A novel method for the automatic generation of cell spheroid with core-shell structure

Abstract

Provided herein are methods of producing spheroids from one or more cells, in a pipette tip. The spheroids may comprise a single cell type, or multiple cell types. Also provided are methods of using the spheroids in fully or partially automated assays, high-throughput screening, and clinical application.

Description

A NOVEL METHOD FOR THE AUTOMATIC GENERATION OF CELL SPHEROID WITH CORE-SHELL STRUCTURE

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 514,865, filed July 21 , 2023, which is incorporated by reference herein in its entirety.BACKGROUND1. FIELD

[0002] Aspects of the disclosure concern at least the fields of cell biology, molecular biology, immunology, and medicine.2. BACKGROUND

[0003] Cell spheroids, also known as 3D cell cultures, represent a more physiologically relevant model compared to traditional 2D cell cultures. Cell spheroids provide several advantages over 2D cultures. Firstly, they offer a more accurate representation of tissue structure and cellular behavior, making them suitable for studying disease mechanisms, drug screening, and tissue engineering applications. Secondly, cell spheroids have shown improved predictive value for assessing drug efficacy and toxicity compared to 2D cultures. Additionally, they allow for the investigation of cell signaling, migration, invasion, and other cellular processes better represented in a three-dimensional context.

[0004] There are multiple methods for cell spheroids formation, and they can be categorized into four groups per the mechanism: 1 Gravity; 2. Micropattern; 3. Microfluidics, and 4. Electrospun. The gravity method includes two types: 1. Hanging drop and 2. Microwells. The hanging drop method is popular for forming cell spheroids in a 3D culture. In this method, cells are suspended in a droplet of culture medium, then placed upside down on the lid of a tissue culture dish. Due to gravity, the cells aggregate and form spheroids within the droplet. The microwell method is used to create cell spheroids in a controlled and reproducible manner. This method uses small microwells or microfabricated molds to confine cells in a specific shape, typically a spherical or cylindrical structure. Cells are seeded into these microwells, allowing them to self-assemble and form spheroids. The microcell method enables precise control over the spheroids' size, shape, and density, making it useful for various applications such as tissue engineering, drug screening, and studying cellular behavior. Those two techniques offer a convenient and versatile approach to generating uniform and well-defined cell spheroids for scientific research and biomedical applications.

[0005] The micropattern method creates cell spheroids with defined shapes and spatial organization. It involves using micropatterned substrates, such as microcontact printing orphotolithography, to create specific adhesive or non-adhesive regions on a culture surface. Cells are seeded onto these patterns, growing, transforming their cell-cell contact and organization into desired spheroid shapes. This method allows precise control over cell positioning, cell-cell interactions, and tissue-like architectures. The micropattern method is valuable for studying cell behavior, tissue development, and creating complex multicellular structures that closely mimic in vivo conditions.

[0006] The microfluidics method is an innovative approach to creating cell spheroids with precise control over their size, shape, and composition. It involves using microscale channels and chambers to manipulate fluids and cells in a highly controlled manner. Cells are introduced into these microfluidic devices, allowing their growth and assembly into spheroids through controlled flow and spatial organization. This method offers advantages such as high throughput, precise manipulation of cell microenvironments, and the ability to create complex multicellular structures.

[0007] The electrospun method creates cell spheroids by utilizing electrospinning, which produces ultrafine fibers from a polymer solution. This method encapsulates cells within the polymer solution, and an electric field is applied to create a charged jet. As the solvent evaporates, the polymer fibers accumulate, creating a three-dimensional scaffold that entangles the cells. This scaffold supports cell-cell interactions and promotes the formation of spheroids.

[0008] The methods above offer versatile approaches to generating uniform and well-defined cell spheroids for scientific research and biomedical applications. However, all the methods based on gravity and micropattern may only form the spheroid with single cell type but not create complex multicellular structures that closely mimic in vivo conditions (i.e., organ contains multiple cell types), hindering broader application for real transplantation in vivo. Although the methods based on microfluidics and electrospun may form complex multicellular structures like core-shell structures that better mimic in vivo conditions, these methods either need a complicated device or an expensive voltage generator, which are common in engineering labs but far less common in hospitals and biotech companies. Of note, all the methods above are incompatible with the current lab automation technique for high-throughput production of high-quality cell spheroids for scientific research and biomedical applications.SUMMARY OF THE INVENTION

[0009] In an aspect the current disclosure encompasses an in vitro method of producing a spheroid from one or more cells in a pipette tip, the method comprising introducing the one or more cells into the pipette tip, wherein the pipette tip optionally comprises a cell culture medium. The one or more cells may comprise one or more cell types. The spheroid may be a complex spheroid comprising a central core and at least one peripheral layer. In an aspect,the one or more cells may comprise a fibroblast. In an aspect, the cell culture medium may comprise a viscosity enhancing agent. The cell culture medium may comprise a basal medium, 0 to 20% of serum, 0 to 5% non-essential amino acids, and 0 to 5% L-glutamine, and 0 to 5% of a viscosity enhancing agent. The viscosity enhancing agent may be methylcellulose. In an aspect, 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 example of chemically inert materials may be polyethylene, polypropylene, or melamine. In some aspects, the tip box may comprise one or more additional pipette tips comprising one or more cells, or spheroid. In some aspects, the pipette tip has a liquid capacity of 10, 20, 100, 200, or 1000 pl. In some aspects, the one or more cells may be cultured at a temperature range of 25-40 degrees centigrade, and an atmosphere comprising 2 to 8% CO2, and 90-98% humidity. In some aspects, the spheroid may comprise 0.1-1 million cells. In some aspects, the spheroid is SO- SOO uM in size.

[0010] In an aspect, the current disclosure also encompasses a method of screening one or more spheroid(s), comprising contacting the one or more spheroid(s) to an agent, wherein the one or more spheroid(s) are cultured in a pipette tip. Non-limiting examples of suitable agents include polypeptide, a peptide, a nucleic acid, a small molecule, or a natural product. In some aspects, the spheroid may be contacted with the agent within the pipette tip or in a separate apparatus, for example a multi-well plate. The method of screening may be fully or partially automated.

