3D Bioprinting with Biomaterial-Related Materials and Related Methods

Abstract

The present disclosure provides a method for bioprinting a 3D structure comprising one or more bio-related materials on a superhydrophobic surface. In one embodiment, the method includes providing a composition having one or more bio-related materials dispersed in a biocompatible medium. A pattern comprising a hydrophilic material is deposited on a defined region of the superhydrophobic surface, the pattern being modeled after a biological structure. A composition having one or more bio-related materials is then bioprinted onto the hydrophilic surface to form a 3D structure, the hydrophilic surface maintaining the 3D structure in a desired position or shape on the superhydrophobic surface.

Description

Technical Field

[0001] The present disclosure generally relates to the field of 3D bioprinting. In certain embodiments, the present disclosure provides compositions and methods of 3D bioprinting structures of defined shapes on superhydrophobic surfaces that include hydrophilic lines or surfaces.

Background Art

[0002] As discussed in a recent review article (Dey and Ozbolat, Sci.Rep. 2020), the first 3D printers capable of making solid objects according to computer-aided design (CAD) were manufactured in the early 1980s. By the late 1990s, 3D printing had made its way into the medical field, and surgeons had started 3D printing dental implants, custom prosthetics, and kidneys and bladders. Subsequently, the term "3D bioprinting" emerged, in which printed materials called "bioinks" consisting of living cells, biomaterials, or bioactive molecules are printed. 3D bioprinting involves the layer-by-layer deposition of bioinks to create 3D structures such as tissues and organs. Apart from organ printing, bioprinting is also used to create in vitro tissue models for drug screening, disease modeling, and several other in vitro applications. A review of bioinks suitable for 3D bioprinting can be found in Williams and Hoying, Bioinks for Bioprinting, K. Turksen (ed.), Bioprinting in Regenerative Medicine, Stem Cell Biology and Regenerative Medicine, Springer International Publishing, 2015.

[0003] 3D bioprinting can be broadly classified into either extrusion, droplet, or laser-based bioprinting. Extrusion-based bioprinting uses mechanical, pneumatic, or solenoid dispenser systems to deposit bioink in a continuous form of filament, and droplet-based bioprinting relies on the generation of bioink droplets by thermal, acoustic, or electrical stimuli. The selection of "bioink" for each of these different bioprinting modalities usually varies based on the ink's fluidity, viscosity, cross-linking chemical reactions, and biocompatibility. Extrusion-based bioprinting mainly requires bioinks that shear thin, and droplet or inkjet bioprinting requires low-viscosity materials. In the past few years, the design and synthesis of bioinks have evolved to meet the increasing needs for new bioprintable materials.

[0004] Although 3D bioprinting is advancing at an appreciable rate and researchers are attempting to develop new printing modalities and improve existing ones, many challenges remain that need to be overcome. Currently, there are a limited number of bioinks that are bioprintable and accurately represent the tissue architecture necessary to restore organ function after printing. Furthermore, the bioprinting process itself needs to be more cell-friendly. There is a need to develop effective techniques for the high-throughput generation of organoids for personalized drug testing and predictive disease models and for bioprinting. Thus, there is a need for improved 3D bioprinting methods in the art. SUMMARY OF THE INVENTION

[0005] The subject matter of the present disclosure meets some or all of the needs identified above, as will become apparent to those skilled in the art after considering the information provided herein.

[0006] This summary describes some embodiments of the subject matter disclosed in this specification and often lists variations and permutations of these embodiments. This summary is merely illustrative of a large number of various embodiments. The mention of one or more representative features of a given embodiment is likewise illustrative. Such embodiments can typically exist regardless of the presence or absence of the recited feature(s), and similarly, those features can apply to other embodiments of the subject matter of this disclosure regardless of whether they are listed in this summary. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.

