such as high throughput, precise manipulation of the cellular microenvironment, and the ability to create complex multicellular structures.

[0007] Electrospun is a method for creating cell spheroids using electrospinning technology, which generates ultrafine fibers from polymer solutions. In this method, cells are encapsulated in a polymer solution, and an electric field is applied to form a charged jet. As the solvent evaporates, the polymer fibers accumulate, forming a three-dimensional scaffold that entangles the cells. This scaffold supports intercellular interactions and promotes spheroid formation. [Overview of the project] [Problems that the invention aims to solve]

[0008] The methods described above offer diverse approaches to producing uniform and clearly defined cell spheroids for scientific research and biomedical applications. However, gravity-based and micropattern-based methods often only produce spheroids consisting of a single cell type, and are unable to create complex multicellular structures (i.e., organ-like structures containing multiple cell types) that more faithfully mimic in vivo conditions, thus limiting their application to actual in vivo transplantation. On the other hand, microfluidic and electrospun-based methods can create complex multicellular structures such as core-shell structures that better mimic in vivo conditions, but both methods require complex equipment or expensive voltage generators, and are common in engineering laboratories but far less common in hospitals and biotechnology companies. More importantly, none of the above methods are compatible with current laboratory automation technologies for high-throughput production of high-quality cell spheroids for scientific research and biomedical applications. [Means for solving the problem]

[0009] Summary of the Invention In one embodiment, the disclosure encompasses an in vitro method for preparing a spheroid from one or more cells in a pipette tip, the method comprising the step of introducing the one or more cells into a pipette tip, the pipette tip optionally containing a cell culture medium. The one or more cells may include one or more cell types. The spheroid may be a composite spheroid comprising a central core and at least one peripheral layer. In one embodiment, the one or more cells may include fibroblasts. In one embodiment, the cell culture medium may contain a viscosity enhancer. The cell culture medium may contain a basal medium, 0-20% serum, 0-5% non-essential amino acids, 0-5% L-glutamine, and 0-5% viscosity enhancer. The viscosity enhancer may be methylcellulose. In one embodiment, the pipette tip may be made of a chemically inert material. The pipette tip may be housed in a tip box made of a chemically inert material. Non-limiting examples of chemically inert materials include polyethylene, polypropylene, or melamine. In some embodiments, the tip box may include one or more additional pipette tips containing one or more cells or spheroids. In some embodiments, the pipette tips have a liquid volume of 10, 20, 100, 200, or 1000 μl. In some embodiments, the one or more cells may be cultured at a temperature range of 25–40°C, in an atmosphere containing 2–8% CO2, and at a humidity of 90–98%. In some embodiments, the spheroids may contain 0.1 to 1 million cells. In some embodiments, the size of the spheroids is 50–500 μm.

[0010] In one embodiment, the disclosure also encompasses a method for screening one or more spheroids, the method comprising the step of contacting the one or more spheroids with a drug, the one or more spheroids being cultured in a pipette tip. Non-limiting examples of suitable drugs include polypeptides, peptides, nucleic acids, small molecules, or natural products. In some embodiments, the spheroids may be contacted with the drug in the pipette tip or in a separate device, such as a multiwell plate. The screening method may be fully or partially automated.

[0011] In one embodiment, the disclosure also encompasses a method for treating a subject in need of treatment, the method comprising administering to the subject a pharmaceutical composition comprising one or more spheroids, or cells, tissues, organoids, or organs obtained from the one or more spheroids, wherein the one or more spheroids are cultured in a pipette tip. A kit is also envisioned that comprises a tip box containing one or more tips for growing one or more spheroids from cells or a population of cells, and instructions on how to use the kit.

[0012] Brief explanation of the drawing The following drawings constitute part of this specification and are included to further illustrate specific embodiments of the invention. The invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments shown herein. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 is a schematic diagram illustrating the formation and scale-out production of single-cell spheroids (1 to a) and a method for producing cell spheroids with a core-shell structure containing two cell types (orange and green, respectively) within a tip (1 to b, and further 2). Both methods utilize an air gap to prevent the liquid from flowing out of the tip due to gravity or the positive pressure generated when the tip is mounted on the pipette.

[0014] [Figure 2] Figure 2 is a schematic diagram showing a method (Steps 1 to 11) of producing a plurality of cell spheroids within a single pipette tip, showing separation of the spheroids and use of an air gap to prevent outflow from the tip by gravity or the positive pressure that occurs when the pipettor is attached to the tip with liquid.

[0015] [Figure 3A] Figure 3A shows a normal tip and tip box used for the formation of cell spheroids. A side view of the tip box containing 1×DPBS to maintain humidity is shown. The box surrounded by the black line is a top view of the entire tip box.

[0016] [Figure 3B] Figure 3B shows four tips each containing 40 μl of medium, which were cultured in a tip box in an incubator for 4 days. No evaporation or decrease in the medium was observed even after 4 days.

[0017] [Figure 4] Figure 4 is a representative microscopic image of a spheroid. The morphology of the human fibroblast spheroid demonstrates that fibroblasts can form spheroids within 4 days by the tip spheroid method.

Embodiments for Carrying Out the Invention

[0018] This disclosure is partly based on the unexpected discovery that viable three-dimensional cell spheroids can be cultured in pipette tips using the methods provided. Surprisingly, these spheroids can be generated from a single cell type, and these methods can also be used to culture complex spheroids containing multiple cell types. This disclosure encompasses novel methods for generating single-cell or multi-cell type organospheroids using pipette tips and is compatible with any liquid handling robot, enabling high-throughput spheroid production. Pipette tips are routinely used in laboratories and manufacturing facilities to dispense and transfer cells into tubes and plates. Aspects of this disclosure encompass compositions, methods, and systems for efficiently preparing three-dimensional spheroids directly in pipette tips, which allow cells to aggregate and form spheroid structures by aspirating and incorporating cells into the pipette tip and incubating the pipette tip under appropriate cell culture conditions. This allows the use of biologically non-toxic pipette tips and offers several advantages, including high-density and large-scale automated spheroid generation. The cell products typically do not require subsequent separation and purification steps, unlike conventional microwell-based methods which often result in unintended aggregates and necessitate additional sorting by cell strainers. Furthermore, the cell spheroid formation process can be operated by liquid processing robots, enabling easy automation and compatibility with high-throughput methods. These spheroids can be directly used in biological and medical scientific research, cell therapy for immunological and degenerative diseases, cell transplantation for tissue and organ damage, and can also meet the requirements of drug screening, yielding immeasurable scientific and socioeconomic benefits. These spheroids can be used in a more physiologically appropriate and reproducible manner for studying cell behavior, tissue engineering, drug testing, disease modeling, clinical training, research, and therapeutic applications.

[0019] In one aspect, the present disclosure encompasses an in vitro method for generating spheroids from one or more cells within a pipette tip, the method including introducing the one or more cells into the pipette tip, which may optionally contain a cell culture medium.

[0020] In one aspect, the one or more cells can include cells of any cell type. Non-limiting examples of suitable cell types include stem cells such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, progenitor cells; fibroblasts such as placenta-derived fibroblasts, omentum-derived fibroblasts, umbilical cord blood-derived fibroblasts, fibroblasts derived from skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, or foreskin; cancer cells and cell lines such as MCF-7, MDA-MB-231, HeLa, A549, HCT116, PC-3, DU145, U87, HepG2, K562, PANC-1, SKOV-3, U251, Caco-2, T47D, SW480, LNCaP, H460, HT-29, A375, PANC-1, T47D, PC-3, MDA-MB-23; and tissue and organ-specific cells such as hepatocytes, cardiomyocytes, pancreatic islet cells, mesenchymal stromal cells (MSC), etc. Various adherent cell types including these can be used for spheroid formation. In one aspect, the one or more cells may include a single cell type. In one aspect, the one or more cells may include at least 2, 3, 4, 5, or more different cell types.