[0011] In an aspect, the current disclosure also encompasses a method of treating a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising one or more spheroids, or cells, tissue, organoid or organ derived from the one or more spheroid, wherein the one or more spheroid is cultured in a pipette tip. Also envisaged are kits comprising a tip box comprising one or more tips, for growing one or more spheroids from a cell or a cell population, and instructions for using the kit.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0013] FIG. 1. is a schematic diagram for making single-cell type spheroid formations and scale-out production (1 to a) and cell spheroids with a core-shell structure containing two cell types (orange and green, respectively) in a tip (1 to b to 2). Both methods use air gaps to prevent the liquid from flowing out of the tip by gravity or positive pressure introduced when the tip is loaded onto a pipettor.

[0014] FIG. 2. is a schematic diagram for making multiple cell spheroids in a single pipette tip (steps 1 to 11) and using air gaps to separate spheroids and also to prevent the liquid from flowing out of the tip by gravity or positive pressure introduced when the tip is loaded onto a pipettor.

[0015] FIG. 3A shows an ordinary tip and tip box used to form the cell spheroids. Side-view of the tip box with 1 X DPBS to maintain humidity is shown. The black line squared box is the top-view of the whole tip box.

[0016] FIG. 3B shows four tips that contained 40 pl medium each, and which were incubated in the tip box for four days in an incubator. No evaporation or media loss was evident after 4 days.

[0017] FIG. 4 are representative microscopic images of the Spheroids. Morphology of human fibroblast cell spheroids validates that the fibroblast cell can form the spheroid with the tipspheroid method within four days.DETAILED DESCRIPTION

[0018] The current disclosure is based in part on the unexpected discovery that viable 3D cell spheroids can be cultured within pipette tips using the methods as provided herein. Surprisingly, not only can these spheroids be generated to comprise a single cell-type, but these methods can also be used to culture complex spheroids with more than one cell type. The current disclosure encompasses novel methods of generating single cell-type or multiple cell-type organ spheroids using pipette tips, and is compatible with all liquid handling robots, enabling high-throughput spheroid production. Pipette tips are routinely used in the laboratory and manufacturing facilities for dispensing and transferring of cells into tubes and plates. Aspects of the disclosure encompass compositions, methods, and systems for the efficient preparation of 3D spheroids directly inside a pipette tip by intake of cells inside the pipette tip and incubating the pipette in the appropriate cell culturing conditions to enable aggregation of the cells into a spheroid structure. A pipette tip with no biological toxicity can be used and provides multiple benefits including high-density and large-scale automatic spheroid generation. Cell products do not need subsequent separation and purification steps compared to conventional method based on microwells which usually have intended aggregate and need extra selection with cell strainer. Moreover, the cell spheroid process can be operated by the liquid handler robot allowing for easy automation and compatibility with high throughput methods. The spheroids can be directly used for biological and medical scientific research, cell therapy for immune diseases and degenerative diseases, cell transplantation for tissue and organ damage, and satisfy the requirements for drug screening, with immeasurable scientific and socioeconomic benefits. These spheroids can be used in studying cell behavior,tissue engineering, drug testing, disease modeling, clinical training, research, and therapeutic use in a more physiologically relevant and reproducible manner.I. Methods

[0019] In an aspect, the current disclosure encompasses an in vitro method of producing a spheroid from one or more cells in a pipette tip, comprising introducing the one or more cells into the pipette tip, wherein the pipette tip may optionally comprise a cell culture medium.

[0020] In an aspect, the one or more cells may comprise cells of any cell-type. Non-limiting examples of suitable cell-types include stem cells, for instance, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, progenitor cells; fibroblasts, for instance, placental fibroblasts, omental tissue derived fibroblasts, cord blood fibroblasts, fibroblasts derived from skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, or foreskin; cancer cells and cell lines for example, 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; tissue and organ specific cells for example, hepatocytes, cardiomyocytes, pancreatic islet cells, mesenchymal stromal cells (MSCs), and various other adherent cell types can be used to form spheroids. In an aspect, the one or more cells may comprise a single cell-type. In an aspect, the one or more cells may comprise at least 2, 3, 4, 5, or more different cell types.

[0021] In an aspect, the one or more cells can be introduced into the pipette tip at a concentration of 0.1 cells / ml to about 1 x 1010cells / ml. In an aspect, the cells can be introduced into the pipette tip at a concentration of 0.1 - 1 , 1 - 10, 10 - 100, 100 - 1 ,000, 1 x 103- 1 x 104, 1 x 104- 1 x 105, 1 x 105- 1 x 106, 1 x 106- 1 x 107, 1 x 107- 1 x 108, 1 x 108- 1 x 109, 1 x 109- 1 x 1010cells / mL or more. In an aspect, the cells may be introduced in one intake step. In an aspect, the cells may be introduced into the pipette tip in one, two, three, four, five or more intake steps. In an aspect, an air gap may be maintained between each intake step, such that more than one spheroid can form within the same tip, separated by air gaps. In an aspect, no air gap is maintained. Thus, in an aspect, a single spheroid may be formed in each pipette tip by drawing in a single cell suspension into the tip. Alternatively, it may be desirable to form more than one spheroid in each tip, such as 2 or 3 or 4 or 5 or 6 or 7 spheroids. The more than one spheroids formed within the tip may have different cell compositions, for example derived from different tissue types.

[0022] Spheroids may be formed at predetermined locations on the tip. For example, particularly when the method is automated, spheroids can be formed at predetermined distances from each other. The spheroids may be formed at regular or non-random spaced intervals. Spheroids may be formed at the same position in each tip.

[0023] Accordingly, when multiple spheroids are produced in a single tip, the spheroids may be formed at least 0.5 mm to 10 mm apart, such as at least 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 apart. However, it may be desirable to study interactions between spheroids in which case the spheroids may be formed less than 2.0 mm apart.

[0024] In an aspect, a first cell type may be introduced into the pipette tip and cultured for a period of time, followed by introduction of a second cell type, and cultured for a period of time. In an aspect, this step may be repeated at least once, at least twice, at least thrice or more. This may result in the formation of a complex spheroid with more than one layer of cells. In an aspect, the first and the second cell type may be the same. In an aspect, the first and the second cell type may be different. In an aspect, the resulting spheroid may comprise a single cell type or more than one, or two, or three, or more cell types. Thus, in an aspect, the spheroid may be a complex spheroid comprising a central core and at least one peripheral layer.