[0007] In some implementations of the subject matter of this disclosure, methods for fabricating 3D structures that include bio-related materials are provided. In one implementation, a method for fabricating a 3D structure that includes one or more bio-related materials, first, a composition is made or provided, the composition including one or more bio-related materials dispersed within a biocompatible medium, is provided. Next, a quantity of hydrophilic material is deposited on the superhydrophobic surface of a suitable substrate in a defined region and / or in a defined quantity. In some embodiments, the hydrophilic material is deposited in a pattern modeled after a biological structure. In some implementations, the hydrophilic material deposited on the superhydrophobic surface includes a polyoxyethylene-polyoxypropylene block copolymer. In some implementations, the superhydrophobic surface utilized in accordance with the subject matter of this disclosure has a water contact angle greater than about 150°, for example, in some implementations, a water contact angle of from about 150° to about 170°.

[0008] Regardless of the specific hydrophilic materials and / or water contact angles of the superhydrophobic surfaces utilized in the exemplary methods of the present disclosure, when the composition and the substrate are fabricated, the composition is then bioprinted (e.g., directly written and printed) directly onto the hydrophilic material positioned on the superhydrophobic surface, thereby fabricating a 3D structure containing bio-related materials. In some embodiments, after bioprinting the composition, the resulting 3D structure can then be incubated at physiological temperature for a period of time while maintaining the shape of the 3D structure. In some implementations, optionally, the 3D structure can then be further cultured in a cell culture medium.

[0009] In some implementations of the methods of the present disclosure, one or more bio-related materials included in the exemplary 3D structure include magnetic beads, stromal vascular fraction cells, stem cells, one or more related cells, a group of cells or tissues, or combinations thereof. For example, in some implementations, a 3D structure can be fabricated that includes stromal vascular fraction cells in combination with one or more related cells such as pancreatic islet cells. In some implementations, one or more bio-related materials can thus include stromal vascular fraction cells. In some implementations, one or more bio-related materials include one or more pancreatic islet cells.

[0010] Regarding the biocompatible medium used to form the suspension utilized in the methods of the present disclosure, in some implementations, the biocompatible medium includes a hydrogel. In some implementations, the hydrogel includes materials selected from the group consisting of agarose, alginate, collagen, polyoxyethylene-polyoxypropylene block copolymers, silicone, polysaccharides, polyethylene glycol, and polyurethane. In some implementations, the hydrogel includes type I collagen.

[0011] These and other aspects of the invention will be understood from the subsequent description of the figures and detailed description of the invention.

[0012] Some embodiments of the present invention are described herein by way of example only with reference to the accompanying drawings. Next, with specific and detailed reference to the drawings, it is emphasized that the details shown are for the purpose of example and for illustrative consideration of embodiments of the present invention. In this regard, the description made in conjunction with the drawings will make it clear to those skilled in the art how embodiments of the present invention can be implemented.

Brief Description of the Drawings

[0013]

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DETAILED DESCRIPTION OF THE INVENTION

[0014] Details of one or more embodiments of the subject matter of the present disclosure are described herein. Modifications to the embodiments described herein and other embodiments will be apparent to those skilled in the art upon consideration of the information provided herein. The information provided herein, particularly the specific details of the exemplary embodiments described, is provided primarily to clarify understanding and should not be construed as imposing unnecessary limitations therefrom. In case of conflict, the present specification, including definitions, will control.

[0015] The terms used herein are considered to be well understood by those skilled in the art, but specific definitions are set forth herein to facilitate the description of the subject matter of the present disclosure.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention(s) belong.

[0017] Unless otherwise stated, all patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other published materials referenced throughout the present disclosure are hereby incorporated by reference in their entirety.

[0018] As used herein, "comprising" is open-ended and means any other element(s) not recited, in addition to the recited elements or their equivalents in structure or function. The terms "having" and "including" are also to be construed as open-ended unless the context otherwise indicates.

[0019] In accordance with longstanding patent law convention, the terms "a," "an," and "the" as used in this application refer to "one or more" including, for example, in the claims. Thus, for instance, a reference to "a cell" includes a plurality of such cells.

[0020] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure.

