Spherical bodies comprising bio-related materials and related methods

Abstract

The present invention provides a method for manufacturing a spheroid, comprising a step of providing a suspension having one or more biologically related materials dispersed within a biocompatible medium. An amount of hydrophilic material is deposited on a defined area of the super-hydrophobic surface, and droplets of the suspension are bio-printed onto the hydrophilic material located on the super-hydrophobic surface, thereby forming spheroids.

Description

[0001] This application is a divisional application of patent application No. 201780050308.9 (international application No. PCT / US2017 / 039483), filed on June 27, 2017, entitled "Spherical bodies containing biologically related materials and related methods".

[0002] Related applications

[0003] This application claims priority based on U.S. Provisional Application No. 62 / 354,929, filed June 27, 2016, and U.S. Provisional Application No. 62 / 422,694, filed November 16, 2016, the entire disclosure of which is incorporated herein by reference. Technical Field

[0004] This disclosure generally relates to spheres comprising bio-related materials and related methods. In particular, certain embodiments of this disclosure relate to spheres comprising bio-related materials and methods for manufacturing such spheres, wherein a suspension comprising one or more bio-related materials is bioprinted onto a superhydrophobic surface. Background Technology

[0005] The use of three-dimensional environments for cell culture provides more physiologically relevant systems for in vitro modeling of cell behavior and for the formation of constructs for subsequent implantation. In vivo, tissues are composed of multiple cell species, and cells are organized in specific spatial arrangements, providing cell orientation to organ-specific geometries. In vitro cell function studies, initially utilizing cells grown on tissue culture surfaces such as glass and plastic, have now shifted to three-dimensional (3D) culture of cells, typically embedded in collagen gels. Coordinated, researchers have evaluated the ability of two-dimensional and three-dimensional cell cultures to undergo spontaneous formation of spheroids during culture. Organoid cultures of epithelial and endothelial cells have been established in this manner. In those procedures, embryonic stem cells are cultured as hanging droplets and allowed to form embryonic bodies (EBs). Spheroid culture strategies have evolved to include endothelial cells representing the vascular system, common cellular components of all complex tissues. Recently, composite three-dimensional tissue constructs containing parenchymal cells and vascular cells have been implanted into experimental models. Each of these studies demonstrates that functional tissue organoids can be constructed in vitro, implanted into tissues, and that there is evidence of vascular integration between implantation and recipient circulation, as well as evidence that organoids can provide restoration of tissue function.

[0006] To facilitate vascular integration in three-dimensional tissue constructs, several studies have utilized adipose-derived stromal vascular fractions (SVFs). Adipose-derived SVFs are heterogeneous populations of cells with diverse regenerative properties for in vivo and clinical applications. The current primary delivery modality for SVFs, as well as many other stem cell and regenerative cell populations, is direct injection of cells suspended in solution. However, a major drawback of direct SVF injection is the observed lack of cell retention at the implantation site. Therefore, it has been proposed to assemble cells into aggregates through direct encapsulation or culture-dependent self-aggregation to improve cell retention at injection. In this regard, SVFs have been pre-embedded in scaffolds or encapsulated in various matrix hydrogels for in vitro applications, and these encapsulated aggregates exhibit improved SVF localization and retention. Previous studies on SVF cell aggregation have also included encapsulation in collagen gels using alginate to form spheroids and other 3D cultures of SVFs.

[0007] Currently, spheroid formation primarily falls under the nominal categories of cell spheroids, surface-inoculated spheroids, and encapsulated spheroids. However, these cell spheroids are still typically produced via hanging drop methods or, conversely, rely on cell self-aggregation mechanisms due to the lack of other adhesion sites. Cells can spontaneously aggregate using adhesion sites to support migration toward a central clump or cell aggregate. This manufacturing method requires several days to produce spheroids and is susceptible to inconsistencies in cell proliferation rates. Surface-inoculated spheroids are pre-formulated with selected biopolymers and then incubated with selected cells to allow cell migration-driven inoculation. This manufacturing strategy has very broad applications, provided that selected biomaterials can be pre-formulated. A drawback of pre-formed spheroids is that cell inoculation is based on migration / adhesion, thus limiting it primarily to the spheroid surface rather than the entire volume of the spheroid, which restricts its load-bearing capacity. A third strategy for encapsulated spheroids revolves around the idea of ​​mixing cells into a suspension and then using that suspension as a scaffold / hydrogel of choice. Encapsulated spheroids have the advantage of being able to transport cells using the entire volume of the spheroid and being produced in less than a day. However, this strategy is very biomaterial-specific, and in the case of collagen, it is often necessary to use carrier materials with altered mechanical properties (such as alginate) and often to substitute (by proxy) some of the biological properties that make the original biological material attractive.

[0008] In short, the formation of three-dimensional cellular and tissue constructs has not yet been fully realized. Therefore, any improvements in the fabrication of such composite biological structures are highly anticipated and beneficial. Summary of the Invention

[0009] This disclosure satisfies some or all of the above-mentioned needs, which will become apparent to those skilled in the art upon studying the information provided herein.

[0010] This summary describes several embodiments of the subject matter of this disclosure and, in most cases, lists variations and alternatives to these embodiments. This summary is merely an example of numerous different embodiments. Mentions of more than one representative feature of a given embodiment are also illustrative. Such embodiments may generally exist with or without the mentioned features; similarly, these features can be applied to other embodiments of the subject matter of this disclosure, whether or not they are listed in this summary. To avoid excessive repetition, this summary does not list or indicate all possible combinations of such features.

[0011] In some embodiments of the subject matter of this disclosure, a method for manufacturing spheres comprising bio-related materials is provided. In one embodiment, a method for manufacturing spheres comprising one or more bio-related materials is provided, wherein a suspension is first formed or provided, wherein the suspension comprises one or more bio-related materials dispersed in a biocompatible medium. Then, a certain amount of hydrophilic material is deposited in a defined region and / or in a defined amount onto a superhydrophobic surface of a suitable substrate. In some embodiments, the hydrophilic material deposited on the superhydrophobic surface comprises a polyoxyethylene-polyoxypropylene block copolymer. In some embodiments, the water contact angle of the superhydrophobic surface utilized according to the subject matter of this disclosure is greater than about 150°, for example, in some embodiments, the water contact angle is about 150° to about 170°.

[0012] Regardless of the specific hydrophilic material and / or water contact angle of the superhydrophobic surface used in the example methods of this disclosure, once the suspension and substrate are prepared, droplets of the suspension are directly bioprinted (e.g., directly written) onto the hydrophilic material located on the superhydrophobic surface, thereby creating spheroids containing bio-related materials. The suspension is then bioprinted, and the resulting spheroids can then be incubated at physiological temperatures for a period of time while maintaining their spherical shape. In some embodiments, the spheroids can then be further cultured in cell culture media, if desired.

[0013] In some embodiments of the methods disclosed herein, the example spheroids contain one or more bio-related materials including magnetic beads, stromal vascular fraction cells, stem cells, one or more related cells, cell populations or tissues, or combinations thereof. For example, in some embodiments, spheroids may be manufactured comprising a combination of stromal vascular fraction cells and one or more related cells (such as pancreatic islet cells). In some embodiments, one or more bio-related materials may thereby comprise stromal vascular fraction cells. In some embodiments, one or more bio-related materials comprise one or more islet cells.

[0014] Regarding the biocompatible medium used to form the suspension used in the methods of this disclosure, in some embodiments, the biocompatible medium comprises a hydrogel. In some embodiments, the hydrogel comprises a material selected from agarose, alginate, collagen, polyoxyethylene-polyoxypropylene block copolymer, silicone, polysaccharides, polyethylene glycol, and polyurethane. In some embodiments, the hydrogel comprises type I collagen.