[0025] In an aspect, the current disclosure encompasses an in vitro method of producing a spheroid from one or more cells in a pipette tip, comprising introducing the one or more cells into the pipette tip, wherein the pipette tip may comprise a cell culture medium. In an aspect, the current disclosure encompasses an in vitro method of producing a spheroid from one or more cells in a pipette tip, comprising introducing the one or more cells into the pipette tip, wherein the pipette tip may comprise a cell culture medium, to produce a spheroid under suitable conditions. In an aspect, a cell culture medium to produce the spheroid, may be present in the tip prior to the introduction of the one or more cells. In an aspect, the cell culture medium to produce the spheroid , may be introduced into the tip with the one or more cells. In an aspect, cell culture medium may be rejuvenated, replenished, or changed at least once during the growth of the spheroid.

[0026] The cell culture medium may comprise a basal medium. Typically, the medium may comprise a basal medium formulation as known in the art. Many basal media formulations can be used to culture spheroids herein, including but not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Leibovitz L-15, DMEM / F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art, and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the cells cultured. In some aspects, a culture medium formulation may be explants medium (CEM) which is composed of IMDM supplemented with 10% fetal bovine serum (FBS), 100 ll / rnl penicillin G, 100 pg / ml streptomycin and 2 mmol / LL-glutamine. Other aspects may employ further basal media formulations, such as chosen from the ones above.

[0027] Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum may be added to the above medium in order to support the growth of cells. A defined medium may however be used if the growth factors, cytokines, and hormones necessary for culturing cells are provided at appropriate concentrations in the medium. In an aspect, the media may be free of fetal bovine serum. In an aspect, the medium may comprise one or more of human platelet rich plasma, platelet lysate, umbilical cord blood serum, autologous serum, one or more cytokines, fibroblast growth factor, epidermal growth factor, leukemia inhibitory factor, insulin like growth factor, angiopoietin, vascular endothelial growth factor, or any combination thereof. Media useful in the methods of the disclosure may further comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of cells.

[0028] For use in the cell culture, media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution. Further antioxidant supplements may be added, e.g., p- mercaptoethanol. While many media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is supplementation of cell culture medium with mammalian plasma or sera. Plasma or sera often contain cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated. One or more of the medium components may be added at a concentration of at least, at most, or about 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 derivable therein.

[0029] The medium in certain aspects can be prepared using a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI-1640, and Fischer's media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as it canbe used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined.

[0030] The medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal as that of the stem cell(s). The serum-free medium refers to a medium with no unprocessed or unpurified serum and accordingly, can include a medium with purified blood-derived components or animal tissue-derived components (such as growth factors).

[0031] The medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid- rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'- thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98 / 30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0032] In certain aspects, the medium may comprise 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: Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha-Tocopherol; Vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; Catalase; Human Recombinant Insulin; Human Transferrin; Superoxide Dismutase; Other Components such as Corticosterone; D-Galactose; Ethanolamine HCI; Glutathione (reduced); L-Carnitine HCI; Linoleic Acid; Linolenic Acid; Progesterone; Putrescine 2HCI; Sodium Selenite; and / or T3 (Triiodothyronine). In specific aspects, one or more of these may be explicitly excluded.

[0033] In some aspects, the medium further comprises vitamins. In some aspects, the medium comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13 of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium includes combinations thereof or salts thereof. In some aspects, the medium comprises or consists essentially 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 aspects, the vitamins include or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some aspects, the medium further comprises proteins. In some aspects, the proteins comprise albumin orbovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some aspects, the medium further comprises one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodothyronine, or combinations thereof. In some aspects, the medium comprises one or more of the following: a B-27® supplement, xeno-free B-27® supplement, GS21™ supplement, or combinations thereof. In some aspects, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some aspects, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some aspects, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some aspects, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain aspects, the medium comprises or consists essentially of one or more vitamins discussed herein and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodothyronine, a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In specific aspects, one or more of these may be explicitly excluded.

[0034] The medium may also contain one or more externally added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and / or inorganic salts. In specific aspects, one or more of these may be explicitly excluded.

[0035] In an aspect, the cell culture medium may further comprise one or more viscosity enhancing agents. Viscosity of the compositions, if desired, can be maintained at the selected level using one or more pharmaceutically-acceptable viscosity enhancing agents. Non-limiting examples of viscosity enhancing agents include agar, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, guar gum, xanthan gum, pectin, collagen, gelatin, methylcellulose, starch, and or any combination thereof. Methylcellulose is preferred because it is readily and economically available and is easy to work with. The preferred concentration of the viscosity enhancing agent will depend upon the agent selected. The important point is to use an amount, which will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such agents. In an aspect, one or more viscosity enhancingagents may be used at a concentration of between 0.001 %-3.0%. In an aspect, the one or more viscosity enhancing agents may be used at a combined concentration of at least about 0.001 % - 0.01%, 0.01 % - 0.1 %, 0.1% - 0.5%, 0.5% - 1%, 1 % - 1.5%, 1.5% - 2%, 2% - 2.5%, 2.5% - 3.0%, 3.0% - 3.5%, 3.5% - 4.0%, 4.0% - 5% or more. In an aspect, the viscosity enhancing agent is methyl cellulose and the concentration is between 0.1%-3%. The pharmaceutically acceptable viscosity enhancing agent does not produce biological toxicity, can be metabolized, has no residue, and is safe for cells. The medium may contain other materials and conditions for optimal culture success. In an aspect, the cell culture medium may comprise a basal medium, 0 to 20% of serum, 0 to 5% non-essential amino acids, and 0 to 5% L-glutamine and 0 to 5% of a viscosity enhancing agent.

[0036] In an aspect, the pipette tip may be placed in any suitable container, for example a tip box. In an aspect, the container, for example the tip-box may comprise any liquid, for example water, saline, or cell culture medium, or a hydration medium, for example a gel, wet cloth, wet paper, or any combination thereof. In an aspect, the container, for example a tip box may be sealed to reduce evaporation of the medium. In an aspect, the container may be placed under conditions that support growth of the spheroid.