[0021] As used herein, the term "about" when referring to a value or to an amount of mass, weight, time, volume, concentration, or percentage, means that in some embodiments it encompasses a variation of ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% for the purposes suitable for carrying out the disclosed method.

[0022] As used herein, a range can be expressed from "about" a particular value and / or to "about" another particular value. Also, several values are disclosed herein, and it is understood that each value is disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0023] Accordingly, in some implementations of the subject matter of the present disclosure, methods of fabricating 3D structures are provided. In some implementations, a method of fabricating a 3D structure is provided, comprising first placing a hydrophilic material on a defined region on a surface of a substrate, wherein the surface of the substrate is superhydrophobic. In some embodiments, the hydrophilic material is deposited in a pattern modeled after a biological structure (e.g., the Purkinje system of the heart). Other examples include the systemic circulation of the heart (i.e., the coronary vascular system of the heart where blood vessels are printed as tubes based on the coronary artery vascular architecture).

[0024] In some embodiments, modeling a biological structure includes computer-aided design (CAD). One or more bio-related materials can then be suspended in a biocompatible medium to form a composition, which can be bioprinted onto a hydrophilic material. For example, in one exemplary implementation of a method for fabricating the subject matter of the present disclosure, direct write printing is used in the form of bioprinting (see, e.g., BioArchitecture Tool (BAT), e.g., U.S. Patent No. 7,857,756, and also Smith, et al., Tissue Eng. 2004;10:1566-1576, both of which are incorporated herein by reference). In some embodiments, the use of a computer-controlled stage is utilized, which allows for independent X-axis and Y-axis translations, as well as Z-axis movement of one or more translational printhead / dispensing systems. In this regard, the bioprinting parameters can first be scripted as print commands and then uploaded to a printing tool (i.e., BAT) so that an accurate structure can be fabricated. In some implementations, by utilizing such a printing tool, the size of the structures printed by such a system can be controlled by controlling the size of the pen used to print the droplets and the pressure at which the droplets are extruded from the pen. In some embodiments, pens of about 15 gauge to about 25 gauge and pressures of about 2 psi to about 7 psi can be used to create droplets. In some implementations, the droplets have a diameter of about 1 mm to about 5 mm, about 2 mm to about 4 mm, or about 3 mm to about 4 mm. In some implementations, the size of the droplets is controlled by adjusting one or more parameters selected from the group consisting of the viscosity of the suspension, the size of the delivery pen tip, the pressure used to extrude the suspension from the delivery pen, and the amount of time pressure applied to the suspension within the delivery pen. Such parameters can be readily adjusted by one of ordinary skill in the art to create droplets or spheroids having a desired size.

[0025] Figures 5A - 5C show one embodiment of a method in which an array of the coronary system of the heart is converted into a "script" CAD design and then this design can be used as a guide to print a collagen solution onto a surface. Here, since the surface is not a hydrophobic surface, the collagen spreads on the surface.

[0026] As one exemplary implementation of a method for creating a 3D structure containing one or more bio - related materials, the 3D structure is first created by placing a suspension in the form of a cell suspension (e.g., a cell suspension containing a mixture of human stromal vascular fraction cell population and type I collagen) into a delivery pen containing a hollow needle or a tube - like structure. Then, the extrusion of the bio - suspension from the delivery pen is controlled by increasing the pressure within the delivery pen to a specific value, whereby droplets are formed. Next, the delivery pen is lowered towards a hydrophilic material placed on a super - hydrophobic surface of a substrate at a predetermined speed (e.g., 5 mm / second). Upon contact with the hydrophilic material, the suspension droplets are then attracted to the hydrophilic material and released from the pen, thereby forming a 3D structure on top of the hydrophilic spot on the super - hydrophobic surface. In some implementations, after bioprinting the suspension, the resulting 3D structure can then be incubated at a physiological temperature (e.g., 37 °C) for a period such as a period sufficient for the biological medium utilized to polymerize. In some implementations, optionally, the 3D structure can then be further cultured in a cell culture medium.