[0021] In one aspect, the one or more cells may be introduced into the pipette tip at a concentration of 0.1 cells / mL to about 1×10 10 cells / mL. In one aspect, the cells are at a concentration of 0.1 - 1, 1 - 10, 10 - 100, 100 - 1,000, 1×10 3 ~1×10 4 、1×10 4 ~1×10 5 、1×10 5 ~1×10 6 、1×10 6 ~1×10 7 、1×10 7 ~1×10 8 、1×108 ~1 × 10 9 , 1 x 10 9 ~1 × 10 10 Cells / mL or higher may be introduced into the pipette tip. In one embodiment, the cells may be introduced in a single aspiration step. In one embodiment, the cells may be introduced into the pipette tip in one, two, three, four, five or more aspiration steps. In one embodiment, an air gap may be maintained between each aspiration step, thereby allowing multiple spheroids separated by the air gap to form within the same tip. In one embodiment, no air gap is maintained. Therefore, in one embodiment, a single spheroid may be formed in each pipette tip by aspirating a single cell suspension into the tip. Alternatively, it may be desirable to form multiple spheroids, such as two, three, four, five, six or seven, in each tip. The multiple spheroids formed in the tip may have different cellular compositions, for example, originating from different tissue types.

[0022] Spheroids may be formed at predetermined locations on the chip. For example, especially if the method is automated, spheroids may be formed at predetermined distances from each other. Spheroids may be formed at regular or non-random intervals. Spheroids may be formed at the same location within each chip.

[0023] Therefore, when multiple spheroids are generated within a single chip, the spheroids may be formed at least 0.5 mm to 10 mm apart from each other, for example, at intervals of 0.5 to 1.0, 1.0 to 2.0, 2.0 to 3.0, 3.0 to 4.0, 4.0 to 5.0, 5.0 to 6.0, 6.0 to 7.0, 7.0 to 8.0, 8.0 to 9.0, 9.0 to 10.0 mm or more. However, if it is desirable to study the interactions between spheroids, the spheroids may be formed at intervals of less than 2.0 mm.

[0024] In one embodiment, a first cell type may be introduced into a pipette tip and cultured for a certain period, and then a second cell type may be introduced and cultured for a certain period. In one embodiment, this step may be repeated at least once, at least twice, at least three times, or more times. This may result in the formation of a composite spheroid having two or more cell layers. In one embodiment, the first cell type and the second cell type may be the same. In one embodiment, the first cell type and the second cell type may be different. In one embodiment, the resulting spheroid may consist of a single cell type or one or more, two or more, or three or more cell types. Therefore, in one embodiment, the spheroid may be a composite spheroid including a central core and at least one peripheral layer.

[0025] In one embodiment, the disclosure comprises an in vitro method for producing spheroids from one or more cells in a pipette tip, the method comprising the step of introducing the one or more cells into the pipette tip, the pipette tip may contain a cell culture medium. In one embodiment, the disclosure comprises an in vitro method for producing spheroids from one or more cells in a pipette tip, the method comprising the step of introducing the one or more cells into the pipette tip, the pipette tip may contain a cell culture medium, and produces spheroids under appropriate conditions. In one embodiment, the cell culture medium for producing spheroids may be present in the tip prior to the introduction of the one or more cells. In one embodiment, the cell culture medium for producing spheroids may be introduced into the tip together with the one or more cells. In one embodiment, the cell culture medium may be reactivated, replenished, or replaced at least once during the growth period of the spheroids.

[0026] The cell culture medium may include a basal medium. Typically, the medium may include basal medium formulations known in the art. Many basal medium formulations can be used for spheroid culture as described herein, and include, but are not limited to, Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), α-Modified Minimum Essential Medium (alpha-MEM), Basic Essential Medium (BME), Iskov's Modified Dulbecco's Medium (IMDM), BGJb Medium, F-12 Nutrient Mixture (Ham), Leibowitz L-15, DMEM / F-12, Essential Modified Eagle Medium (EMEM), RPMI-1640, and their modifications and / or combinations. The compositions of the above basal mediums are generally known in the art, and it is within the skill of those skilled in the art to modify or adjust the concentrations of the medium and / or medium additives as needed depending on the cells being cultured. In some embodiments, the culture medium formulation may be Explant Medium (CEM), which consists of IMDM supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin G, 100 μg / mL streptomycin, and 2 mmol / L L-glutamine. In other embodiments, further basal medium formulations selected from the above may be used.

[0027] Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum may be added to the above medium to support cell proliferation. However, the defined medium may be used if the growth factors, cytokines, and hormones necessary for cell culture are supplied to the medium at appropriate concentrations. In one embodiment, the medium may not contain fetal bovine serum. In one embodiment, the medium may contain one or more of the following: human platelet-rich plasma, platelet lysate, umbilical cord blood serum, autologous serum, one or more cytokines, fibroblast growth factor, epidermal growth factor, leukemia suppressor, insulin-like growth factor, angiopoietin, vascular endothelial growth factor, or any combination thereof. A medium useful in the method of the present disclosure may further contain one or more target compounds, including but not limited to antibiotics, mitotic compounds, or differentiation-inducing compounds useful for cell culture.

[0028] For use in cell culture, one or more additional components may be added to the culture medium. For example, additional supplements can be used to supply cells with trace elements and substances necessary for optimal growth and amplification. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components may, but are not limited to, be included in salt solutions such as Hanks equilibrium salt solution (HBSS) or Earl's salt solution. Furthermore, antioxidant supplements, such as β-mercaptoethanol, may be added. While many culture media already contain amino acids, some amino acids, such as L-glutamine, which is known to be unstable in solution, may be added later. The culture medium may also be supplemented with antibiotics and / or antifungal compounds, typically a mixture of penicillin and streptomycin, as well as amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeosin, among others. The addition of mammalian plasma or serum to the cell culture medium is also conceivable. Plasma or serum often contains cytofactors and components necessary for cell survival and proliferation. The use of appropriate serum substitutes is also conceivable. One or more of the culture medium components may be added at a concentration of at least, at most, or at a maximum of approximately 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / mL, pg / mL, mg / mL, or any range derived therefrom.

[0029] In one embodiment, the culture medium may be prepared using as a base medium any medium used for animal cell culture, such as AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer medium, or any combination thereof, but is not particularly limited to these, as long as it is suitable for use in culturing animal cells. In particular, the medium may be a xeno-free medium or a chemically defined medium.

[0030] The culture medium may be a serum-containing medium, a serum-free medium, or a xeno-free medium. From the viewpoint of preventing contamination by components from other animals, the serum may be derived from the same animal as the stem cells. A serum-free medium refers to a medium that does not contain untreated or unpurified serum, and therefore may include a medium containing purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0031] The culture medium may or may not contain a serum substitute. The serum substitute may include substances appropriately containing albumin (e.g., albumin substitutes such as lipid-rich albumin, bovine albumin, recombinant albumin or humanized albumin, vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalent substances. The serum substitute can be prepared, for example, by the method disclosed in International Publication No. 98 / 30679 (which is incorporated herein by reference in its entirety). Alternatively, commercially available materials can be used more conveniently. Examples of commercially available materials include KnockOut Serum Replacement (KSR), Chemically-defined Lipid Concentrate (Gibco), and Glutamax (Gibco).