[0037] Conditions that support growth of the spheroid may comprise a temperature range of 25-40 degrees centigrade, and an atmosphere comprising 2 to 8% CO2, and 90-98% humidity. The cells may be grown at temperatures between 27° C to 30° C, 30° C to 33°, 33° C to 36° C, 36° C to 39° C, for example 31 ° C to 37° C, and may be in a humidified incubator. The carbon dioxide content may be maintained between 2% to 10% and the oxygen content may be maintained between 1 % and 22%. The culturing conditions can vary depending on the specific method and components of the culture. In some aspect, the fibroblasts may be cultured at a fixed or variable temperature ranging from about 20° to about 40 °C, or about 25 °C to about 38 °C, or about 37 °C. In an aspect, the cells may be cultured at a fixed, or variable carbon dioxide concentration ranging from 1-10%, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% carbon dioxide concentration. In an aspect, the oxygen tension can range from 1-20%, 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% and any combination of ranges derivable therein. Cells may be grown under suitable condition / 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 aspects, cells may be cultured for at least between about 2 days and about 40 days, for at least between about 2 days and about 10 days, for at least between about 10 days and 21 days, such as for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 days. In some aspects, the cells of the disclosure may be cultured for no longer than 10 days, no longer than 15 days, or no longer than 45 days.The 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 an aspect, any suitable pipette tip may be used in the methods disclosed herein. In an aspect the chemically inert material may be polyethylene, polypropylene, or melamine. Pipette tips may be constructed of other materials which do not adversely affect cells. In an aspect, the tip may be further coated with suitable coating materials which reduce cell adherence and / or promote formation of spheroids. Also contemplated herein are pipette tips of different geometrical shapes, and / or volumes. For instance, the pipette tip may be of any suitable capacity, for example 10, 20, 100, 200, or 1000 pl. Similarly, the container, for example tip box, for housing the pipette tips may be of any suitable shape and size and may be made of any suitable material.

[0039] The disclosure should in no way be construed to be limited to any one method of isolating and culturing cells. Rather, any method of isolating and culturing cells should be construed to be included in the present disclosure. Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed. In particular aspects, cells are cultured in a cell culture system comprising a cell culture medium, preferably in a culture vessel, in particular a cell culture medium supplemented with a substance suitable and determined for protecting the cells from in vitro aging and / or inducing in an unspecific or specific reprogramming.

[0040] In an aspect, the current disclosure also encompasses method of using the cultured spheroids in various clinical and non-clinical applications. In an aspect, the spheroid(s) may be used in screening assays. In an aspect, the screening assay may include contacting the one or more spheroids formed in the pipette tip to an agent. In an aspect, the contacting can be done within the pipette tip, or the spheroids may be released into a suitable apparatus (for example a petri-dish, multi-well plate or any such suitable apparatus) and contacted with the agent. In an aspect, the cells of the spheroid may be dissociated (for example trypsinzed with 0.25% trypsin) prior to use. In an aspect, the spheroids may be used for therapeutic applications, as a source or cells, tissues, organoids for administration to a subject in need thereof. The spheroids may be derived from cells autogenic, allogeneic, xenogeneic, or syngenetic to the subject. In an aspect, the spheroids may be further cultured in a suitable apparatus and under suitable conditions to obtain tissues, organoids or organs for research and therapeutic use. These methods and applications are elaborated herein.

[0041] As discussed previously, in one aspect, a single spheroid may be formed in each pipette tip by drawing in a single cell suspension into the tip. This allows the effect of various agents on a spheroid to be assayed, for example by subjecting each tip to a different agent. Alternatively, it may be desirable to form more than one spheroid in each tip, such as 2 or 3 or 4 or 5 or 6 or 7 spheroids. The more than one spheroids formed within the tip may have different cell compositions, for example derived from different tissue types, which may be useful in assaying how different cells interact with each other, i.e., how they affect each other’s growth, survival and / or migration. In a particular example, pro-angiogenic properties of cancer cells may be studied by combining a 'cancer cell' spheroid with an 'endothelial cell' spheroid, and the system may be used to screen for anti-angiogenic drugs.

[0042] In an aspect, the spheroids formed by the methods of the invention, may exhibit characteristics that substantially mimic those of the tissue of origin. Thus, at least one of the antigen profile, genetic profile, tumor biology, tumor architecture, cell proliferation rate(s), tumor microenvironment, therapeutic resistance, cell composition, gas concentrations, cytokine expression, growth factor expression and cell adhesion profile of the one or more spheroids may be substantially identical to that of the tissue of origin. Accordingly, the spheroids exhibit a substantially similar or identical behavior to that of natural cell systems, for example with respect to organization, growth, viability, cell survival, cell death, metabolic and mitochondrial status, oxidative stress, and radiation response as well as drug response. The spheroids produced by the methods of the invention may exhibit a substantially similar or identical behavior to that of natural cell systems, making them particularly useful for 3D cell assays. In an aspect, the disclosed spheroids may be assessed for properties such as survival, growth, proliferation, differentiation, migration, morphology, signaling, metabolic activity, gene expression and cell-cell interaction using known methods in the art. Assessing one or more properties of a cell may be carried out using any suitable method known in the art, either from cell spheroids or from spheroids fixed by snap-freezing or chemical fixation techniques. For example, any of cell survival, growth, proliferation, differentiation, migration and morphology may be assessed by microscopy or image analysis. Properties may be detected using appropriate markers. For example, expression of detectably-labelled proteins, reporters and / or single-step labelling of cell components and markers can enable cell architecture, multicellular organization, and other readouts to be directly visualized, for example by fluorescence microscopy. Gene expression may be assessed by functional genomic (e.g., microarray) techniques, and so on. Any of immunofluorescence, Hoechst staining or Annexin-V assays may be used. It is appreciated that the skilled person may select the appropriate technique to assess a given property.

[0043] In an aspect, the method may also allow the assessment of how a particular cell type affects the function of another cell type, for example where different cell types are present inthe same spheroid or where two or more spheroids with at least two different cellular compositions are in close proximity to each other.

[0044] In an aspect, the current disclosure also encompasses a method of assessing the effect of an agent on the property of a cell selected from any of survival, growth, proliferation, differentiation, migration, morphology, signaling, metabolic activity, gene expression and cellcell interaction, the method comprising (i) producing a spheroid in a pipette tip, and (ii) assessing the effect of the agent on the property of a cell in the spheroid. It will be appreciated that the method allows the assessment of the effect of an agent on how a particular cell type affects the function of another cell type, for example where different cell types are present in the same spheroid or where two or more spheroids with at least two different cellular compositions are in close proximity to each other. The term agent, as used herein, includes any of a polypeptide, a peptide, a nucleic acid, a small molecule, or a natural product. Thus, the agent may be a drug or a biologically active agent. The agent may be an inhibitor of a particular cellular function.