[0015] Other features and advantages of the subject matter of this disclosure will become apparent to those skilled in the art after a study of the description, drawings, and non-limiting embodiments herein. Attached Figure Description

[0016] Figures 1A-1D Includes: Images of type I collagen spheroids loaded with stromal vascular fraction (SVF) immediately after bioprinting on parental surfaces ( Figures 1A-1C ), and a schematic diagram showing the cross-section of the parental surfaces and type I collagen spheroids ( Figure 1D ).

[0017] Figure 2A-2H These include: images showing the morphology of spherical bodies as observed visually using phase contrast microscopy. Figure 2A and Figure 2E ); shows targeting live cells ( Figure 2B and Figure 2F ), dead cells ( Figure 2C and Figure 2G Images showing the morphology of encapsulated SVFs as observed visually using epifluorescence at 4x magnification; and images showing the cell distribution as observed visually using calcein AM, ethidium homodimer-1, and Hoechst 33258 bis(benzyl)imide, respectively. Figure 2D and Figure 2H The spheroids were cultured in a rotating culture for 2 days. Figures 2A-2D ) and 6 days ( Figure 2E-2H ). Figure 2H The micrograph shown is composed of and Figures 2E-2GThe spheres shown are obtained from different spheres.

[0018] Figure 3 It is a graph showing cell viability in the spheroids on days 0, 2, 6, 9, and 13.

[0019] Figures 4A-4D Including displaying the second day of rotation culture ( Figure 4A Day 6 Figure 4B Day 9 Figure 4C ) and the 13th day ( Figure 4D A 4x magnified phase-contrast photomicrograph of SVF-loaded type I collagen spheroids.

[0020] Figure 5 This is a graph showing a significant reduction in the mean diameter of SVF-loaded collagen spheroids after 6–13 days of rotational culture (n=12). Statistical significance is indicated at P<0.05. , The following measurements were taken.

[0021] Figures 6A-6B Includes a 10x magnification confocal micrograph showing SVF-loaded type I collagen spheroids after 14 days of rotational culture and staining with endothelial-specific lectin Griffoniasimplicifolia (GS-1 (green)), α-smooth muscle cell actin (α-SMA (red)), and nuclear stain (RedDot (blue)). Tubular structures are highlighted with arrows. Figure 6B Display and Figure 6A The top view shown represents a perspective shift of approximately 20°.

[0022] Figures 7A-7B Images showcasing bioprinted human fat SVF / islet spheres illustrate the feasibility of forming prevascularized islet implants using a 3D bioprinting system and superhydrophobic surfaces. Figure 7A This is a phase contrast image illustrating SVF / islet spheroids immediately after printing and polymerization. Figure 7B This image illustrates the spheroids of SVF / islet spheroids on day 7. The spheroids undergo shrinkage during culture. Scale bar = 500 micrometers (μm).

[0023] Figure 8 This is a graph showing the ability of pancreatic islets / SVF globules to correct hyperglycemia in diabetic animal models.

[0024] Figures 9A-9G Including phase contrast and confocal microscopy images of SVF spheres. Figure 9A This is a phase-contrast image after 11 days of culture, illustrating the distribution of SVF cells within the spheroids. Scale bar = 100 micrometers. Figure 9B This is a phase contrast image after 12 days of culture, illustrating the onset of shrinkage. Scale bar = 100 micrometers. Figure 9C This is a phase contrast image after 8 days of cultivation. Scale bar = 50 micrometers. Figure 9D These are phase contrast images after 11 days of culture, illustrating more extensive branching compared to the spheroids on day 8. Scale bar = 50 micrometers. Figure 9E This is a confocal micrograph of type I collagen spheroids loaded with SVF after 14 days of staining with endothelial-specific lectin Ghana seed (GS-1 (green)), α-smooth muscle cell actin (α-SMA (red)), and nuclear stain (RedDot (blue)). Scale bar = 100 μm. Figure 9F and 9G It is co-labeled with GS-1 and α-SMA. Figure 6B Two higher magnification perspective views of the vascular-like structure, showing the branching structure divided by arrows. Scale bar = 50 micrometers. Detailed Implementation

[0025] Details of more than one embodiment of the subject matter of this disclosure are set forth in this text. Variations and other embodiments of the embodiments described herein will be apparent to those skilled in the art upon review of the information provided herein. The information provided herein, and in particular the details of the exemplary embodiments described, are intended primarily for clarity and should not be construed as unnecessarily limiting. In the event of conflict, the description (including definitions) in this text shall prevail.

[0026] While the terminology used herein is believed to be well understood by those skilled in the art, certain definitions are set forth in order to facilitate the interpretation of the subject matter of this disclosure.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] Unless otherwise stated, all patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other public materials mentioned in this document are incorporated herein by reference in their entirety.

[0029] In cases involving URLs or other such identifiers or addresses, it is understood that such identifiers can change and specific information can be exchanged on the internet, but equivalent information can be found by searching the internet. The cited literature demonstrates the availability and public dissemination of such information.

[0030] Although any methods, apparatuses and materials similar to or equivalent to those described herein may be used in the practice or experimentation of the subject matter of this disclosure, representative methods, apparatuses and materials are described herein.

[0031] This application may refer to the components / parts of the invention as well as other ingredients or elements / elements described herein as either "comprising / including / containing" (open-ended) or "consisting substantially of...". As used herein, "comprising / including / containing" is open-ended and refers to the stated elements / elements, or their structural or functional equivalents, plus any other elements / elements not described. Unless the context otherwise requires, the terms "having" and "possessing" should also be interpreted as open-ended.

[0032] According to long-established patent law practice, the terms "a / an" and "the" when used in this application (including the claims) mean "more than one." Therefore, for example, references to "a cell" include multiple such cells, and so on.

[0033] Unless otherwise stated, all figures used in this specification and claims to indicate the amount of an ingredient, properties such as reaction conditions, etc., should be understood to be modified by the term "about" in all cases. Therefore, unless stated to the contrary, the numerical parameters listed in this specification and claims are approximate values ​​that may vary depending on the desired properties sought to be obtained from the subject matter of this disclosure.

[0034] When referring to values ​​or quantities of mass, weight, time, volume, concentration, or percentage, the term “about” as used herein means including variations from the specified amount by ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments, because such variations are suitable for implementing the methods of this disclosure.

[0035] The scope used herein may be expressed as starting “about” of a particular value and / or as ending “about” of another particular value. It should also be understood that many values ​​are disclosed herein, and each value is also disclosed herein as “about” of that particular value, in addition to being the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It should also be understood that units between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0036] As used herein, “optional” or “optionally” means that the event or situation described below occurs or does not occur, and the description includes examples of the event or situation occurring and examples of it not occurring. For example, an optional variant section means that the section is a variant or non-variant.

[0037] This disclosure is based, at least in part, on the finding that three-dimensional (3D) bioprinting techniques can be combined with substrates having superhydrophobic surfaces to support the automated production of spheroids containing bio-related materials, including spheroids loaded with stromal vascular fractions (SVFs) in type I collagen gels. A limiting factor in the use of bulk collagen gels is the handling before and during implantation. Type I collagen polymerization from liquid to crosslinked hydrogels typically occurs within shape maintainers such as porous plates. These polymerized constructs are then typically removed and manually manipulated using sterile equipment. However, this manipulation, and any potential subsequent gel damage, is difficult to control. Furthermore, while collagen gels possess a degree of elasticity, in vivo applications often require irregular shapes, thus necessitating the cutting of bulk gels into slices or significant deformation to fit the desired shape. Without being bound by any particular mechanistic theory, it is believed that combining collagen gels into small spheroids can alleviate these problems compared to injecting cell suspensions alone, and further, it is believed that such collagen spheroids offer the advantage of simplified handling of fluid suspensions and allow for precise spatial and dosing control, thus adapting well to the requirements of in vivo applications.