[0032] In certain embodiments, the culture medium may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: namely, as vitamins, biotin; DL-alpha-tocopherol acetate; DL-alpha-tocopherol; vitamin A (acetate); as proteins, BSA (bovine serum albumin) or human albumin, fatty acid-free fraction V; catalase; human recombinant insulin; human transferrin; superoxide dismutase; and as other components, corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and / or T3 (triiodothyronine). In certain embodiments, one or more of these may be expressly excluded.

[0033] In some embodiments, the medium may further contain vitamins. In some embodiments, the medium may contain biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 (and any range derived therefrom), or a combination thereof or salts thereof. In some embodiments, the medium may contain, or essentially consist of, biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamin may include biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamin A, or a combination or salt thereof, or may be essentially composed of these. In some embodiments, the medium may further include protein. In some embodiments, the protein may include albumin or bovine serum albumin, BSA fraction, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In some embodiments, the medium may further include one or more of corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, triiodothyronine, or a combination thereof. In some embodiments, the medium may contain B-27® supplement, xeno-free B-27® supplement, GS21 TMThe medium may further contain one or more supplements or combinations thereof. In some embodiments, the medium may further contain amino acids, monosaccharides, and inorganic ions. In some embodiments, the amino acids may include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, valine, or combinations thereof. In some embodiments, the inorganic ions may include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations thereof or salts thereof. In some embodiments, the medium may further contain one or more of molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In a particular embodiment, the medium contains one or more vitamins and / or one or more proteins as discussed herein, and / or corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, triiodothyronine, B-27® supplement, xeno-free B-27® supplement, GS21 TM The supplement may contain, or be essentially composed of, one or more of the following: supplements, amino acids (e.g., arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (e.g., sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In certain embodiments, one or more of these may be explicitly excluded.

[0034] The culture medium may also contain one or more externally added fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvate, buffers, and / or inorganic salts. In certain embodiments, one or more of these may be explicitly excluded.

[0035] In one embodiment, the cell culture medium may further contain one or more viscosity enhancers. If necessary, the viscosity of the composition can be maintained at a desired level using one or more pharmaceutically acceptable viscosity enhancers. Non-limiting examples of viscosity enhancers include agar, carboxymethylcellulose, hydroxypropylcellulose, carbomer, guar gum, xanthan gum, pectin, collagen, gelatin, methylcellulose, starch, or any combination thereof. Methylcellulose is preferred because it is readily and economically available and easy to handle. The preferred concentration of the viscosity enhancer depends on the agent selected. The important point is to use an amount that achieves the desired viscosity. Viscous compositions are usually prepared from a solution by adding such agents. In one embodiment, one or more viscosity enhancers may be used in concentrations of 0.001% to 3.0%. In one embodiment, the one or more viscosity enhancers may be used in a total concentration of at least about 0.001% to 0.01%, 0.01% to 0.1%, 0.1% to 0.5%, 0.5% to 1%, 1% to 1.5%, 1.5% to 2%, 2% to 2.5%, 2.5% to 3.0%, 3.0% to 3.5%, 3.5% to 4.0%, 4.0% to 5%, or higher. In one embodiment, the viscosity enhancer is methylcellulose, and its concentration is 0.1% to 3%. Pharmaceutically acceptable viscosity enhancers do not cause biological toxicity, are metabolizable, leave no residue, and are safe for cells. The culture medium may contain other substances and conditions for optimal culture success. In one embodiment, the cell culture medium may contain a basal medium, 0 to 20% serum, 0 to 5% non-essential amino acids, 0 to 5% L-glutamine, and 0 to 5% viscosity enhancer.

[0036] In one embodiment, the pipette tip may be placed in any suitable container, such as a tip box. In one embodiment, the container, such as a tip box, may contain any liquid, such as water, saline solution, or cell culture medium, or a hydration medium, such as a gel, a damp cloth, a damp paper, or any combination thereof. In one embodiment, the container, such as a tip box, may be sealed to reduce evaporation of the medium. In one embodiment, the container may be placed under conditions that support the growth of spheroids.

[0037] Conditions supporting spheroid growth include a temperature range of 25–40°C and an atmosphere containing 2–8% CO2 and 90–98% humidity. Cells may be cultured at temperatures in the range of 27–30°C, 30–33°C, 33–36°C, 36–39°C, for example, 31–37°C, or in a humidified incubator. The carbon dioxide concentration may be maintained in the range of 2%–10%, and the oxygen concentration may be maintained in the range of 1%–22%. Culture conditions may vary depending on the specific method and components of culture. In some embodiments, fibroblasts may be cultured at fixed or variable temperatures in the range of approximately 20°C–40°C, approximately 25°C–38°C, or approximately 37°C. In one embodiment, the cells may be cultured at a fixed or variable carbon dioxide concentration in the range of 1 to 10%, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In one embodiment, the oxygen partial pressure may be in the range of 1 to 20%, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%, or any combination of these ranges. The cells may be cultured under suitable conditions for less than 24 hours, or at least 1, 2, 3, 4, 5, or 6 days, or at least 1, 2, 3, or 4 weeks, or at least 1 or 2 months. In some embodiments, cells may be cultured for at least about 2 to about 40 days, at least about 2 to about 10 days, or at least about 10 to 21 days, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the cells of the Disclosure may be cultured for a period not exceeding 10 days, not exceeding 15 days, or not exceeding 45 days. Cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days.

[0038] In one embodiment, any suitable pipette tip may be used in the method disclosed herein. In one embodiment, the chemically inert material may be polyethylene, polypropylene, or melamine. The pipette tip may be composed of other materials that do not adversely affect cells. In one embodiment, the tip may be further coated with a suitable coating material that reduces cell adhesion and / or promotes spheroid formation. Also herein, pipette tips having different geometric shapes and / or capacities are envisioned. For example, the pipette tip may have any suitable capacity such as 10, 20, 100, 200, or 1000 μl. Similarly, a container for housing the pipette tips, such as a tip box, may be of any suitable shape and size and may be made of any suitable material.

[0039] This disclosure should not be construed as being limited to any one method of isolating and culturing cells. Rather, it should be construed as encompassing all methods relating to the isolation and culture of cells. References to specific buffers, media, reagents, cells, culture conditions, etc., or their subclasses, should not be intended as limitation but should be construed as including all relevant materials that a person skilled in the art would recognize as of interest or value in the particular context in which such discussion is presented. For example, it is often possible to substitute one buffer system or culture medium with another, and different but known means can be used to achieve the same objectives as those directed toward by the use of the proposed method, material or composition. In certain embodiments, cells are cultured in a cell culture system including cell culture media, preferably in a culture vessel, and in particular in cell culture media supplemented with substances appropriately and determined to protect cells from in vitro senescence and / or induce nonspecific or specific reprogramming.

[0040] In one embodiment, the disclosure also encompasses methods for utilizing cultured spheroids for various clinical and non-clinical applications. In one embodiment, the spheroids may be used in a screening assay. In one embodiment, the screening assay may include contacting one or more spheroids formed in a pipette tip with a drug. In one embodiment, the contact may be performed in a pipette tip, or the spheroids may be released into a suitable apparatus (e.g., a Petri dish, a multiwell plate, or any such suitable apparatus) and contacted with the drug. In one embodiment, the cells of the spheroids may be dissociated prior to use (e.g., they may be trypsinized with 0.25% trypsin). In one embodiment, the spheroids may be used for therapeutic purposes as a source of cells, tissues, or organoids for administration to a subject in need of treatment. The spheroids may be obtained from autologous, allogeneic, heterogeneic, or syngeneic cells of the subject. In one embodiment, the spheroids may be further cultured in a suitable apparatus and under suitable conditions to obtain tissues, organoids, or organs for research and therapeutic applications. These methods and applications are described in detail herein.