[0045] In an aspect, the method allows for the assessment of the function of a cellular gene or protein, for example by using one or more agents that is an inhibitor of that gene or protein. For instance, the agent may be any of a chemical inhibitor, a peptide inhibitor, a siRNA molecule or a shRNA construct or any agent capable of effecting a gene knockdown. It may also be desirable to use more than one inhibitor (e.g., with different selectivity) to provide further insight into the function of a cellular gene or protein. Similarly, by exposing multiple spheroids to a range of respective agents (e.g., RNAi inhibitors), the methodology can be scaled up to investigate the cellular functions of genome or proteome arrays on a larger scale. In an aspect, the agent may be an infectious agent such as a bacterium or virus. In this way, the method may be used to study infection related processes such as whether cells are infected by bacteria or viruses and, if so, how the infection progresses and spreads. It may also be desirable to include a further agent so as to assess the effect of the further agent on the infection. In an aspect, the agent is a further cell. Thus, cells may be introduced to a tip containing one or more spheroids, and the effect of the further cell on the cells within the spheroid assessed. For example, the spheroid may comprise macrophages and the agent comprises skin cells, or vice versa, such that the interaction between macrophages and skin cells can be studied.

[0046] The agent may be applied to the spheroid after formation or may be present in the medium in which the spheroid is formed, or it may be injected along with the cell suspension. In one aspect, the agent may be contained within a bead so as to control the rate at which the agent is released (e.g., slow-release). Alternatively, the agent may be one that is expressed in the cells of the spheroid, such as a polynucleotide. A particular example is the screening ofcDNA libraries or siRNA libraries, for example to screen for genes involved in particular signaling pathways.

[0047] It is appreciated that the method includes identifying an agent that modulates one or more properties of a cell selected from any of survival, growth, proliferation, differentiation, migration, morphology, signaling, metabolic activity, gene expression and cell-cell interaction.

[0048] In an aspect, the agent is a drug-like compound or 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 a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesized by the techniques of organic chemistry, or by techniques of molecular biology or biochemistry, and may be a small molecule, which may be of less than 5000 Daltons, and which may be water-soluble. A druglike compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and / or able to penetrate target cellular membranes or the bloodbrain barrier, but it will be appreciated that these features are not essential. The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesize or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.

[0049] In an aspect, the method further comprises modifying an agent which has been shown to modulate at least one of the properties listed above, and testing the ability of the modified agent to modulate at least one of the properties listed above.

[0050] As well as being useful in drug development, the method may also be useful in personalized medicine regimes. For example, the method may be used to test the safety or efficacy of potential drug treatments, and so may aid the customization of treatments for individual patients, for example by using organoids with allogenic cells. It is appreciated that assays which are capable of high throughput operation are particularly preferred. Thus, the assays may be performed simultaneously on multiple spheroids in multiple tips or on multiwell plates as described above.

[0051] Further, the assays may be preferably automated or semi-automated. As explained above, one advantage of the present method to produce spheroids is that the spheroids may be formed at predefined locations in tips present in a pipette tip box. These spheroids can be transferred to a cell culture plate. Thus, automatic detection of cell properties, for example, by automated microscopy, is much easier. Thus, in one aspect, the assessment of the one or more of the cell properties listed above is automated. Application of the agent to the spheroidmay be automated. Alternatively, where present in the medium prior to spheroid formation, the formation of the spheroids may be analyzed.

[0052] Spheroids may comprise cells from any organ, tissue, or part of the body, and similarly represent cells from any organ, tissue, or part of the body. In an aspect, cell may be a cancer cell. Thus, the methods may be used to assess various properties of cancer cells or to determine the effects of particular agents (e.g., candidate drugs) on, for example, cancer cell invasion or migration. However, it is appreciated that the properties of other cell types may also be assessed including stem cells, endothelial cells, and immune cells and that the methods have equal application in any one or more of stem cell biology, angiogenesis, immune biology, toxicity studies and tissue engineering. For example, it is within the scope of the invention to rebuild a metastatic microtumor e.g., tumor cells with hepatocytes, or tumor cells with bone marrow cells.

[0053] In an aspect, the spheroids of the invention may be used for research, diagnostic and / or therapeutic purposes, for example pharmacokinetic profiling, pharmacodynamic profiling, efficacy studies, cytotoxicity studies, penetration studies of compounds, therapeutic resistance studies, antibody generation, personalized or tailored therapies, RNA / DNA 'drug' testing, small molecule identification and / or testing, biomarker identification, tumor profiling, hyperthermia studies, radio resistance studies, tissue engineering and the like.

[0054] In a particular aspect, the methods of producing spheroids according to the invention are used in tissue engineering. For example, tissues can be produced by aspirating cells into a pipette tip and growing them for 1-15 days. This has two main advantages compared to conventional tissue engineering approaches. Firstly, cells are directly placed together such that cell-cell contacts form rapidly. Secondly, different types of cells can be placed at predefined spots with higher precision as the cells may be applied sequentially or simultaneously. Thus, the method is particularly suitable for tissues which feature multiple cell types that are in close proximity.

[0055] Another application of the method in tissue engineering is in organ printing. This involves the layer-by-layer robotic bio fabrication of three-dimensional functional living microtissues and organ constructs as described in Mironov et al (Biomaterials 30: 2164-2174, 2009) and in Moon et al (Tissue Engineering 16(1): 157, 2010). Using the method of producing spheroids of the invention, tips may be seeded with a high density concentration of cells. Preferably, the spheroids are less than 300 micrometers in diameter so as to prevent necrosis. The tip may then be used to dispense cells in a controlled manner so as to print three- dimensional tissue structures. This has the advantage that cell-cell contacts form immediately and start migrating and / or forming tissue after printing. Alternatively, the method of producing spheroids of the invention may be used to form spheroids at defined positions so as to print a three-dimensional tissue structure. Preferably, the spheroids are less than 300 micrometersin diameter so as to prevent necrosis. Also, to enhance nutrition delivery to the cells / spheroids, the tips may be aspirated multiple times, in order to enable perfusion of nutrient-rich medium or blood. It will be appreciated that the combination of known bioprinting techniques with the injection method of the invention may also be desirable. For example, the method of the invention may be used to place specific cells only where they are needed once a three- dimensional structure has been formed (e.g., by printing or seeding a scaffold).