[0038] In some embodiments of the subject matter of this disclosure, a method for manufacturing spherical bodies is thus provided. In some embodiments, a method for manufacturing spherical bodies is provided, wherein a hydrophilic material is first placed on a defined region on a substrate surface, wherein the substrate surface is superhydrophobic. Then, one or more bio-related materials are suspended in a biocompatible medium to form a suspension, and droplets of the suspension are bioprinted onto the hydrophilic material. For example, in an exemplary embodiment of the method for manufacturing spherical bodies of the subject matter of this disclosure (using direct writing as the form of bioprinting), the BioArchitecture Tool (BAT; see, for example, U.S. Patent No. 7,857,756; see also Smith et al.) is utilized. Tissue Eng2004;10:1566-1576, both of which are incorporated herein by reference), using a computer-controlled stage that allows not only independent X-axis and Y-axis translation, but also Z-axis movement of more than one translation printhead / dispensing system. In this regard, bioprinting parameters can first be scripted as printing instructions and then uploaded to the printing tool, so that the printing tool (i.e., BAT) can be used to manufacture precise structures containing suspensions. In some embodiments, by using such a printing tool, the size of the droplets printed by this system can be controlled by controlling the size of the pen used to print the droplets and by controlling the pressure used to expel the droplets from the pen. In some embodiments, droplets or resulting spheres having a diameter of about 0.2 mm to about 5 mm can be manufactured using pens of about 15 to about 25 gauge and pressures of about 2 psi to about 7 psi. In some embodiments, the diameter of the droplets or resulting spheres is about 1 mm to about 5 mm, or about 2 mm to about 4 mm, or about 3 mm to about 4 mm. In some embodiments, the size of the droplets or spheres is controlled by adjusting one or more parameters selected from the following: the viscosity of the suspension, the size of the delivery pen tip, the pressure used to expel the suspension from the delivery pen, and the amount of time for which pressure is applied to the suspension in the delivery pen. Those skilled in the art can readily adjust these parameters to produce droplets or spheres with the desired size.

[0039] As an exemplary embodiment of a method for manufacturing spherical bodies comprising one or more bio-related materials, the spherical bodies are manufactured by the following method: First, a suspension in the form of a cell suspension (e.g., a cell suspension containing a population of human stromal vascular cells mixed with type I collagen) is placed in a delivery pen comprising a hollow needle or tubular structure. Then, the bio-suspension is controlled to be extruded from the delivery pen by increasing the pressure in the delivery pen to a specific value, thereby forming droplets. The delivery pen is then lowered at a predetermined rate (e.g., 5 mm / s) toward a hydrophilic material placed on a superhydrophobic surface of a substrate. Upon contact with the hydrophilic material, the suspension droplets are subsequently attracted to the hydrophilic material and released from the pen, thereby forming a spherical body on top of a hydrophilic spot on the superhydrophobic surface. In some embodiments, after bioprinting the suspension, the resulting spherical bodies can then be incubated at a physiological temperature (e.g., 37°C) for a period of time, such as a period sufficient for polymerization of the bio-medium used. In some embodiments, the spherical bodies can then be further cultured in a cell culture medium, if necessary.

[0040] As used herein, the term "suspension" refers to a composition containing bio-related materials, including magnetic particles, cells, tissues, proteins, etc., dispersed within a biocompatible medium. Suitable biocompatible media used in this disclosure can generally be formed from any biocompatible material that is a gel, semi-solid, or liquid, such as a low-viscosity liquid, at room temperature (e.g., 25 °C), and can be used as a three-dimensional substrate for cells, tissues, proteins, and other biomaterials of interest. Exemplary materials that can be used to form biocompatible media of this disclosure include, but are not limited to, polymers and hydrogels containing collagen, fibroin, chitosan, MATRIGEL™ (BD Biosciences, San Jose, CA), polyethylene glycol, dextran (including chemically crosslinkable or photocrosslinkable dextran, etc.), and electrospun bio, synthetic, or biosynthetic blends. In some embodiments, the biocompatible medium contains materials that support endothelialization, see, for example, U.S. Patent Nos. 5,744,515 and 7,220,276, both of which are incorporated herein by reference. In some implementations, the biocompatible medium contains a hydrogel.

[0041] As used herein, the term "hydrogel" refers to a two- or multi-component gel containing a three-dimensional network of polymer chains, wherein water acts as a dispersion medium and fills the spaces between the polymer chains. The hydrogels used in this disclosure are typically selected for a specific application (e.g., a particular spheroid) based on the intended use of the structure, taking into account the printing parameters to be used and the desired effects. The selected hydrogel will exhibit the behavior and activity of biomaterials (e.g., cells) incorporated into a biological suspension to be placed within the structure. Exemplary hydrogels of this disclosure may contain polymeric materials, including but not limited to: alginate, collagen (including type I and type VI collagen), fibrinogen, elastin, keratin, fibronectin, proteoglycans, glycoproteins, polylactide, polyethylene glycol, polycaprolactone, polyglycolide, polydioxanone, polyacrylate, polyurethane, polysulfone, peptide sequences, proteins and their derivatives, oligopeptides, gelatin, elastin, fibroin, laminin, polymethacrylate, polyacetate, polyester, polyamide, polycarbonate, polyanhydride, polyamino acid carbohydrates, polysaccharides and modified polysaccharides, and their derivatives and copolymers; and inorganic materials, such as glass (e.g., bioactive glass), ceramics, silica, alumina, calcite, hydroxyapatite, calcium phosphate, bone, and combinations thereof. Additional information regarding materials that may incorporate the subject matter of this disclosure can be found, for example, in U.S. Patent Nos. 7,919,11, 6,991,652, and 6,969,480, all of which are incorporated herein by reference.

[0042] Further regarding the hydrogel used to manufacture spherical bodies, in some embodiments, the hydrogel comprises agarose, alginate, type I collagen, and polyoxyethylene-polyoxypropylene block copolymers (e.g., Pluronic). ® Materials in the form of F127 (BASF Corporation, Mount Olive, New Jersey), silicone, polysaccharides, polyethylene glycol, and polyurethane. In some embodiments, the hydrogel contains alginate. In some embodiments, the hydrogel contains type I collagen.

[0043] Turning now to exemplary suspensions and biorelated materials used in the subject matter of this disclosure, the term "biorelated material" as used herein is used to describe materials that can be contained in the biocompatible media described herein and subsequently interact with and / or influence biological systems. For example, in some embodiments, the biorelated material is magnetic beads (i.e., beads that are magnetic themselves or contain materials responsive to magnetic fields, such as iron particles), which can be combined with a hydrogel and then bioprinted together with the hydrogel to create spherical bodies of a defined size (which can be used for instrument calibration or for the isolation and purification of cells and tissues according to methods known to those skilled in the art). As another example, in other embodiments, the biorelated material includes more than one type of cell and tissue such that combining the cells or tissue with a suitable biocompatible media results in the formation of a cell or tissue suspension. In some embodiments, the biorelated material includes stromal vascular fraction cells, stem cells, more than one type of related cells, or combinations thereof. In some embodiments, the biorelated material includes stromal vascular fraction cells.

[0044] Regarding the stromal vascular fraction cells used in the methods of this disclosure, these stromal vascular fraction cells are those typically obtained by enzymatic digestion of a quantity of adipose tissue obtained from a subject, followed by centrifugation for a period of time to granulate the stromal vascular fraction of the adipose tissue. In this regard, the stromal vascular fraction comprises a variety of cell types, including endothelial cells, smooth muscle cells, pericytes, preadipocytes, mesenchymal stem cells (MSCs), endothelial progenitor cells, T cells, B cells, mast cells, and adipose tissue macrophages, as well as small blood vessels or microvascular fragments found in the stromal vascular fraction. Further description and guidance on adipose tissue separation for the manufacture of stromal vascular fractions can be found, for example, in U.S. Patent No. 4,820,626, the contents of which are incorporated herein by reference. In some embodiments, incomplete digestion of adipose tissue can also be used to generate adipose microvascular fragments; see, for example, U.S. Patent No. 7,029,838, which is also incorporated herein by reference.