[0041] As mentioned above, in one embodiment, a single spheroid may be formed in each pipette tip by aspirating a single cell suspension into the tip. This makes it possible to evaluate the effects of various drugs on spheroids by, for example, applying different drugs to each tip. Alternatively, it may be desirable to form multiple spheroids, such as two, three, four, five, six, or seven, in each tip. The multiple spheroids formed in the tip may have different cellular compositions, for example, originating from different tissue types, which is useful for evaluating how different cells interact with each other, i.e., how they affect each other's proliferation, survival, and / or migration. As a specific example, the proangiogenic properties of cancer cells can be studied by combining "cancer cell" spheroids and "endothelial cell" spheroids, and this system can be used for screening anti-angiogenic drugs.

[0042] In one embodiment, spheroids formed by the method of the present invention may exhibit features that substantially mimic the properties of the source tissue. Therefore, at least one of the following characteristics of one or more spheroids may be substantially identical to those of the source tissue: antigen profile, gene profile, tumor biology, tumor structure, cell proliferation rate, tumor microenvironment, treatment resistance, cell composition, gas concentration, cytokine expression, growth factor expression, and cell adhesion profile. Thus, the spheroids exhibit behavior substantially similar to or identical to that of the native cell line, for example, with respect to organization, proliferation, viability, cell survival, cell death, metabolism and mitochondrial state, oxidative stress, radiation response, and drug response. Because spheroids produced by the method of the present invention may exhibit behavior substantially similar to or identical to that of the native cell line, they are particularly useful in three-dimensional cell assays. In one embodiment, the disclosed spheroids may be evaluated using methods known in the art for properties such as viability, proliferation, proliferative capacity, differentiation, migration, morphology, signaling, metabolic activity, gene expression, and intercellular interactions. The evaluation of one or more characteristics of cells can be carried out from cell spheroids, or from spheroids fixed by rapid freezing or chemical fixation techniques, using any suitable method known in the art. For example, cell viability, proliferation, proliferative capacity, differentiation, migration, and morphology can all be evaluated by microscopic observation or image analysis. Characteristics can be detected using appropriate markers. For example, the expression of detectably labeled proteins, reporters, and / or one-step labeling of cellular components and markers makes it possible to directly visualize cellular structure, multicellular organization, and other readouts, for example, by fluorescence microscopy. Gene expression can be evaluated by functional genomics (e.g., microarray) techniques. Immunofluorescence, Hoechst staining, or Annexin-V assays can all be used. It will be understood that those skilled in the art can select appropriate techniques to evaluate a given characteristic.

[0043] In one embodiment, this method also makes it possible to evaluate how a particular cell type affects the function of other cell types, for example, when different cell types exist within the same spheroid, or when two or more spheroids having at least two different cell compositions are located in close proximity to each other.

[0044] In one embodiment, the disclosure also encompasses a method for evaluating the effects of a drug on a cellular property selected from among survival, proliferation, proliferative capacity, differentiation, migration, morphology, signaling, metabolic activity, gene expression, and intercellular interactions, the method comprising (i) preparing a spheroid in a pipette tip, and (ii) evaluating the effects of the drug on the cellular properties within the spheroid. It is understood that this method makes it possible to evaluate the effects of a drug on how a particular cell type affects the function of other cell types, for example, when different cell types are present within the same spheroid, or when two or more spheroids having at least two different cellular compositions are located in close proximity to each other. As used herein, the term “drug” includes polypeptides, peptides, nucleic acids, small molecules, or natural products. Thus, the drug may be a drug or a biologically active drug. The drug may be an inhibitor of a particular cellular function.

[0045] In one embodiment, the method allows for the evaluation of the function of a cell's gene or protein by using one or more agents, for example, inhibitors of the gene or protein. For example, the agent may be a chemoinhibitor, a peptide inhibitor, an siRNA molecule, an shRNA construct, or any agent capable of performing gene knockdown. To gain further insights into the function of the cell's gene or protein, it may be desirable to use multiple inhibitors (e.g., with different selectivity). Similarly, this methodology can be extended to investigate the cellular function of a genome or proteome array on a larger scale by exposing multiple spheroids to a range of each agent (e.g., RNAi inhibitors). In one embodiment, the agent may be an infectious agent, such as a bacterium or virus. Thus, the method may be used to study infection-related processes, such as whether cells are infected with bacteria or viruses and how the infection progresses and spreads. It may also be desirable to include additional agents to evaluate the effect of additional agents on infection. In one embodiment, the agent is additional cells. Therefore, cells can be introduced into a chip containing one or more spheroids, and the effect of these additional cells on the cells within the spheroids can be evaluated. For example, when the spheroid contains macrophages and the drug is skin cells, or vice versa, the interaction between macrophages and skin cells can be studied.

[0046] The agent may be applied to the spheroid after spheroid formation, or it may be present in the culture medium in which the spheroid is formed, or it may be injected together with the cell suspension. In one embodiment, the agent may be contained in beads to control its release rate (e.g., sustained release). Alternatively, the agent may be expressed within the spheroid cells, such as a polynucleotide. A specific example is the screening of a cDNA library or siRNA library for screening genes involved in a particular signaling pathway.

[0047] This method is understood to involve identifying agents that modulate one or more cellular properties selected from among survival, proliferation, proliferative capacity, differentiation, migration, morphology, signaling, metabolic activity, gene expression, and intercellular interactions.

[0048] In one embodiment, the drug is a drug-like compound or a lead compound for the development of a drug-like compound. The term "drug-like compound" is well known to those skilled in the art and may mean a compound that possesses properties that make it potentially suitable for use as an active ingredient in a medicine. For example, a drug-like compound may be a molecule that can be synthesized by organic chemistry, molecular biology, or biochemistry, may be a small molecule with a weight of less than 5000 daltons, and may be water-soluble. A drug-like compound may also exhibit selective interaction with a particular protein or group of proteins, be bioavailable, and / or be able to cross a target cell membrane or the blood-brain barrier, although these characteristics are not essential. The term "lead compound" is also well known to those skilled in the art and may include a compound that, even if the compound itself is not suitable for use as a medicine (for example, because it exhibits weak activity against the intended target, has low selectivity of action, is unstable, has low solubility, is difficult to synthesize, or has low bioavailability), can provide a starting point for designing other compounds with more desirable properties.

[0049] In one embodiment, the method further includes the steps of modifying a drug that has been shown to modulate at least one of the properties listed above, and testing the ability of the modified drug to modulate at least one of the properties listed above.

[0050] This method is useful not only for drug development but also for personalized medicine regimens. For example, it may be used to test the safety or efficacy of potential drug therapies, and therefore, by using organoids derived from allogeneic cells, for instance, it can support the customization of treatment to individual patients. It is understood that assays that allow for high-throughput operation are particularly preferred. Therefore, the assay may be performed simultaneously on multiple spheroids in multiple chips or on a multi-well plate, as described above.