[0056] In an aspect, the current disclosure also encompasses method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject, a spheroid cultured using the methods disclosed herein, or a cell, population of cells, tissue, organ or organoid derived from the spheroid. In an aspect, the spheroid may be derived from autologous, allogenic, xenogeneic, or syngeneic cell.

[0057] In particular cases, the subject being treated with methods and compositions of the disclosure also receives one or more other therapies. In specific cases, the one or more other therapies may or may not treat one or more symptoms of the medical condition rather than the underlying cause. Examples include one or more of surgery; rehabilitation, including physical therapy, occupational therapy, and / or speech therapy; dopaminergic treatment; antiinflammatory; analgesic; cholinesterase; antipsychotic, etc. The one or more other therapies may be provided to the subject before, during, and / or after the methods and compositions of the disclosure. The one or more other therapies and the methods and compositions of the disclosure may or may not be administered via the same route. The one or more other therapies and the methods and compositions of the disclosure may or may not be in the same composition.

[0058] The compositions of the disclosure (for example, a spheroid cultured using the methods disclosed herein, or a cell, population of cells, tissue, organ or organoid derived from the spheroid and optionally one or more excipients) may be administered by any route of administration. In some aspects, the composition(s) may be administered intravenously, intrathecally, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the subject, the subject's clinical history and response to the treatment, and the discretion of the attending physician.

[0059] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single delivery but may comprise continuous delivery over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0060] In some aspects, the composition is administered at a dose of between 100 cells and 10 million cells. In some aspects, the therapy is administered at a dose of at least, at most, or about 100-1 million, 100-100,000, 100-10,000, 100-1000, 1000-10 million, 1000-1 million, 1000-100,000, 1000-10,000, 10,000-10 million, 10,000-1 million, 10,000-100,000, 100,000-10 million, 100,000-1 million, or 1 million -10 million cells, or products therefrom, or any range derivable therein.

[0061] In some aspects, a single dose of the therapy is administered. In some aspects, multiple doses of the therapy are administered. In some aspects, the therapy is administered at a dose of between 100 cells-10 million cells, or products therefrom. In some aspects, the therapy is administered at a dose of at least, at most, 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, 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 quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain aspects, it is contemplated that doses in the range from 100 cells to 10 million cells can affect the protective capability of these agents. Furthermore, such doses can be administered at multiple times during a day, and / or on multiple days, weeks, or months.

[0063] Precise amounts of the therapeutic composition may also depend on the judgment of the practitioner and may be peculiar to each subject. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0064] It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0065] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1 , 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11 , or 12 week intervals, including all ranges there between.

[0066] Also disclosed is a kit of parts comprising a tip box, suitable media, and cells. The kit may also include instructions for making and / or using the spheroids. The kit of parts may further comprise a means for assessing the property of a cell selected from any of survival,growth, proliferation, differentiation, migration, morphology, signaling, metabolic activity, gene expression and cell-cell interaction.II. Spheroid

[0067] Cell spheroids are three-dimensional cellular aggregates that more closely mimic in vivo tissue architecture and cell-cell interactions compared to traditional two-dimensional cell cultures. In an aspect, the current disclosure also encompasses cell spheroids generated using the methods disclosed herein. Aspects of the current disclosure encompass a cell spheroid developed in a pipette tip, using the methods disclosed herein. The disclosed spheroids can be generated from one or more cell types, for example stem cells, for instance, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, progenitor cells; fibroblasts, for instance, placental fibroblasts, omental tissue derived fibroblasts, cord blood fibroblasts, fibroblasts derived from skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, or foreskin, cancer cells and cell lines for example 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; tissue specific cells for example hepatocytes, cardiomyocytes, pancreatic islet cells, mesenchymal stromal cells (MSCs), and various other adherent cell types can be used to form spheroids. In an aspect, the spheroid may comprise a single celltype. In an aspect, the spheroid may comprise at least 2, 3, 4, 5, or more different cell types. In an aspect, the cells may be genetically manipulated prior to or during the development of spheroids. In an aspect, the cells may be genetically engineered to impact their immune modulation potential. In an aspect, the cells may be genetically engineered to target one or more genes or gene products to attenuate their immune modulation potential. Genetic modification methods or compositions may be used to introduce nucleic acids into cells, edit genomic DNA or transcripts using gene editing, homologous and non-homologous recombination, TALENS, CRISPR, zinc finger nucleases or any combination thereof. These genetic modifications may introduce exogenous genes, delete, or mutate endogenous genes, introduce selection or identification markers or any combination thereof. These methods are known in the art and may be used in isolation, or a combination of these methods may be used to obtain cells with desired properties.

[0068] In an aspect, the cells are chemically, physically, or epigenetically activated with one or more of nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or any combination thereof, prior to or during the development of spheroids, or prior to administration. In an exemplary aspect, isolated cells may be cultured in the presence of other immune cells. Non-limiting examples include macrophages, microglia, dendritic cells, Treg cells etc.