[0045] Regarding stem cells that can be used in the methods of this invention, the term "stem cell" as used herein refers broadly to conventional stem cells, progenitor cells, preprogenitor cells, precursor cells, reserve cells, etc. Exemplary stem cells include, but are not limited to: embryonic stem cells, adult stem cells, pluripotent stem cells, neural stem cells, liver stem cells, muscle stem cells, muscle precursor stem cells, endothelial progenitor cells, bone marrow stem cells, chondrogenic stem cells, lymphoid stem cells, mesenchymal stem cells, hematopoietic stem cells, central nervous system stem cells, peripheral nervous system stem cells, etc. Descriptions of stem cells (including methods for isolating and culturing them) can be found in the following literature: Embryonic Stem Cells, Methods and Protocols, Turksen, ed., Humana Press, 2002; Weisman et al. Annu. Rev. Cell. Dev. Biol. , 17:387-403; Pittinger et al., Science , 284:143-47, 1999; Animal Cell Culture, Masters (ed.), Oxford University Press, 2000; Jackson et al., PNAS 96(25):14482-86, 1999; Zuk et al., Tissue Engineering , 7:211-228,2001; and U.S. Patent Nos. 5,559,022, 5,672,346, and 5,827,735. Descriptions of stromal cells (including methods for isolating them) can be found in the following literature, Prockop, Science , 276:71-74, 1997; Theise et al., Hepatology References: 31:235-40, 2000; Current Protocols in Cell Biology, eds. Bonifacino et al., John Wiley & Sons, 2000; and U.S. Patent No. 4,963,489. Those skilled in the art will understand that stem cells and / or stromal cells are typically selected for inclusion in tissue constructs when such cells are suitable for the intended use of a particular construct.

[0046] Finally, regarding the relevant cells that can be used in the methods of the present invention, the term "relevant cell" as used herein refers to cells suitable for incorporation into the spheroids of the subject matter of this disclosure, based on the intended use of the spheroid. In some embodiments, the term "relevant cell" may be used interchangeably with the term "regenerative cell" because the relevant cells described herein have the ability to form functional tissues after implantation. For example, relevant cells suitable for the repair, reconstruction, or reproliferation of a particular damaged tissue or organ typically include cells or cell populations commonly found in that tissue or organ. In this regard, exemplary relevant cells that can be incorporated into the spheroids of the subject matter of this disclosure include: neurons, cardiomyocytes, myocytes, vascular and / or gastrointestinal smooth muscle cells, chondrocytes, pancreatic acinar cells, Langerhans islets, pancreatic β cells, osteocytes, hepatocytes, Kupffer cells, fibroblasts, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, bile duct epithelial cells, etc. These types of cells can be isolated and used immediately or cultured using conventional techniques known in the art. Exemplary techniques can be found in the following literature: Freshney, Culture of Animal Cells: A Manual of Basic Techniques, 4th Edition, Wiley Liss, John Wiley & Sons, 2000; Basic Cell Culture: A Practical Approach, edited by Davis, Oxford University Press, 2002; Animal Cell Culture: A Practical Approach, edited by Masters, 2000; and U.S. Patents 5,516,681 and 5,559,022. In some embodiments, the biologically related cells comprise pancreatic islet cells (e.g., β cells) or whole, intact islets.

[0047] Regardless of the specific type of bio-related material combined with the biocompatible medium of this disclosure, as described above, once the bio-related material is combined with the biocompatible medium, droplets of the resulting suspension are then bioprinted onto a hydrophilic material placed on a superhydrophobic surface. In this regard, the suspension typically gels and forms spheres with a more stable geometry when it reaches room temperature. However, in order to maintain the geometry of the droplets or spheres after extrusion but before polymerization or gelation, as described above, the method of this disclosure uses a substrate with a superhydrophobic surface.

[0048] As used herein, the term "superhydrophobic" refers to a substrate that exhibits minimal attraction to water. Superhydrophobic surfaces often exhibit the lotus effect, such as the effect that occurs when a water droplet comes into contact with, for example, a lotus or taro leaf. Other examples of naturally occurring superhydrophobic surfaces formed by water droplets can be found, for example, in the stunt beetle *Stenocara gracilipes* found in the Namib Desert. In this respect, such superhydrophobic substrates or surfaces typically have a water contact angle or angle greater than about 150° (wherein, when measured by liquid, the angle at which a liquid or vapor interface meets a solid surface). In some embodiments, the superhydrophobic surfaces used herein have a water contact angle greater than 150°. In some embodiments, the water contact angle of exemplary superhydrophobic surfaces is about 150° to about 170°. Numerous superhydrophobic surfaces having such water contact angles are known to those skilled in the art and can exist as a result of a particular substrate used or as a result of a coating applied to the substrate. For example, in some implementations, a superhydrophobic surface can be created by spraying a waterproof coating (e.g., NEVERWET™ (Rust Oleum, Mount Vernon, Illinois)) onto a suitable substrate. Other examples of superhydrophobic surface coatings include, but are not limited to: silica, manganese oxide polystyrene (MnO2 / PS), zinc oxide polystyrene (ZnO / PS), precipitated calcium carbonate, perfluorobutane sulfonic acid, carbon nanotube structures, paraffin wax, polytetrafluoroethylene, waxes, etc.

[0049] As described above, in some embodiments of the methods described herein, a certain amount of hydrophilic material (i.e., a material with increased affinity for water and typically a water contact angle of less than about 90°) is placed on a defined region of the hydrophobic surface. Of course, the amount of hydrophilic material and the region where it is placed can vary depending on the spheroids being prepared. However, in some embodiments, about 2 μl to about 5 μl of hydrophilic material is placed on the hydrophobic surface to ensure that the spheroids adhere to the superhydrophobic surface rather than remain attached to the printing pen. In some embodiments, block copolymers with amphiphilic block structures (such as Pluronic) can be used. ® (F127), because this copolymer is both hydrophilic and hydrophobic, thus enabling it to adhere to both hydrophobic surfaces and aqueous biocompatible media, such as collagen. Other hydrophilic materials that can be used in this invention include, but are not limited to, other copolymers such as P188, and other materials such as urethanes and silanes. In some embodiments, the hydrophilic material used to form the spheres provides reversible adhesion properties to allow for the removal of the spheres. This reversal can also be caused by changes in temperature or the dissolution of the hydrophilic substance in the aqueous phase of the spheres, among other reasons / factors.

[0050] In some embodiments of the subject matter of this disclosure, spheroids prepared according to the methods described herein are further provided. Without wishing to be bound by any particular theory or mechanism, such spheroids are considered advantageous for: an in vitro assay of angiogenesis and vasculogenesis to screen drugs; a device (spheroid) that can be implanted in a patient to provide new blood flow to ischemic tissue; and a device (spheroid) that can be constructed using adipose-derived stem cells and regenerative cells, and in combination with other parenchymal cells (including hepatocytes, muscle cells, adipocytes, pancreatic cells (including islets), brain cells, germ cells, kidney cells, etc.). Furthermore, the spheroids and methods of this disclosure are considered to allow the manufacture of devices that can be formed and immediately implanted without requiring tissue culture of the material and without the use of other additives (e.g., alginate) to support the formation of stable spheroids. Additionally, the spheroids described herein are considered to support in vitro angiogenesis.