[0051] Furthermore, the assay may preferably be automated or semi-automated. As described above, one advantage of producing spheroids by this method is that the spheroids can be formed at predetermined positions on the tips present in the pipette tip box. These spheroids can then be transferred to a cell culture plate. This makes automated detection of cell characteristics, for example, by automated microscopy, much easier. Thus, in one embodiment, the evaluation of one or more of the cell characteristics listed above is automated. The application of drugs to the spheroids may also be automated. Alternatively, if the drug is present in the culture medium before spheroid formation, spheroid formation may be analyzed.

[0052] The spheroid may contain cells derived from any organ, tissue, or part of the body, or may represent cells from any organ, tissue, or part of the body. In one embodiment, the cells may be cancer cells. Therefore, the method may be used to evaluate various properties of cancer cells or to determine the effect of a particular drug (e.g., a candidate drug) on ​​the invasion or migration of cancer cells, for example. However, it is understood that the properties of other cell types, including stem cells, endothelial cells, and immune cells, can also be evaluated, and that the method is equally applicable to any of the fields of stem cell biology, angiogenesis, immunobiology, toxicity studies, and tissue engineering. For example, reconstructing metastatic microtumors using tumor cells and hepatocytes, or tumor cells and bone marrow cells, is also within the scope of the present invention.

[0053] In one embodiment, the spheroids of the present invention may be used for research, diagnostic and / or therapeutic purposes, for example, in pharmacokinetic profiling, pharmacodynamic profiling, efficacy testing, cytotoxicity testing, compound penetration testing, treatment resistance testing, antibody production, personalized or tailor-made therapy, RNA / DNA "drug" testing, small molecule identification and / or testing, biomarker identification, tumor profiling, hyperthermia testing, radiation resistance testing, tissue engineering, and the like.

[0054] In certain embodiments, the method for producing spheroids according to the present invention can be used in tissue engineering. For example, tissue can be produced by aspirating cells into a pipette tip and culturing them for 1 to 15 days. This has two main advantages compared to conventional tissue engineering approaches. First, intercellular contact is rapidly formed because the cells are directly positioned relative to each other. Second, different types of cells can be placed in predetermined positions with greater precision because the cells can be applied sequentially or simultaneously. Therefore, this method is particularly suitable for tissues characterized by multiple cell types that are in close proximity.

[0055] Another application of this method in tissue engineering is organ printing. This involves the robotic biofabrication of three-dimensional functional biological microtissues and organ structures layer by layer, as described by Mironov et al. (Biomaterials 30: 2164-2174, 2009) and Moon et al. (Tissue Engineering 16(1): 157, 2010). Using the spheroid fabrication method of the present invention, cells can be seeded at high density onto a chip. Preferably, the spheroids are less than 300 micrometers in diameter to prevent necrosis. The cells can then be extruded in a controlled manner using the chip to print a three-dimensional tissue structure. This has the advantage that intercellular contacts are immediately formed and migration and / or tissue formation begin after printing. Alternatively, the spheroid fabrication method of the present invention can also be used to form spheroids at predetermined locations to print a three-dimensional tissue structure. Preferably, the spheroids are less than 300 micrometers in diameter to prevent necrosis. Furthermore, to enhance nutrient supply to cells / spheroids, the tip may be aspirated multiple times to allow perfusion of nutrient-rich culture medium or blood. It is understood that it may be desirable to combine the injection method of the present invention with known bioprinting techniques. For example, the method of the present invention may be used to position specific cells only where needed after a three-dimensional structure has been formed (e.g., by printing or seeding a scaffold).

[0056] In one embodiment, the disclosure also encompasses a method for treating a disease or condition in a subject requiring treatment, the method comprising administering to the subject a spheroid cultured using the method disclosed herein, or cells, cell populations, tissues, organs, or organoids derived from such spheroid. In one embodiment, the spheroid may be obtained from autologous, allogenic, xenogeneic, or syngeneic cells.

[0057] In certain cases, subjects treated by the methods and compositions of the Disclosure may receive one or more additional treatments. In certain cases, the one or more additional treatments may or may not treat one or more symptoms of a medical condition, rather than the underlying cause. Examples include one or more surgical procedures; rehabilitation including physiotherapy, occupational therapy, and / or speech therapy; dopaminergic treatments; anti-inflammatory drugs; analgesics; cholinesterase inhibitors; and antipsychotics. The one or more additional treatments may be provided to the subject before, concurrently with, and / or after the methods and compositions of the Disclosure. The one or more additional treatments and the methods and compositions of the Disclosure may or may not be administered via the same route of administration. The one or more additional treatments and the methods and compositions of the Disclosure may or may not be included in the same composition.

[0058] The compositions of this disclosure (for example, spheroids cultured using the methods disclosed herein, or cells, cell populations, tissues, organs or organoids derived from such spheroids, and optionally one or more excipients) may be administered by any route of administration. In some embodiments, the compositions may be administered intravenously, intrathecally, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, by implantation, by inhalation, intraventricularly, or intranasally. The appropriate dose may be determined based on the type of disease being treated, the severity and course of the disease, the clinical condition of the subject, the clinical history and response to treatment of the subject, and the discretion of the attending physician.

[0059] The aforementioned treatment may include various "unit doses." A unit dose is defined as a predetermined amount of the therapeutic composition. The amount to be administered, as well as the specific route of administration and formulation, are within the scope of the clinical technician's judgment. A unit dose does not need to be administered as a single dose, but may include continuous administration over a certain period. In some embodiments, a unit dose includes a single administerable dose.

[0060] In some embodiments, the composition is administered in doses of 100 to 10 million cells. In some embodiments, the treatment may be administered in doses of at least, up to, or about 1 million to 1 million, 1 million to 100,000, 100 to 10,000, 10 million to 10 million, 10 million to 10 million, 10 million to 100,000, 10 million to 10,000, 10 million to 10,000, 10 million to 10 million, 10 million to 1 million, 10 million to 100,000, 100,000 to 10 million, 100,000 to 1 million, or 1 million to 10 million cells, or products obtained therefrom, or any range of doses derived therefrom.

[0061] In some embodiments, the treatment is administered as a single dose. In some embodiments, the treatment is administered multiple times. In some embodiments, the treatment is administered in doses of 100 to 10 million cells, or products obtained therefrom. In some embodiments, the treatment is administered in doses of at least, up to, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 It may be administered in doses of 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, 99, or 100 mg / kg.

[0062] The amount to be administered depends on the desired therapeutic effect, both in terms of the number of treatments and the unit dose. The effective dose is understood to mean the amount necessary to achieve a particular effect. In certain implementations, doses ranging from 100 to 10 million cells may be expected to affect the protective capacity of these drugs. Furthermore, such doses may be administered multiple times a day and / or over multiple days, weeks, or months.

[0063] The precise amount of therapeutic composition may depend on the judgment of the administering physician and may be specific to each individual. Factors that may influence the dosage include the patient's physical and clinical condition, the route of administration, the intended goal of the treatment (symptom relief or cure), and the potency, stability, and toxicity of the particular therapeutic substance, or other treatments the individual is receiving.

[0064] It is also understood that uptake is species and organ / tissue dependent. The conversion factors and physiological assumptions applicable to uptake and concentration measurements are well known, enabling those skilled in the art to convert one concentration measurement to another and to derive reasonable comparisons and conclusions regarding the doses, effects, and results described herein.

[0065] In certain cases, it may be desirable to administer the composition multiple times, for example, two, three, four, five, six or more times. Such administrations may be carried out at intervals of 1, 2, 3, 4, 5, 6, 7, or 8 weeks to 5, 6, 7, 8, 9, 10, 11, or 12 weeks, and include all ranges in between.