[0069] Spheroids of the current disclosure typically range in size from 20 -1000 pm in diameter and can be composed of a single cell type or multiple cell types to create more complex spheroids. Thus, the spheroids may range in size from about 25 - 50 pm, about 50 - 75 pm, about 75 - 100 pm, about 100 - 125 pm, about 125 - 150 pm, about 150 - 175 pm, about 175 - 200 pm, about 200 - 250 pm, about 250 - 300 pm, about 300 - 400 pm, about 400 - 500 pm in size. Preferably, the spheroids are less than 300 micrometers in diameter so as to prevent necrosis. The spheroids may be on any shape, for example spherical, oval or irregular in size. The spheroid may comprise an irregular or layered architecture. Further, the layers may comprise a single cell type or at least 2, at least 3, at least 4 or more different cell types. These cell types may differ in survival, growth, proliferation, differentiation, migration, morphology, signaling, metabolic activity, gene expression and cell-cell interaction. The disclosed spheroids may comprise 10 - 100, 100 - 1 ,000, 1 x 103- 1 x 104, 1 x 104- 1 x 105, 1 x 105- 1 x 106, 1 x 106- 1 x 107, 1 x 107- 1 x 108, 1 x 108- 1 x 109, 1 x 109- 1 x 1010cells or more of the same cell-type or more than one cell type. When meant for clinical use, the spheroids may be derived from an autologous, allogenic, xenogeneic, or syngeneic cell. The spheroids formed by the methods of the invention may exhibit characteristics that substantially mimic those of the tissue of origin. Thus, at least one of the antigen profile, genetic profile, tumor biology, tumor architecture, cell proliferation rate(s), tumor microenvironment, therapeutic resistance, cell composition, gas concentrations, cytokine expression, growth factor expression and cell adhesion profile of the spheroid may be substantially identical to that of the tissue of origin. Accordingly, the spheroid may exhibit a substantially similar or identical behavior to that of natural cell systems, for example with respect to organization, growth, viability, cell survival, cell death, metabolic and mitochondrial status, oxidative stress, and radiation response as well as drug response. The spheroids produced by the methods of the invention may exhibit a substantially similar or identical behavior to that of natural cell systems, making them particularly useful for 3D cell assays. In an aspect, the disclosed spheroids may be assessed for properties such as survival, growth, proliferation, differentiation, migration, morphology, signaling, metabolic activity, gene expression and cell-cell interaction using known methods in the art. Assessing one or more properties of a cell may be carried out using any suitable method known in the art, either from cell spheroids or from spheroids fixed by snap-freezing or chemical fixation techniques. For example, any cell survival, growth, proliferation, differentiation, migration, and morphology may be assessed by microscopy or image analysis. Properties may be detected using appropriate markers. For example, expression of detectably-labelled proteins, reporters and / or single-step labelling of cell components and markers can enable cell architecture, multicellular organization, and other readouts to be directly visualized, for example by fluorescence microscopy. Gene expression may be assessed by functional genomic {e.g.,microarray) techniques, and so on. Any of immunofluorescence, Hoechst staining or Annexin- V assays may be used. It is appreciated that the skilled person may select the appropriate technique to assess a given property.

[0070] In an aspect, the current disclosure also encompasses pharmaceutical compositions comprising the spheroids disclosed herein. In an aspect, the pharmaceutical composition may comprise one or more cells, tissues, organs, and organoids derived from the spheroids disclosed herein. The pharmaceutical composition may further comprise one of more carriers or excipients. The composition can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0071] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0072] The pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0073] Sterile injectable solutions are prepared by incorporating the active composition (for example composition comprising spheroids as disclosed herein, or cells, population of cells, tissue, organ or organoids derived from the spheroid) in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methodsof preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0074] III. Definitions

[0075] In keeping with long-standing patent law convention, the words “a” and “an” when used in the present specification in concert with the word comprising, including the claims, denote “one or more.” Some aspects of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0076] The phrase “and / or” means “and” or “or.” To illustrate, 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” operates as an inclusive or.

[0077] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular aspects, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1 %. With respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Unless otherwise stated, the term 'about' means within an acceptable error range for the particular value.

[0078] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ means including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0079] Reference throughout this specification to “one aspect,” “an aspect,” “a particular aspect,” “a related aspect,” “a certain aspect,” “an additional aspect,” or “a further aspect” or combinations thereof means that a particular feature, structure, or characteristic described in connection with the aspect is included in at least one aspect of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more aspects.

[0080] The term "administered" or "administering", as used herein, refers to any method of providing a composition to a subject such that the composition has its intended effect on the subject. For example, one method of administering is by an indirect mechanism using a medical device such as, but not limited to a catheter, applicator gun, syringe etc.

[0081] As used herein, “allogeneic” refers to tissues or cells from another body that in a natural setting are immunologically incompatible or capable of being immunologically incompatible, although from one or more subjects of the same species.

[0082] As used herein, “autologous” refers to tissues or cells that are derived or transferred from the same subject's body (i.e., autologous blood donation; an autologous bone marrow transplant).

[0083] As used herein, “agent” refers to a polypeptide, a peptide, a nucleic acid, a small molecule, natural product, cytokines, chemokines, transcription factors, epigenetics factors, growth factors, or hormones.

[0084] As used herein, “xenogeneic” refers to tissues or cells from a species different from the patient.

[0085] “Cell culture" is an artificial in vitro system containing viable cells, whether quiescent, senescent or (actively) dividing. In a cell culture, cells are grown and maintained at an appropriate temperature, typically a temperature of 37°C and under an atmosphere typically containing oxygen and CO2. Culture conditions may vary widely for each cell type though, and variation of conditions for a particular cell type can result in different phenotypes being expressed. The most commonly varied factor in culture systems is the growth medium. Growth media can vary in concentration of nutrients, growth factors, and the presence of other components. The growth factors used to supplement media are often derived from animal blood, such as calf serum.

[0086] The term "subject", or “individual” as used herein, refers to a human or animal that may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The subject may be receiving one or more medical compositions via the internet. A subject may comprise any age of a human or non-human animal and therefore includes both adults and juveniles (i.e., children) and infants. It is not intended that the term "subject" connotes a need for medical treatment, therefore, a subject may voluntarily or involuntarily bepart of experimentation whether clinical or in support of basic science studies. The term “subject” or “subject” may be used interchangeably and refers to any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic nonhuman animals.

[0087] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0088] The terms "reduce," "inhibit," "diminish," "suppress," "decrease," "prevent" and grammatical equivalents (including "lower," "smaller," etc.) when in reference to the expression of any symptom in an untreated subject relative to a treated subject, mean that the quantity and / or magnitude of the symptoms in the treated subject is lower than in the untreated subject by any amount that is recognized as clinically relevant by any medically trained personnel. In one aspect, the quantity and / or magnitude of the symptoms in the treated subject is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and / or at least 90% lower than the quantity and / or magnitude of the symptoms in the untreated subject.