[0051] Unless otherwise stated, the practice of this disclosure may employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are well known in the art. These techniques are fully described in the literature.Please see, for example, *Molecular Cloning: A Laboratory Manual* (1989), 2nd edition, edited by Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Patent No. 4,683,195; *DNA Cloning*, Volumes I and II, edited by Glover, 1985; *Oligonucleotide Synthesis*, edited by M.J. Gait, 1984; *Nucleic Acid Hybridization*, edited by D. Hames & S.J. Higgins, 1984; *Transcription and Translation*, edited by BD. Hames & S.J. Higgins, 1984; *Culture of Animal Cells*, by R.R. Freshney, Alan R. Liss, Inc., 1987; *Immobilized Cells and Enzymes*, IRL Press, 1986; *Perbal* (1984), A Practical Guide to Molecular Cloning; see also Methods in Enzymology (Academic Press, New York); Gene Transfer Vectors for Mammalian Cells, eds. JH Miller and MP Calos, Cold Spring Harbor Laboratory, 1987; Methods in Enzymology, Vols. 154 and 155, eds. Wu et al., Academic Press, New York; Immunochemical Methods in Cell and Molecular Biology (eds. Mayer and Walker, Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I-IV, eds. D.M. Weir and C.C. Blackwell, 1986.

[0052] This disclosure is illustrated by the following specific, not limiting, embodiments. These embodiments may include data compilations representing data collected at different times during the development and experimentation process related to this invention.

[0053] Example

[0054] Example 1 – Formation of cell-loaded spheroids of adipose matrix blood vessels using a 3D bioprinter and superhydrophobic surfaces

[0055] Materials and methods.

[0056] Fabrication of Superhydrophobic Surfaces. Superhydrophobic surfaces were formed on 48-well polystyrene plates (Corning Corporation, Corning, NY) and 35mm Petri dishes using a two-step aerosol application of NEVERWET™ (Rust Oleum, Mount Vernon, Illinois). The first step involved applying an adhesive to the surface as a basecoat and air-drying it at room temperature for at least 1 hour. Subsequently, a top sheet consisting of polydimethylsiloxane modified with hexamethyldisilazane was applied to form a superhydrophobic layer. The superhydrophobic layer thickness was measured to be 0.07 mm. The top sheet was then air-dried at room temperature for an additional 1 hour. NEVERWET™ reported a contact angle of 165°, exceeding 150°, for which the surface was considered superhydrophobic. The contact angles of water and unpolymerized collagen in solution were measured using side-view photographs and subsequent ImageJ contact angle measurements.

[0057] Hydrophilic sites were formed. A 3D bioprinter (Bio-Assembly Tool (BAT) 3D printer; nScrypt, Orlando, FL) was used to extrude Pluronic F-127 (Sigma, St. Louis, Missouri) to form hydrophilic sites on a superhydrophobic surface. For each hydrophilic site, BAT extruded 2 μL of the target volume of 3.8% (w / w) Pluronic F-127 in 1X phosphate-buffered saline (PBS). The BAT time-pressure extrusion system, requiring 2.5 PSI (100 ms exposure time through a 25G needle), was used to generate the appropriate extrusion force to distribute the target volume. These sites were then air-dried for 30 minutes prior to use.

[0058] Stroma vascular fraction (SVF) cells. SVF cells were isolated from the epididymal fat pad of mice using a previously disclosed enzyme-based method. All animal studies were conducted with approval from the IACUC (Institutional Animal Care and Use Committee) at the University of Louisville, Louisville, Kentucky. Briefly, fat samples were obtained from the epididymal fat pad of mice during aseptic surgical procedures, manually minced for 3 minutes, washed with PBS containing 0.1% bovine serum albumin (BSA), and resuspended in 2 mg / mL type IV collagenase (Worthington Biochemical, Freehold, NJ). The fat was digested at 37°C for 35 minutes using an enviro-genie (Scientific Industries, Bohemia, NY). SVF cells were separated from the fat cells by centrifugation (350 × g, 4 minutes), and the supernatant was discarded. The particles were washed twice with PBS, filtered through a 250 μm filter, and resuspended in endothelial cell medium for immediate use in spheroid bioprinting.

[0059] Fabrication of SVF-loaded collagen spheres. Freshly isolated SVF was suspended in unpolymerized rat tail collagen I mixed with 1X Dulbecco modified Eagle medium (DMEM) (Sigma, St. Louis, Missouri) and titrated to a final pH of 7.4 to form a mixture of 3 mg / mL collagen containing 1.6 × 10⁵ SVF cells / mL of the final solution. This mixture was maintained at 4°C until printing, and a cooling system on the bioprinter was used to maintain an equivalent temperature throughout the printing process to prevent gel polymerization. The SVF-collagen solution was transferred to a 3cc printing syringe (EFD, Nordson, Westlake, Ohio) and placed in a 3D bioprinter (nScrypt LLC, Orlando, Florida). Initial printing conditions were based on previously published data for continuous roller printing. Once the spheres are printed, they are incubated at 37°C in a tissue culture incubator (5% CO2) for 10 minutes to initiate collagen gel polymerization.

[0060] Spheroid Culture Method. After preparation, the spheroids were transferred to a spinneret (125-mL MagnaFlex microcarrier spinneret, Wheaton Industries, Millville, NJ) for cell suspension culture containing endothelial cell growth medium consisting of DMEM, 10% FBS, 5 mM HEPES buffer, 2 mM L-glutamine, heparin-containing endothelial growth supplement, penicillin (45 U / mL), and streptomycin (45 μg / mL). The spinneret was placed on a magnetic stirrer platform (MCS 104-L bio-stirrer, Techne LLC, Burlington, NJ) to provide continuous suspension of the spheroids, and the magnetic impeller speed was set to 40 rpm throughout the culture period (14 days). The spheroids required for analysis were removed via a 50 mL serum pipette. For the study of spherical shrinkage, individual spherical bodies were inoculated into the separation wells of a 96-well ultra-low adhesion plate (Corning Corporation, Corning, NY).

[0061] Spheroid size measurement. Spheroid size measurements were calculated from images of spheroids cultured in 96-well plates obtained using an optical microscope (CKX41, Olympus, Tokyo, Japan). Diameter measurements from these 4X images were obtained using ImageJ software.

[0062] Live / dead analysis. SVF viability was assessed using a live / dead fluorescent dye (Live / DeadViability / Toxicity Kit, Life Technologies LLC, Carlsbad, CA). Spheroids were washed with PBS, incubated for 45 min at room temperature with 10 μM calcein AM (live) and 10 μM etidium homodimer-1 (dead), and imaged using an epifluorescence microscope (IX71, Olympus, Tokyo, Japan). Images were captured at 4X magnification. To quantify cell viability in the prepared spheroids, SVF cells were isolated and viability was measured using a Nucleocounter to generate live and dead standard curves. Viability was measured on days 0, 2, 6, 9, and 13 based on the established standard curves. To supplement the live / dead kit, cell distribution was assessed using Hoechst 33258 bis-benzylimine nuclear stain (Anaspec, California) after permeation for 15 minutes in 0.1% Triton-X100 (Sigma, St. Louis, Missouri).