[0027] The term "suspension" is used herein to refer to a composition that includes a bio-related material, such as magnetic particles, cells, tissues, proteins, etc., dispersed in a biocompatible medium. Suitable biocompatible media for use in accordance with the subject matter of the present disclosure can typically be formed at room temperature (e.g., 25 °C) from any biocompatible material that is a gel, semi-solid, or liquid (e.g., a low-viscosity liquid) and can be used as a three-dimensional substrate for the cells, tissues, proteins, and other biological materials of interest. Exemplary materials that can be used to form a biocompatible medium in accordance with the subject matter of the present disclosure include, but are not limited to, collagen, fibrin, chitosan, MATRIGEL™ (BD Biosciences, San Jose, Calif.), polyethylene glycol, dextran (including chemically crosslinkable or photo-crosslinkable dextran, etc.), and polymers and hydrogels, including electrospun biological, synthetic, or biosynthetic blends. In some implementations, the biocompatible medium includes materials that assist with endothelialization. See, e.g., U.S. Patent No. 5,744,515 and U.S. Patent No. 7,220,276, both of which are incorporated herein by reference. In some implementations, the biocompatible medium includes a hydrogel.

[0028] The term "hydrogel" is used herein to refer to a two - or multi - component gel that includes a three - dimensional network of polymer chains in which water acts as the dispersion medium and fills the spaces between the polymer chains. Hydrogels used in accordance with the subject matter of the present disclosure are generally selected for a particular use based on the intended use of the structure, taking into account the printing parameters used, as well as the effect of the selected hydrogel on the behavior and activity of biological materials (e.g., cells) incorporated into the biological suspension in which the structure is placed. Exemplary hydrogels of the subject matter of the present disclosure can be composed of polymer materials, including but not limited to alginates, collagens (including types I and VI), fibrinogen, elastin, keratin, fibronectin, proteoglycans, glycoproteins, polylactides, polyethylene glycols, polycaprolactones, polyglycolides, polydioxanones, polyacrylates, polyurethanes, polysulfones, peptide sequences, proteins and derivatives, oligopeptides, gelatin, elastin, fibrin, laminin, polymethacrylates, polyacetates, polyesters, polyamides, polycarbonates, polyanhydrides, polyamino acids, carbohydrates, polysaccharides and modified polysaccharides, and derivatives and copolymers thereof, as well as inorganic materials (e.g., glasses such as bioactive glasses, ceramics, silica, alumina, calcite, hydroxyapatite, calcium phosphate, bone, and all combinations of the foregoing). For further information regarding materials that can be included in the hydrogels of the subject matter of the present disclosure, reference is made, for example, to U.S. Patent No. 7,919,11, U.S. Patent No. 6,991,652, and U.S. Patent No. 6,969,480, each of which is hereby incorporated by reference into this specification.

[0029] Furthermore, with respect to the hydrogel, in some embodiments, the hydrogel comprises a material selected from the group consisting of agarose, alginate, type I collagen, polyoxyethylene-polyoxypropylene block copolymer (e.g., Pluronic® F127 (BASF Corporation, Mount Olive, N.J.)), silicone, polysaccharide, polyethylene glycol, and polyurethane. In some embodiments, the hydrogel comprises alginate. In some embodiments, the hydrogel comprises type I collagen.

[0030] As used herein, the phrase “biologically relevant material” is used to describe a material that is included in a biocompatible medium as defined herein and that can then interact with and / or affect a biological system. For example, in some embodiments, the biologically relevant material is a magnetic bead (i.e., a bead that is itself magnetic or a bead that contains a material that responds to a magnetic field, such as iron particles), which is combined with a hydrogel and then bioprinted with the hydrogel to create a structure having a defined size, which can be used for calibration of an instrument or separation and purification of cells and tissues according to methods known to those of skill in the art. As another example, in other embodiments, the biologically relevant material comprises one or more cells and tissues, such that when the cells or tissues are combined with a suitable biocompatible medium, a cell or tissue suspension is formed. In some embodiments, the biologically relevant material comprises stromal vascular fraction cells, stem cells, one or more associated cells, or combinations thereof. In some embodiments, the biologically relevant material comprises stromal vascular fraction cells.