[0066] Also disclosed are component kits comprising a chip box, a suitable culture medium, and cells. The kit may further include instructions for the preparation and / or use of spheroids. The component kit may further include means for evaluating cellular properties selected from among viability, proliferation, proliferative capacity, differentiation, migration, morphology, signaling, metabolic activity, gene expression, and intercellular interactions.

[0067] II. Spheroids Cell spheroids are three-dimensional cell aggregates that more faithfully mimic in vivo tissue structures and cell-cell interactions compared to conventional two-dimensional cell cultures. In one embodiment, this disclosure also includes cell spheroids produced using the methods disclosed herein. Aspects of this disclosure include cell spheroids prepared in a pipette tip using the methods disclosed herein. The disclosed spheroids can be produced from one or more cell types. For example, stem cells (embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, progenitor cells, etc.); fibroblasts (placental fibroblasts, omental fibroblasts, umbilical cord blood fibroblasts, skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, or foreskin-derived fibroblasts, etc.); cancer cells and cell lines (e.g., MCF-7, MDA-MB-231, HeLa, A549, HCT116, PC-3, DU145, U87, HepG2, K562, PANC-1, SKOV-3, U251, Caco-2, T47D, SW480, LNCaP, H460, HT-29, A375, PANC-1, T47D, PC-3, MDA-MB-23 Spheroids can be formed using various adherent cell types, including tissue-specific cells (e.g., hepatocytes, cardiomyocytes, islet cells, mesenchymal stromal cells (MSCs), etc.). In one embodiment, the spheroid may comprise a single cell type. In one embodiment, the spheroid may comprise at least two, three, four, five, or more different cell types. In one embodiment, the cells may be genetically modified before or during spheroid formation. In one embodiment, the cells may be genetically modified to affect their immunomodulatory capacity. In one embodiment, the cells may be genetically modified to target one or more genes or gene products to reduce their immunomodulatory capacity. Genetic modification methods or compositions for editing genomic DNA or transcripts may be used, using intracellular introduction of nucleic acids, gene editing, homologous or non-homologous recombination, TALENs, CRISPR, zinc finger nucleases, or any combination thereof.These genetic modifications may include the introduction of foreign genes, the deletion or mutation of endogenous genes, the introduction of selection markers or identification markers, or any combination thereof. These methods are known in the art and may be used individually or in combination to obtain cells with desired properties.

[0068] In one embodiment, the cells may be chemically, physically, or epigenetically activated by one or more nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or any combination thereof before or during spheroid formation, or before administration. In exemplary embodiments, the isolated cells may be cultured in the presence of other immune cells. Examples, but not limited to, include macrophages, microglia, dendritic cells, and regulatory T cells (Treg cells).

[0069] The spheroids of this disclosure typically have a size in the range of 20 to 1000 μm in diameter and may consist of a single cell type or multiple cell types to form more complex spheroids. Therefore, the spheroids may have a size in the range of about 25 to 50 μm, about 50 to 75 μm, about 75 to 100 μm, about 100 to 125 μm, about 125 to 150 μm, about 150 to 175 μm, about 175 to 200 μm, about 200 to 250 μm, about 250 to 300 μm, about 300 to 400 μm, or about 400 to 500 μm. To prevent necrosis, the spheroids are preferably less than 300 micrometers in diameter. The spheroids may take any shape, such as spherical, elliptical, or irregular. The spheroids may have an irregular or layered structure. Furthermore, the layer may contain a single cell type, or at least two, at least three, at least four, or more different cell types. These cell types may differ in terms of survival, proliferation, proliferative capacity, differentiation, migration, morphology, signaling, metabolic activity, gene expression, and intercellular interactions. The spheroids of this disclosure may contain 10 to 100, 100 to 1000, or 1 × 10¹⁶ cells of the same or multiple cell types. 3~1×10 4 1×10 4 ~1×10 5 1×10 5 ~1×10 6 1×10 6 ~1×10 7 1×10 7 ~1×10 8 1×10 8 ~1×10 9 1×10 9 ~1×10 10The spheroids may contain one or more cells. When intended for clinical use, the spheroids may be obtained from autologous, allogenic, xenogeneic, or syngeneic cells. Spheroids formed by the method of the present invention may exhibit features that substantially mimic the characteristics of the source tissue. Therefore, at least one of the following characteristics of the spheroids may be substantially identical to those of the source tissue: antigen profile, gene profile, tumor biology, tumor structure, cell proliferation rate, tumor microenvironment, treatment resistance, cell composition, gas concentration, cytokine expression, growth factor expression, and cell adhesion profile. Thus, the spheroids may exhibit behavior substantially similar to or identical to that of the native cell line, for example, with respect to organization, proliferation, viability, cell survival, cell death, metabolism and mitochondrial state, oxidative stress, radiation response, and drug response. Because spheroids produced by the method of the present invention may exhibit behavior substantially similar to or identical to that of the native cell line, they are particularly useful for three-dimensional cell assays. In one embodiment, the disclosed spheroids may be evaluated for properties such as survival, proliferation, proliferative capacity, differentiation, migration, morphology, signaling, metabolic activity, gene expression, and intercellular interactions using methods known to those skilled in the art. Evaluation of one or more cell properties can be carried out directly from cell spheroids or from spheroids fixed by rapid freezing or chemical fixation techniques using any suitable method known in the art. For example, cell survival, proliferation, proliferative capacity, differentiation, migration, and morphology can be evaluated by microscopic observation or image analysis. Properties can be detected using appropriate markers. For example, the expression of detectably labeled proteins, reporters, and / or one-step labeling of cellular components or markers allows for the direct visualization of cellular structure, multicellular organization, and other indicators, for example, by fluorescence microscopy. Gene expression can be evaluated by functional genomics (e.g., microarray) techniques. Immunofluorescence, Hoechst staining, or Annexin-V assays may also be used. It will be understood that those skilled in the art can select appropriate techniques to evaluate a given property.

[0070] In one embodiment, the disclosure also encompasses a pharmaceutical composition comprising a spheroid disclosed herein. In one embodiment, the pharmaceutical composition may comprise one or more cells, tissues, organs, or organoids derived from a spheroid disclosed herein. The pharmaceutical composition may further comprise one or more carriers or excipients. The composition may be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as a liquid solution or suspension. They can also be prepared as a solid form suitable for preparing a solution or suspension by adding liquid before injection. Furthermore, these formulations can be emulsified.

[0071] Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions; for example, formulations containing aqueous propylene glycol; and sterile powders for sterile injectable solutions or dispersions prepared at the time of use. In all cases, the form must be sterile and have sufficient fluidity to be easily injected. It must also be stable under manufacturing and storage conditions and have preservation properties against contamination by microorganisms such as bacteria and fungi.

[0072] The pharmaceutical composition may include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and a solvent or dispersion medium containing vegetable oil. Appropriate fluidity can be maintained, for example, by the use of coating agents such as lecithin, maintenance of the required particle size in the case of dispersion, and the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Absorption can be prolonged by using agents that delay the absorption of the injectable composition, such as aluminum monostearate or gelatin, in the composition.

[0073] A sterile injection solution is prepared by compounding an active composition (for example, a composition containing spheroids disclosed herein, or cells, cell populations, tissues, organs, or organoids derived from such spheroids) in a required amount in a suitable solvent, along with various other components as listed above as needed, and then sterilizing by filtration or equivalent means. Generally, dispersions are prepared by compounding sterile active ingredients in a sterile carrier containing a basic dispersion medium and other necessary components as listed above. In the case of sterile powders for preparing sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield powders containing the active ingredients from a pre-sterilized filtered solution and additional components as needed.