[0063] Confocal microscopy. For in vitro vascularization analysis, spherical specimens were imaged using an MPE FluoView 1000 confocal microscope with 10X water immersion objectives (Olympus, Tokyo, Japan). Confocal image stacks were reconstructed and displayed as z-axis projections using AMIRA 3D visualization software (Thermo, Waltham, MA). Prior to imaging, spherical specimens were fixed with 4% paraformaldehyde for 10 minutes at room temperature and then permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature (Sigma, St. Louis, Missouri). After permeabilization, the spherical specimens were stained with FITC (GS-1)-conjugated Griffonia Simplicifolia-1 Isolectin 4 diluted 1:500 (Vector Laboratories, Burlingame, CA). Simultaneously, a primary antibody (α-SMA) of mouse monoclonal α-smooth muscle actin (Santa Cruz Biotechnology, Dallas, Texas) was added at a dilution of 1:250. The spheroids were incubated overnight at 4°C. The next day, the spheroids were washed three times with PBS and incubated for 2 hours at room temperature with RedDot nuclear staining agent (Biotium, Fremont, California) and goat anti-mouse IgG Alexa Fluor 594 secondary antibody (Thermo Fisher, Waltham, Massachusetts) at dilutions of 1:200 and 1:1000, respectively. The samples were then washed with PBS and imaged as described above. Endothelial components were stained green with GS-1 FITC. Perivascular supporting cells and fibroblast components were stained red with Alexa Fluor 594-conjugated α-SMA, and the nuclei were stained with RedDot, with artificial staining in blue.

[0064] Statistical analysis. The statistical significance of the mean sphere diameter at day 2 and all other subsequent time points was determined using one-way ANOVA with Dunnet multiple comparison post-test via GraphPad Prism 7 for Windows (GraphPad Software LLC, La Jolla, CA).

[0065] result

[0066] The 3D bioprinters used in these studies were capable of extruding materials of varying viscosities using a series of extrusion tips with adjustable time and pressure. The contact angles of water and cell suspensions, culture media, and collagen at the printed concentrations were measured at 155° and 156°, respectively. Table I shows the range of printing parameters tested. The selected parameters represent conditions for consistent extrusion of spheroids with uniform size at a rate of 10.3 spheroids / min. Injector pressure, pressurization duration, and needle gauge were all combined to determine the extrusion speed, thereby determining the spheroid size. Higher pressure and larger needle gauges yielded more consistent results and were chosen for these reasons. The pressurization duration was kept low to maintain spheroid volume. The cartridge size was selected to optimally match the batch volume compared to unprinted collagen gel, and the collagen concentration was selected to maintain spheroid properties. The previously published conditions required for extruding cell-loaded collagen cylinders (where cell viability is maintained) were used as the starting point for this study.

[0067] Table I. 3D Printing Conditions for Forming Spherical Objects

[0068]

[0069] To maintain the spherical shape after collagen extrusion but before collagen polymerization, a superhydrophobic coating was used on tissue culture polystyrene plates, and 3D spherical bodies were directly bioprinted onto those surfaces. Figure 1A-Figure 1B As shown, during the entire stage of printing, the spherical object is fixed to the superhydrophobic surface by a Pluronic® F127 during movement (XY axis), and then transferred to an incubator for gelation. The Pluronic® F127 discs on the superhydrophobic surface... Figure 1D The figure shows this. Then, by incubating in an aqueous solution or by cooling the surface to 10°C, the spheroids can be easily extracted from the Pluronic... ® Released in F127. Throughout the process, the spherical bodies retain their shape on those superhydrophobic surfaces and after removal.

[0070] Batch production of spheroids was achieved using 2 mL of unpolymerized collagen I containing suspended SVF cells, with 100–150 spheroids produced per batch, depending on size. The average diameter of the spheroids was 3.54 mm, with a standard deviation of 0.195 mm. Batch production required 20 minutes for instrument preparation and 5 minutes for extruding every 48 spheroids. After extrusion, the spheroids were incubated at 37°C for 10 minutes, resulting in a total production time of 30–35 minutes before use.

[0071] Typical morphology of 3D bioprinted SVF spheroids after initial collagen gelation is as follows: Figures 2A-2HAs shown. The spherical morphology of the SVF load, as observed visually by phase contrast microscopy, is uniform. Figure 2A ), and as indicated by calcein AM live staining, most of the transplanted cells were viable. Figure 2B ).like Figure 2C As shown, some non-viable cells were present, labeled with ethidium homodimer-1. Cell distribution was assessed by nuclear staining with bis-benzylimine. Figure 2D The cells were homogeneous, indicating that collagen gelation maintained cell encapsulation throughout the globules, with no evidence of cell sedimentation within any specific location. Cell distribution and viability within the globules were reassessed after 6 days of rotational culture. Live cells (calcein AM positive) are shown in Figure 2, while dead cells (ethidium homodimer-1 positive) are shown in Figure 3. Figure 2G As shown, the total cell distribution by nuclear staining (bis-benzylimine positive) is as follows. Figure 2H As shown in the figure. At this culture time point, note the morphological changes in the encapsulated SVF, including microvascular structures with slender shoots and complex geometries, indicating in vitro angiogenesis in 3D collagen I.

[0072] exist Figure 3 In this study, SVF activity was quantified immediately after printing on day 0 and throughout the entire incubation period on days 2, 6, 9, and 13. The activity of the day 0 printed SVF (68.24% ± 3.59%) was on average 5.96% lower than that of the pipetting and encapsulating control group and 27.8% lower than that of the pre-encapsulation Nucleocounter-based activity.

[0073] Studies of spheroids during suspension culture showed that the spheroids underwent shrinkage accompanied by a decrease in diameter. This shrinkage of the spheroids was achieved through… Figures 4A-4D The phase contrast microscopy technique in the text is shown, and... Figure 5 The results provided were quantitatively evaluated. Early collagen shrinkage occurred at an average rate of 0.0083 ± 0.0875 mm / day from day 2–3 of rotational culture, and the shrinkage rate increased to its peak rate of 0.3675 ± 0.1359 mm / day from day 6–7 of rotational culture. After this peak, the shrinkage rate decreased to 0.042 ± 0.0463 mm / day from day 9–13 of rotational culture. The average spheroid diameter differed significantly between day 2 of culture and all subsequent days. Figure 5 (p < 0.0001).

[0074] The general morphology of SVF spheroids was assessed after 14 days in rotating culture (the time point when the spheroids underwent significant shrinkage). Due to the high density of cells in the shrinking spheroids, epifluorescence was insufficient to produce clear images of cell morphology. Therefore, to accurately observe cell distribution and characterize the vascular cell phenotype in the shrinking spheroids, the spheroids at day 14 were stained for endothelial (GS-1 FITC positive) and α-smooth muscle actin (α-SMA-Alexa Fluor 594 positive) components and evaluated by confocal microscopy. Subsequently, Amira software was used for 3D volume rendering to form the entire construct. Cells not only remained viable throughout the culture time, but they also underwent phenotypic changes in vascular structures, as previously seen in 3D collagen I in vitro analysis using SVF. Examples showing tubular formation of cellular components (containing endothelial cells surrounded by α-SMA-positive cells on the outer membrane) provide evidence of in vitro angiogenesis and, Figures 6A-6B Highlighted with an arrow.

[0075] discuss

[0076] The delivery of stem cells and regenerative cells (including stromal vascular fractions) for the treatment of various diseases has reached human clinical trials; however, limited cell retention at the implantation site has been associated with observed poor therapeutic effects. Alternatives to improve retention include cell self-aggregation in culture and cell encapsulation within biomaterials. These studies have identified improvements in cell retention at the implantation site. Matrix vascular fractions have been encapsulated in various materials (including alginate) to produce spheroids containing SVF. Retention of SVF in alginate maintains cell viability; however, alginate gels do not show any morphological changes, meaning they do not support transplanted cell migration and proliferation, thus limiting in vitro angiogenesis and potential in vivo applications for cell-based therapies. For this reason, the use of type I collagen for spheroid fabrication has been investigated, as SVF cell populations have been shown to exhibit in vitro angiogenesis within this extracellular matrix. SVF-loaded collagen gels can be formed in sheets and are cumbersome to handle. Additionally, collagen spheroids printed on conventional tissue plates flatten rapidly due to the hydrophilic properties of polystyrene. To maintain the spherical shape of type I collagen after extrusion and polymerization, a superhydrophobic coating was applied to tissue culture polystyrene plates and these surfaces were directly 3D bioprinted.