[0031] Regarding the stromal vascular fraction cells used in accordance with the methods of the subject matter disclosed herein, the stromal vascular fraction cells are typically obtained by enzymatically digesting a quantity of adipose tissue obtained from a subject and then performing a period of centrifugation to pellet the stromal vascular fraction of the adipose tissue. In this regard, the stromal vascular fraction contains several 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 within the stromal vascular fraction. For further explanation and guidance regarding the dissociation of adipose tissue to produce the stromal vascular fraction, reference is made, for example, to U.S. Patent No. 4,820,626, the entire content of which is incorporated herein by reference. In some embodiments, incomplete digestion of adipose tissue can also be used to obtain adipose microvascular fragments; see, for example, U.S. Patent No. 7,029,838, which is incorporated herein by reference.

[0032] Regarding stem cells that can be utilized according to the method of the present invention, the term "stem cell", as used herein, broadly refers 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, hepatic stem cells, muscle stem cells, muscle progenitor stem cells, endothelial progenitor cells, bone marrow stem cells, chondral 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 stem cells, can be found, inter alia, in 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 No. 5,559,022, U.S. Patent No. 5,672,346, and U.S. Patent No. 5,827,735. Descriptions of stromal cells, including methods for isolating stromal cells, can be found, inter alia, in Prockop, Science, 276:71-74, 1997; Theise et al., Hepatology, 31:235-40, 2000; Current Protocols in Cell Biology, Bonifacino et al., eds., John Wiley & Sons, 2000; and U.S. Patent No. 4,963,489. Those skilled in the art will understand that the stem cells and / or stromal cells selected for inclusion in a tissue construct are selected when such cells are typically appropriate for the intended use of a particular construct.

[0033] Finally, with regard to the related cells that can be utilized in accordance with the method of the present invention, the term "related cells", as used herein, refers to cells that are appropriate for incorporation into the structures of the subject matter of the present disclosure based on the intended use of their structure. In some embodiments, the term "related cells" can be used interchangeably with the term "regenerative cells" because the related cells described herein have the ability to form functional tissue after transplantation. For example, related cells suitable for the repair, reconstruction, or reconstitution of a particular damaged tissue or organ typically include cells or cell populations commonly found in that tissue or organ. In that regard, exemplary related cells that can be incorporated into the subject matter of the present disclosure include neurons, cardiomyocytes, muscle cells, vascular cells and / or gastrointestinal smooth muscle cells, chondrocytes, pancreatic acinar cells, islets of Langerhans, pancreatic beta cells, osteocytes, hepatocytes, Kupffer cells, fibroblasts, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, bile duct epithelial cells, and the like. These types of cells can be isolated, used immediately, or subjected to culture by conventional techniques known in the art. Exemplary techniques can be found, inter alia, in Freshney, Culture of Animal Cells, A Manual of Basic Techniques, 4th ed., Wiley Liss, John Wiley & Sons, 2000; Basic Cell Culture: A Practical Approach, Davis, ed., Oxford University Press, 2002; Animal Cell Culture: A Practical Approach, Masters, ed., 2000; and U.S. Pat. Nos. 5,516,681 and 5,559,022. In some implementations, the biologically relevant cells include pancreatic islet cells (e.g., beta cells) or intact pancreatic islets.

[0034] As indicated above, regardless of the particular type of biomaterial combined with the biocompatible medium according to the subject matter of the present disclosure, when the biomaterial 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, when the suspension reaches room temperature, the suspension typically gels and forms a structure with a more stable shape. However, in order to maintain the geometry of the droplets prior to polymerization or gelation after extrusion, as described above, the method of the present disclosure utilizes a substrate having a superhydrophobic surface.