[0089] “Treatment,” “treat,” or “treating” means a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms. The treatment can be any reduction from pre-treatment levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition. Therefore, in the disclosed methods, treatment” can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or the disease progression, including reduction in the severity of at least one symptom of the disease. For example, a disclosed method for reducing the immunogenicity of cells is considered to be a treatment if there is a detectable reduction in the immunogenicity of cells when compared to pre-treatment levels in the same subject or control subjects. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. It is understood and hereincontemplated that “treatment” does not necessarily refer to a cure of the disease or condition, but an improvement in the outlook of a disease or condition. In specific aspects, treatment refers to the lessening in severity or extent of at least one symptom and may alternatively or in addition refer to a delay in the onset of at least one symptom.EXAMPLES

[0090] The following examples are included to demonstrate particular aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the subject matter of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0091] The current disclosure encompasses a method of growing spheroids in pipette tips, suitable for automation, high throughput, and scale-out production. FIG. 1 and FIG. 2 provide a schematic of exemplary aspects of the disclosed methods. While FIG. 1 top panel provides a static single cell type spheroid production method, the bottom panel elaborates methods for growing complex spheroids. Additionally, the same pipette tip can be used for growing multiple spheroids by introducing air gaps during aspiration as shown in FIG. 2. The examples further describe some representative experiments to validate the methods provided herein.Example 1 : Obtaining Spheroids from Human Fibroblasts

[0092] Skin fibroblast cells or fibroblast cells from other organs were isolated from humans. Cells were grown in typical cell culture environment (37 degrees Celsius, 5% carbon dioxide, 90% humidity) to 80% confluency for experiments. The practical steps in each case are as described:1. Remove the medium and wash the cells twice with 1X PBS.2. Dissociate cells with 0.25% trypsin for 3 minutes.3. Use stem cell medium (low glucose DMEM, 10% human serum, 1% non-essential amino acids, 5% L-glutamine) to collect cells.4. Adjust the cell concentration to 1 x 106cells / mL and use a 200 pl pipette tip and aspirate 30 pl of the cell suspension inside the pipette tip to prepare stem cell spheroids.5. As an option, a volume of anywhere from 1-50 ul airgap can be aspirated to form a cushion between the tip of the pipette tip and the liquid containing the cells.6. Place the pipette tip in a pipette tip box with suitable media, and gently eject the tips into the pipette tip box.7. Place pipette tip box in an incubator set to specific culturing conditions for 3 days.8. Load pipette tip in the pipettor and dispense spheroids into the tube or single well plate post three days in the incubator.9. Collect stem cell spheroid from the tube or plate.10. Centrifuge at 800 rpm for 3 minutes and remove the supernatant.11. Resuspend cells in stem cell medium (low glucose DMEM, 10% human serum, 1% non-essential amino acids, 5% L-glutamine).

[0093] The experimental set-up is shown in FIGs. 3A and 3B. The cells were tested, applied, or returned to the typical culture environment for continued growth depending on the requirements of the individual experiments.

[0094] With this method, human skin fibroblasts were shown to obtain 100% survival rate and have normal cell morphology, cell proliferation, and migration ability. FIG. 4 provides representative microscopy images of the spheroids showing the morphology of human spheroids after 4 days of culture.Example 2: Obtaining Complex Spheroids from Human Skin Fibroblasts

[0095] Next, it was tested to see if the method as provided in Example 1 , can be used to develop complex spheroids with more than one cell type. The skin fibroblasts were used for validation experiments. Cells were grown in a typical cell culture environment (37 degrees Celsius, 5% carbon dioxide, 90% humidity) to 80% confluency for experiments. The practical steps are as described:1. Remove the medium and wash the cells twice with 1X PBS.2. Dissociate cells with 0.25% trypsin for 3 minutes.3. Use stem cell medium (low glucose DMEM, 10% human serum, 1% non-essential amino acids, 5% L-glutamine) to collect cells.4. Adjust the cell concentration to 1 x 106cells / mL and use a 200 l pipette tip and aspirate 30 pl of the cell suspension inside the pipette tip to prepare stem cell spheroids.5. As an option, a volume of anywhere from 1-50 ul airgap can be aspirated to form a cushion between the tip of the pipette tip and the liquid containing the cells.6. Place the pipette tip in a pipette tip box with suitable media, and gently eject the tips into the pipette tip box.7. Place pipette tip box in an incubator set to specific culturing conditions for 48 hours.8. Prepare the Dil-labeled Human fibroblast suspension and adjust the cell concentration to 1 x106cells / mL.9. Reload the pipette tip containing the now formed spheroids with the first type of cell, and aspirate 15 pl Dil-labeled human fibroblasts from the cell suspension.10. If there was an air-gap, then dispense the air-gap first prior to aspirating the liquid containing the second type of cell.11. As an option, a volume of anywhere from 1-50 ul airgap can be aspirated to form a cushion between the tip of the pipette tip and the liquid containing the cells.12. Place the pipette back in the pipette tip box, and gently eject the tip into the pipette tip box.13. Place the tip box in typical cell culture environment for 48 hours to form the shell structure of the cell spheroid.14. Dispense the spheroids into the tube or single well plate post three days in the incubator.15. Collect the spheroid into a tube or a plate, then centrifuge at 1000 rpm for 3 minutes and remove the supernatant.16. Resuspend cells in stem cell medium (low glucose DMEM, 10% human serum, 1% non-essential amino acids, 5% L-glutamine).

[0096] The cells were tested, applied, or returned to the everyday culture environment to continue culturing depending on the experimental needs. This method allowed human dermalfibroblasts to obtain a 97% survival rate and have normal cell morphology, cell proliferation, and migration ability.

Claims

CLAIMS1. An in vitro method of producing a spheroid from one or more cells in a pipette tip, comprising introducing the one or more cells into the pipette tip, wherein the pipette tip optionally comprises a cell culture medium.

2. The method of claim 1 , wherein the one or more cells comprise one or more cell types.

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

4. The method of claim 1, wherein the one or more cells comprise a fibroblast.

5. The method of claim 1, wherein the cell culture medium comprises a viscosity enhancing agent.

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

7. The method of claim 5 or claim 6 wherein the viscosity enhancing agent is methylcellulose.

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

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

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

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

12. The method of claim 1-11, wherein the one or more cells are cultured at a temperature range of 25-40 degrees centigrade, and an atmosphere comprising 2 to 8% CO2, and 90-98% humidity.

13. The method of claim 9, wherein the tip box comprises one or more additional pipette tips comprising one or more cells, or spheroid.

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

15. The method of claim 14, wherein the spheroid is 50-500 uM in size.

16. A method of screening one or more spheroid(s), comprising contacting the one or more spheroid(s) to an agent, wherein the one or more spheroid(s) are cultured in a pipette tip.

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

18. The method of claim 16, wherein the spheroid are contacted with the agent within the pipette tip or in a separate apparatus.

19. The method of claim 18, wherein the separate apparatus is a multi-well plate.

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

21. A method of treating a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising one or more spheroids, or cells, tissue, organoid or organ derived from the one or more spheroid, wherein the one or more spheroid is cultured in a pipette tip.

22. A kit comprising a tip box comprising one or more tips, for growing one or more spheroids from a cell or a cell population, and instructions for using the kit.

Images