[0077] Initial attempts to print on superhydrophobic surfaces were unsuccessful because the spheroids failed to adhere, with most remaining attached to the printhead tip. To overcome this lack of adhesion, Pluronic F-127 discs were printed onto the superhydrophobic surface. Pluronic F-127 is a triblock copolymer with an amphiphilic block structure, giving it both hydrophilic and hydrophobic properties. This allows Pluronic F-127 to adhere to both superhydrophobic surfaces and aqueous materials such as collagen. With the amphiphilic surface established, collagen spheroids of various sizes were fabricated using conditions that provided a range of dimensions. The average diameter could be adjusted by varying the applied pressure, allowing for the fabrication of spheroids ranging in size from 1 mm to 3.5 mm.

[0078] Compared to self-assembly-based spheroid manufacturing methods, amphiphilic spheroid manufacturing is an order of magnitude faster, with spheroids exhibiting similar consistency in size and shape. When using automated time-pressure extrusion systems (such as BAT), the primary source of spheroid size variation is the viscosity of the dispensed fluid. However, all parameters, including a stable neutral pH, low temperature, and thorough mixing of the unpolymerized collagen solution prior to BAT extrusion, are programmed to minimize batch-to-batch variation in spheroids.

[0079] These studies demonstrate that globule integrity and cell distribution are maintained throughout the 14-day cell culture cycle, whether under static or more dynamic rotational culture conditions. Cell viability, measured by Nucleocounter, was 96% prior to encapsulation in collagen, thus showing a decrease of less than 30% on day 0 during collagen encapsulation. However, viability in pipette collagen was only 5.96% higher than in printed collagen, leading to the conclusion that collagen encapsulation, rather than bioprinting, is the root cause of the reduced viability. This conclusion conflicts with previous studies on viability in pipette collagen (showing >90% viability for both pipette and printed collagen). This is especially true considering that others used the same markers for their viability analyses, using cells of the same origin encapsulated in the same concentration of collagen. The two most likely reasons for the observed viability differences are: others cultured their cells prior to printing, rather than using the fresh isolates and rat tail collagen of this invention immediately, without listing their collagen source. This discrepancy can be attributed to the fact that others performed their viability-based cell counts by eye rather than by fluorescence values. Eye-based viability counting makes the data susceptible to user bias towards darker objects. This problem may be exaggerated by the fact that, in terms of relative fluorescence units (RFU), live cells are an order of magnitude brighter than an equivalent number of dead cells, and that brightness compensation for image-based viability counts is often unregulated. This seems particularly likely because others have clarified that cells must be brightly stained with any color to be counted. This is further supported by the fact that, for both studies, the differences in viability between pipette and printed cells were similar. A potential problem with fluorometer-based viability is the possibility of non-specific staining skewing the results; however, immediate use of stained, cell-free collagen spheroids as a baseline should help compensate for the cell-free signal, and the standard curve should compensate for non-specific cell staining. Viability remained high throughout the rotational culture period after day 0, although a decrease in viability consistent with later spheroid shrinkage occurred on day 9.

[0080] Contrary to the results obtained using alginate to form spheroids, significant changes in cell morphology were observed, indicating the cells' ability to undergo the initial steps of angiogenesis, including but not limited to tip cell formation and budding from preserved microvascular fragments. Contraction of SVF-collagen spheroids was also observed after 6 days of cell culture. Collagen contraction in the presence of fibroblasts and endothelial cells has been well-documented and has shown that cell-dependent contraction provides an assessment of the active metabolism, microenvironmental remodeling, and cell motility of these embedded cells. Confocal microscopic analysis of the cellular composition and phenotype within the contracted spheroids revealed the presence of organized, elongated tubular structures that were positive for staining with the endothelial cell-specific lectin Griffonia simplificiolia-1 (GS-1), which binds to α-smooth muscle actin. This phenotypic change over time provides evidence for either SVF self-organization into microvascular-like structures (vasculogenesis) or endothelial cell budding from pre-existing microvascular fragments (angiogenesis). In vitro SVF angiogenesis and neovascularization offer therapeutic potential for treating in vivo ischemic conditions using prevascularized type I collagen spheroids.

[0081] Automation through direct writing to computer-aided design and fabrication offers the opportunity to manufacture spheroids in a controlled, precise, and efficient manner using amphipathic surfaces. This system also enables the design of assays based on high-throughput microenvironments for a wide range of purposes, including drug delivery, cell delivery, or immunotherapy delivery. Numerous in vitro applications of spheroids remain to be investigated. The ability to fabricate a variety of extracellular matrix-based spheroids composed of biomaterials such as fibrin, hyaluronic acid, and other unstudied hydrogels will allow for the creation of microenvironments suitable for a variety of cell-based therapies. Indeed, phenotypic changes and cellular functions can vary depending on the ECM used. In vivo studies are also essential for addressing and characterizing the therapeutic benefits of encapsulated cells, including their functional interactions with the surrounding host tissue, compared to direct injection of liquid suspensions of cells alone.

[0082] In summary, the above studies demonstrate that amphiphilic surfaces can be used to form viable SVF-loaded spheroids with uniform size and shape. Increased automation via 3D bioprinters enables high throughput and sufficiently low production times to suit the demands of clinical care settings. Furthermore, the use of water-soluble hydrophilic points and the phase-change properties of Pluronic F-127 allows for minimal removal of broken spheroids for any application requiring spheroid manipulation. Through improved cell localization and retention, these SVF-loaded collagen spheroids could provide a strategy to enhance the therapeutic efficacy of cell infusions for regenerative purposes.

[0083] Example 2 - Fabrication of pancreatic islet cell-loaded spheroids.

[0084] method

[0085] Fabrication of the superhydrophobic surface, formation of hydrophilic sites, and isolation of SVF cells. The fabrication of the superhydrophobic surface and the formation of hydrophilic sites were performed essentially as described in Example 1 above. Also as described in Example 1 above, SVF cells were again isolated from the mouse epididymal fat pad according to the previously disclosed enzyme-based method.

[0086] Islet isolation. Islets were isolated according to the Balamarughan method.

[0087] Fabrication of islet / SVF-loaded collagen spheres. Freshly isolated SVF and islets were suspended in unpolymerized rat tail collagen I mixed with 1X Dulbecco modified Eagle medium (DMEM) (Sigma, St. Louis, Missouri) and titrated to a final pH of 7.4 to form a mixture of 3 mg / mL collagen containing 1.6 × 10⁵ SVF cells / mL of the final solution. This mixture was maintained at 4°C until printing, and a cooling system on the bioprinter was used to maintain the equivalent temperature throughout the printing process to prevent gel polymerization. The islet / SVF-collagen solution was transferred to a 3cc printing syringe (EFD, Nordson, Westlake, OH) and placed in a 3D bioprinter (nScrypt LLC, Orlando, FL). Initial printing conditions were based on our previously published data for continuous roller printing [4,5,31,42]. Once the spheres are printed, they are incubated at 37°C in a tissue culture incubator (5% CO2) for 10 minutes to initiate collagen gel polymerization.

[0088] Spheroid culture method. After preparation, the spheroids were transferred to a spinner flask (125-mL MagnaFlex microcarrier spinner flask, Wheaton Industries, Millville, NJ) for cell suspension culture containing endothelial cell growth medium

[85] consisting of DMEM, 10% FBS, 5 mM HEPES buffer, 2 mM L-glutamine, heparin-containing endothelial growth supplement, penicillin (45 U / mL), and streptomycin (45 μg / mL). The spinner flask was placed on a magnetic stirrer platform (MCS 104-L bio-stirrer, Techne LLC, Burlington, NJ) to provide a continuous suspension of the spheroids, and the magnetic impeller speed was set to 40 rpm throughout the culture period (14 days). The spheroids required for analysis were removed through a 50 mL serum pipette. For the study of spherical shrinkage, individual spherical bodies were inoculated into the separation wells of a 96-well ultra-low adhesion plate (Corning Corporation, Corning, NY).