[0074] III. Definition

[0075] In accordance with long-standing patent law practice, the words “a” and “an” (including claims) used herein with the word “comprising” mean “one or more.” Some aspects of this disclosure may consist of, or substantially consist of, one or more elements, process steps and / or methods of this disclosure. Any method or composition described herein is intended to be practiced in relation to any other method or composition described herein.

[0076] The phrase "and / or" means "and" or "or." For example, A, B, and / or C includes A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C. In other words, "and / or" functions as an inclusive "or."

[0077] As used herein, the terms “about” or “approximately” mean a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies by up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a given quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length. In certain embodiments, the terms “about” or “approximately” preceding a numerical value indicate a range of ±15%, ±10%, ±5%, or ±1% of that value. With respect to biological systems or processes, the terms may mean a range of one order of magnitude, preferably five times, and more preferably two times. Unless otherwise stated, the term “about” means within the allowable margin of error relative to that value.

[0078] Throughout this specification, unless otherwise required by context, the terms “comprise,” “comprises,” and “comprising” are understood to mean that they include the described process or element or group of processes or elements, and not to exclude other processes or elements or groups of processes or elements. “Consisting of” means that it includes and is limited to the matters that follow it. Thus, the phrase “consisting of” indicates that the enumerated elements are essential or necessary, and other elements may not be present. “Consisting essentially of” means that it includes the elements that follow it, and is limited to other elements that do not interfere with or contribute to the activity or action of the enumerated elements as identified in this disclosure. Thus, the phrase “consisting essentially of” indicates that the enumerated elements are essential or necessary, but other elements may or may not be present, depending on whether they affect the activity or action of the enumerated elements.

[0079] Throughout this specification, any reference to “one aspect,” “an aspect,” “a particular aspect,” “a related aspect,” “a certain aspect,” “an additional aspect,” “a further aspect,” or any combination thereof, means that the specific features, structures, or properties described in relation to that aspect are included in at least one aspect of this disclosure. Therefore, the appearance of these terms in various places in this specification does not necessarily refer to the same aspect. Furthermore, the specific features, structures, or properties can be combined in any appropriate manner in one or more aspects.

[0080] As used herein, the terms “administered” or “administering” mean any method by which a composition is provided to a subject so that the composition exerts the intended effect on that subject. For example, one example of an administration method is administration by an indirect mechanism using a medical device such as a catheter, applicator gun, or syringe.

[0081] As used herein, "allogeneic" refers to tissues or cells derived from other individuals that, although originating from one or more subjects belonging to the same species, are immunologically incompatible or may become immunologically incompatible under natural conditions.

[0082] As used herein, "autologous" refers to tissue or cells derived from or transplanted from the same body (e.g., autologous blood donation, autologous bone marrow transplant).

[0083] As used herein, "agent" refers to polypeptides, peptides, nucleic acids, small molecules, natural products, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, or hormones.

[0084] As used herein, "xenogeneic" refers to tissue or cells derived from a species different from that of the patient.

[0085] Cell culture refers to an artificial in vitro system containing viable cells, whether quiescent, senescent, or actively dividing. In cell culture, cells are cultured and maintained at an appropriate temperature, usually 37°C, and in an atmosphere that typically contains oxygen and CO2. However, culture conditions can vary considerably depending on the cell type, and changes in conditions for a particular cell type can result in the expression of different phenotypes. The most commonly variable factor in a culture system is the culture medium. The medium can differ in the concentration of nutrients, growth factors, and the presence of other components. Growth factors used to supplement the medium are often derived from animal blood, such as calf serum.

[0086] As used herein, the terms “subject” or “individual” refer to a human or animal, whether or not it is housed in a medical facility, and including those receiving treatment as outpatients of a medical facility. The subject may receive one or more medical compositions via the Internet. The subject includes any age of human or non-human animals, and therefore includes adults and young people (i.e., children) as well as infants. The term “subject” does not imply a need for medical treatment. Therefore, the subject may be part of an experiment, either voluntarily or involuntarily, whether it is a clinical study or support for basic scientific research. The terms “subject” or “individual” may be used interchangeably and refer to any organism or animal that is the subject of a method or material, including mammals (e.g., humans, laboratory animals [e.g., primates, rats, mice, rabbits]), livestock [e.g., cattle, sheep, goats, pigs, turkeys, and chickens], domestic pets [e.g., dogs, cats, and rodents]), horses, and transgenic non-human animals.

[0087] The terms "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that, when administered to animals or humans, do not produce adverse reactions, allergic reactions, or other undesirable reactions. In this specification, "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating agent, antimicrobial agent, antifungal agent, isotonic agent, absorption retarder, etc. The use of such media and agents with pharmaceutically active substances is well known to those skilled in the art. Unless conventional media or agents are incompatible with the active ingredient, they are intended to be used in immunogenic compositions and therapeutic compositions. Furthermore, other auxiliary active ingredients, such as other anti-infective agents and vaccines, may also be incorporated into the compositions.

[0088] When used in relation to the manifestation of symptoms in a treated subject compared to an untreated subject, the terms "reduce," "inhibit," "diminish," "suppress," "decrease," "prevent," and their grammatical synonyms (including "lower," "smaller," etc.) mean that the amount and / or severity of symptoms in the treated subject is lower than the amount and / or severity of symptoms in the untreated subject to any degree that is clinically significant to a medically trained person. In one embodiment, the amount and / or severity of symptoms in the treated subject is at least 10%, at least 25%, at least 50%, at least 75%, and / or at least 90% lower than in the untreated subject.

[0089] "Treatment," "treat," or "treating" means a method of reducing the effects of a disease or condition. Treatment may also refer to a method of reducing the disease or condition itself, not just the symptoms. Treatment may include, but is not limited to, any reduction from a pre-treatment level, and may include the complete disappearance (ablation) of the disease, condition, or their symptoms. Therefore, in the methods of this disclosure, "treatment" may mean a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or the severity of disease progression, which includes a reduction in the severity of at least one symptom. For example, a method of this disclosure for reducing the immunogenicity of cells is considered a treatment if the immunogenicity of cells is detected to be reduced compared to a pre-treatment level in the same subject or a control subject. Therefore, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage in between, compared to the native level or the control level. It is understood and assumed here that “treatment” does not necessarily mean a cure for the disease or condition, but rather an improvement in the prognosis of the disease or condition. In certain embodiments, treatment may mean a reduction in the severity or degree of at least one symptom, or, in lieu of or in addition to, a delay in the onset of at least one symptom. [Examples]

[0090] The following embodiments are included to illustrate specific aspects of the Disclosure. Those skilled in the art will understand that the techniques disclosed in the following embodiments are techniques that the inventors have found to function well in carrying out the subject matter of the Disclosure, and therefore may be considered to constitute preferred embodiments for carrying out the Disclosure. However, those skilled in the art will understand that, in light of the Disclosure, similar or comparable results can be obtained without departing from the spirit and scope of the Disclosure, even with many modifications to the specific embodiments disclosed.

[0091] This disclosure encompasses a method for culturing spheroids in a pipette tip, which is suitable for automated, high-throughput, and scale-out production. Figures 1 and 2 show schematic diagrams of exemplary embodiments of the method of this disclosure. The upper part of Figure 1 shows a static preparation method for single-cell spheroids, and the lower part details a method for culturing composite spheroids. Furthermore, as shown in Figure 2, it is also possible to culture multiple spheroids in the same pipette tip by introducing an air gap during aspiration. The following examples further illustrate representative experiments to verify the method described herein.