[0089] Spheroid size measurement. Spheroid size measurements were calculated from images of spheroids cultured in 96-well plates obtained using an optical microscope (CKX41, Olympus, Tokyo, Japan). Diameter measurements from these 4X images were obtained using ImageJ software.

[0090] Live / dead analysis. Islet and SVF viability was assessed using a live / dead fluorescent dye (Live / Dead Viability / Toxicity Kit, Life Technologies LLC, Carlsbad, CA). Spheroids were washed with PBS, incubated for 45 min at room temperature with 10 μM calcein AM (live) and 10 μM etidium homodimer-1 (dead), and imaged using an epifluorescence microscope (IX71, Olympus, Tokyo, Japan). Images were captured at 4X magnification. To quantitatively assess the viability of the prepared spheroids, SVF cells were isolated, and viability was measured using a Nucleocounter to calculate known numbers of live and dead cells. From these cells, pipetting, staining, and analysis with a fluorometer were performed to obtain live and dead fluorescence values ​​for each cell mass. A standard curve generated from these values ​​is shown below (n=3). Based on the established standard curve, cell viability was measured on days 0, 2, 6, 9, and 13. To supplement the live / dead kit, cell distribution was assessed using Hoechst 33258 bis-benzylimine nuclear stain (Anaspec, California) after permeabilization to 0.1% Triton-X100 (Sigma, St. Louis, Missouri) for 15 minutes.

[0091] Insulin release. ELISA is used to quantify insulin release.

[0092] Prevascularized islet implantation study. Prevascularized islets were implanted into immunocompromised mice that had developed diabetes through streptozotocin infusion. Subcutaneous implantation of prevascularized islets. Blood glucose levels were measured after islet transplantation.

[0093] Confocal microscopy. For in vitro vascularization analysis, spherical specimens were imaged using an MPE FluoView 1000 confocal microscope with 10X water immersion objectives (Olympus, Tokyo, Japan). Confocal image stacks were reconstructed and displayed as z-axis projections using AMIRA 3D visualization software (Thermo, Waltham, MA). Prior to imaging, spherical specimens were fixed with 4% paraformaldehyde for 10 minutes at room temperature and then permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature (Sigma, St. Louis, Missouri). After permeabilization, the spherical specimens were stained with Griffonia Simplicifolia-1 Isolectin 4 conjugated to FITC (GS-1) (Vector Laboratories, Burlingame, CA). Simultaneously, mouse monoclonal α-smooth muscle actin primary antibody (α-SMA) (Santa Cruz Biotechnology, Dallas, Texas) was added at a dilution of 1:250. The spheroids were incubated overnight at 4°C. The next day, the spheroids were washed three times with PBS and incubated for 2 hours at room temperature with RedDot nuclear stain (Biotium, Fremont, California) and goat anti-mouse IgG Alexa Fluor 594 secondary antibody (Thermo Fisher, Waltham, Massachusetts) at dilutions of 1:200 and 1:1000, respectively. Subsequently, the samples were washed with PBS and imaged as described above. Endothelial components were stained green with GS-1 FITC. Perivascular supporting cells and fibroblast components were stained red with Alexa Fluor 594-conjugated α-SMA, and nuclei were stained with RedDot, with artificial staining as blue.

[0094] Statistical analysis. The statistical significance of the mean sphere diameter at day 2 and all other subsequent time points was determined using one-way ANOVA with Dunnet multiple comparison post-test via GraphPad Prism 7 for Windows (GraphPad Software LLC, La Jolla, CA).

[0095] Results and discussion

[0096] 3D bioprinting represents a novel approach to the development of implantable tissue constructs. Current research has been completed, demonstrating the feasibility of printing adipose-derived SVF cells and adipose-derived microvascular fragments and subsequently implanting these constructs to assess their viability and retention. Another enabling technology for islet construct bioprinting has been developed, utilizing a technique to form cell-encapsulated spheroids of defined sizes. The first study utilized alginate as the encapsulating biomaterial. While these studies established the ability to co-localize adipose SVFs and islets, and the islets remained viable in culture for up to 14 days, a lack of host vascular integration with the spheroid circulation was observed. Therefore, alternative methods for generating spheroids were explored, focusing on the use of type I collagen as the matrix molecule. As mentioned above, a technical solution using superhydrophobic and hydrophilic surfaces to maintain the spheroid shape in non-viscous solutions was found. In this regard, similar to that shown in Figure 1, a bioprinting system was used to bioprint spheroids containing islets and adipose SVFs. Figure 7 shows an image of such a spheroid and demonstrates the ability to co-print islets and adipose-derived regenerative cells. Figure 7 illustrates the cell distribution within these spheroids and the ability to print and maintain the structural integrity of islet / SVF spheroids for over 7 days in rotational culture. Furthermore, as... Figure 8 and Figures 9A-9G As shown, the study has established the ability to treat hyperglycemia in diabetic animal models and has demonstrated the formation of vascular structures in SVF-loaded spheroids.

[0097] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference, including the references listed below:

[0098] References

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[0240] It should be understood that various details of this disclosure may be changed without departing from the scope of the subject matter. Furthermore, the foregoing description is for illustrative purposes only and not for limiting purposes.

Claims

1. A method for manufacturing a sphere, the sphere comprising one or more bio-related materials, the method comprising: Provide a suspension containing one or more bio-related materials dispersed in a biocompatible medium; A certain amount of hydrophilic material is deposited on a defined region of a superhydrophobic surface, wherein the hydrophilic material includes a polyoxyethylene-polyoxypropylene block copolymer; as well as Bioprinting of droplets from a suspension onto a hydrophilic material to form spherical bodies; The spheroids are incubated in an aqueous solution to dissolve the hydrophilic material, thereby releasing the spheroids from the superhydrophobic surface.

2. The method according to claim 1, wherein, One or more bio-related materials include magnetic beads, matrix vascular cells, stem cells, one or more related cells, or a combination thereof.

3. The method according to claim 1, wherein, Biocompatible media include hydrogels.

4. The method according to claim 3, wherein, The hydrogel contains materials selected from agarose, alginate, collagen, fibrinogen, fibrin, laminin, polyoxyethylene-polyoxypropylene block copolymer, organosilicon, polysaccharide, polyethylene glycol and polyurethane.

5. The method according to claim 4, wherein, The hydrogel contains type I collagen.

6. The method according to claim 1, wherein, One or more bio-related materials include stromal vascular cells.

7. The method according to claim 1, wherein, One or more bio-related materials contain tissue-derived microvascular fragments.

8. The method according to claim 1, wherein, One or more biologically related materials contain one or more pancreatic islet cells.

9. The method according to claim 1, wherein, The steps of bioprinting droplets of suspension include directly writing the suspension.

10. The method according to claim 1, wherein, The water contact angle of the superhydrophobic surface is greater than approximately 150°.

11. The method according to claim 10, wherein, The water contact angle is approximately 150° to approximately 170°.

12. The method according to claim 1, wherein, The steps of bioprinting droplets include bioprinting droplets with a diameter of approximately 0.2 mm to approximately 5 mm.

13. The method according to claim 1, wherein, The method further includes the step of incubating the spheroid at physiological temperature for a period of time after the bioprinting droplet.

14. The method according to claim 1, wherein, The method further includes the step of culturing the spheroids in a cell culture medium after the bioprinting droplets.

Images