[0092] Example 1: Obtaining spheroids from human fibroblasts Skin fibroblasts or fibroblasts derived from other organs were isolated from humans. The cells were cultured to 80% confluence in a typical cell culture environment (37°C, 5% carbon dioxide, 90% humidity) and then used for experiments. The specific procedures for each step are as follows. 1. Remove the culture medium and wash the cells twice with 1×PBS. 2. Treat with 0.25% trypsin for 3 minutes to dissociate the cells. 3. Cells are harvested using stem cell culture medium (low glucose DMEM, 10% human serum, 1% non-essential amino acids, 5% L-glutamine). 4. Set the cell concentration to 1 × 10⁻⁶ 6 Prepare the cell suspension by adjusting the cell / mL concentration and aspirating 30 μL of the cell suspension into a 200 μL pipette tip to prepare stem cell spheroids. 5. Optionally, an air gap of 1 to 50 μL may be aspirated to create a cushion between the pipette tip and the liquid containing the cells. 6. Place the pipette tip into the pipette tip box containing the appropriate culture medium, and gently discharge the tip into the tip box. 7. Place the pipette tip box in an incubator set to specific culture conditions for 3 days. 8. After 3 days of incubation, attach the pipette tip to the pipette and dispense the spheroid into a tube or single-well plate. 9. Collect the stem cell spheroids from the tube or plate. Centrifuge at 10,800 rpm for 3 minutes and remove the supernatant. 11. Resuspend the cells in stem cell culture medium (low glucose DMEM, 10% human serum, 1% non-essential amino acids, 5% L-glutamine).

[0093] The experimental setup is shown in Figures 3A and 3B. The cells were returned to a normal culture environment for evaluation, application, or continued culture, depending on the requirements of each experiment.

[0094] This method demonstrated that human dermal fibroblasts exhibited a 100% viability rate and possessed normal cell morphology, proliferative capacity, and migratory ability. Figure 4 provides a representative microscopic image showing the morphology of human spheroids after 4 days of culture.

[0095] Example 2: Obtaining composite spheroids from human dermal fibroblasts Next, we verified whether the method described in Example 1 could be used to produce complex spheroids containing multiple cell types. Skin fibroblasts were used for the verification experiment. The cells were cultured to 80% confluence in a typical cell culture environment (37°C, 5% carbon dioxide, 90% humidity) and then used in the experiment. The specific procedure is as follows. 1. Remove the culture medium and wash the cells twice with 1×PBS. 2. Treat with 0.25% trypsin for 3 minutes to dissociate the cells. 3. Cells are harvested using stem cell culture medium (low glucose DMEM, 10% human serum, 1% non-essential amino acids, 5% L-glutamine). 4. Set the cell concentration to 1 × 10⁻⁶ 6 Prepare the cell suspension by adjusting the cell / mL concentration and aspirating 30 μL of the cell suspension into a 200 μL pipette tip to prepare stem cell spheroids. 5. Optionally, an air gap of 1 to 50 μL may be aspirated to create a cushion between the pipette tip and the liquid containing the cells. 6. Place the pipette tip into the pipette tip box containing the appropriate culture medium, and gently discharge the tip into the tip box. 7. Place the pipette tip box in an incubator set to specific culture conditions for 48 hours. 8. Prepare a suspension of Dil-labeled human fibroblast cells and adjust the cell concentration to 1 × 10⁻⁶. 6 Adjust to the number of cells / mL. 9. Reattach the pipette tip containing the spheroid already formed from the first cell type to the pipette and aspirate 15 μL of Dil-labeled human fibroblasts from the cell suspension. 10. If an air gap is present, expel the air gap first before aspirating the fluid containing the second cell type. 11. Optionally, an air gap of 1 to 50 μL may be aspirated to create a cushion between the pipette tip and the liquid containing the cells. 12. Return the pipette tip to the tip box and gently eject the tip into the tip box. 13. Place the chip box in a normal cell culture environment for 48 hours to allow the spheroid shell structure to form. 14. After culturing in an incubator for 3 days, extrude the spheroids into tubes or single-well plates. 15. Collect the spheroids in a tube or plate, centrifuge at 1000 rpm for 3 minutes, and then remove the supernatant. 16. Resuspend the cells in stem cell culture medium (low glucose DMEM, 10% human serum, 1% non-essential amino acids, 5% L-glutamine).

[0096] The cells were evaluated, applied, or returned to a normal culture environment for continued culture, depending on the experimental objective. This method confirmed that human dermal fibroblasts exhibited a 97% viability rate and possessed normal cell morphology, proliferative capacity, and migratory ability.

Claims

1. An in vitro method for producing spheroids from one or more cells in a pipette tip, comprising the step of introducing the one or more cells into the pipette tip, wherein the pipette tip optionally contains a cell culture medium.

2. The method according to claim 1, wherein the one or more cells include one or more cell types.

3. The method according to claim 1, wherein the spheroid is a composite spheroid comprising a central core and at least one peripheral layer.

4. The method according to claim 1, wherein the one or more cells include fibroblasts.

5. The method according to claim 1, wherein the cell culture medium contains a viscosity enhancer.

6. The method according to claim 1 or claim 5, wherein the cell culture medium comprises a basal medium, 0-20% serum, 0-5% non-essential amino acids, 0-5% L-glutamine, and 0-5% viscosity enhancer.

7. The method according to claim 5 or claim 6, wherein the viscosity enhancer is methylcellulose.

8. The method according to claim 1, wherein the pipette tip is made of a chemically inert material.

9. The method according to any one of claims 1 to 8, wherein the pipette tip is housed in a tip box made of a chemically inert material.

10. The method according to claim 8 or 9, wherein the chemically inert material is polyethylene, polypropylene, or melamine.

11. The method according to any one of claims 1 to 10, wherein the pipette tip has a liquid capacity of 10, 20, 100, 200, or 1000 μl.

12. The above one or more cells are subjected to a temperature range of 25-40°C and 2-8% CO2. ２ The method according to any one of claims 1 to 11, wherein the culture is performed in an atmosphere containing and at a humidity of 90 to 98%.

13. The method according to claim 9, wherein the tip box includes one or more additional pipette tips containing one or more cells or spheroids.

14. The method according to any one of claims 1 to 13, wherein the spheroid comprises 0.1 to 1 million cells.

15. The method according to claim 14, wherein the size of the spheroid is 50 to 500 μm.

16. A method for screening one or more spheroids, comprising the step of contacting the one or more spheroids with a drug, wherein the one or more spheroids are cultured in a pipette tip.

17. The method according to claim 16, wherein the drug is a polypeptide, peptide, nucleic acid, small molecule, or natural product.

18. The method according to claim 16, wherein the spheroid is brought into contact with the drug in a pipette tip or in a separate device.

19. The method according to claim 18, wherein the separate device is a multiwell plate.

20. The method according to any one of claims 16 to 19, wherein the screening method is fully or partially automated.

21. A method for treating a subject in need of treatment, comprising the step of administering to the subject a pharmaceutical composition containing one or more spheroids, or cells, tissues, organoids, or organs obtained from the one or more spheroids, wherein the one or more spheroids are cultured in a pipette tip.

22. A kit comprising a chip box containing one or more chips for growing one or more spheroids from one or more cells or cell populations, and instructions on how to use the kit.

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