[0035] The term "superhydrophobic" is used herein to refer to a substrate that exhibits a minimal attraction to water. Superhydrophobic surfaces typically exhibit the lotus effect, such as when water droplets come into contact with the leaves of a lotus or taro plant. Other naturally occurring examples of superhydrophobic surfaces that assist in the formation of water droplets can be found, for example, in the fogstand beetle (Stenocara gracilipes) found in the Namib Desert. In this regard, such superhydrophobic substrates or surfaces typically have a water contact angle greater than about 150°, or an angle at which the liquid or vapor interface contacts the solid surface as measured through the liquid. In some implementations, the superhydrophobic surfaces used herein have a water contact angle greater than 150°. In some implementations, the water contact angle of an exemplary superhydrophobic surface is from about 150° to about 170°. A number of superhydrophobic surfaces having such water contact angles are known to those skilled in the art, and those superhydrophobic surfaces can exist as a result of the particular substrate being utilized or as a result of a coating applied to the substrate. For example, in some implementations, a superhydrophobic surface can be fabricated by spraying a water-repellent coating, such as NEVERWET™ (Rust Oleum, Vernon Hills, Ill.), onto a suitable substrate. Further 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, perfluorobutanesulfonic acid, carbon nanotube structures, paraffin, polytetrafluoroethylene, waxes, and the like.

[0036] Also, as described above, in some implementations of the methods described herein, an amount of hydrophilic material, i.e., a material having an increased affinity for water and typically having a water contact angle of less than about 90°, is placed on a defined region of the hydrophobic surface. The amount of hydrophilic material and the region on which the hydrophilic material is placed can, of course, vary depending on the structure being fabricated. In some implementations, about 2 μl to about 5 μl of hydrophilic material is placed on the hydrophobic surface to ensure that the spheroid does not remain attached to the printing pen but adheres to the superhydrophobic surface. In some implementations, block copolymers such as Pluronic® F127 having an amphiphilic block structure can be utilized. The reason is that such copolymers have both hydrophilic and hydrophobic properties and can thus adhere to both the hydrophobic surface and an aqueous biocompatible medium such as collagen. Other hydrophilic materials that can be used in accordance with the present invention include other copolymers such as P188, as well as other materials such as urethane and silane, but are not limited thereto. In some embodiments, the hydrophilic material useful for forming the 3D structure provides adhesive properties that are reversible to enable removal of the 3D structure. Such reversal can be caused, inter alia, by a change in temperature in the aqueous phase of the 3D structure or solubilization of the hydrophilic substance.

[0037] Furthermore, in some embodiments of the subject matter of the present disclosure, 3D structures produced according to the methods described herein are provided. Without wishing to be bound by any particular theory or mechanism, such 3D structures can be used, for example, in in vitro assays of angiogenesis and vasculogenesis for screening drugs, devices that can be implanted into a patient to provide new blood flow to ischemic tissue, and devices constructed using adipose-derived stem cells and regenerative cells and incorporating other parenchymal cells that can include, for example, hepatocytes, myocytes, adipocytes, pancreatic cells including islets, brain cells, germ cells, kidney cells, and the like. Further, the 3D structures and methods of the present disclosure are believed to enable the manufacture of devices that can be formed and implanted immediately without the need to subject materials to tissue culture and without the need to utilize other additives (such as alginates) to assist in the formation of stable structures.

[0038] The practice of the subject matter of this disclosure, unless otherwise indicated, can employ conventional techniques in the fields of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the scope of the relevant art. Such techniques are well explained in the literature.For example, see Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Patent No. 4,683,195; DNA Cloning, Volumes I and II, Glover, ed., 1985; Oligonucleotide Synthesis, M.J. Gait, ed., 1984; Nucleic Acid Hybridization, D.Hames & S.J. Higgins, eds., 1984; Transcription and Translation, B.D. Hames & S.J. Higgins, eds., 1984; Culture Of Animal Cells, R.I. Freshney, Alan R. Liss, Inc., 1987; Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), A Practical Guide To Molecular Cloning; See Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells, J.H. Miller and M.P. Calos, eds. Cold Spring Harbor Laboratory, 1987; Methods In Enzymology, Vols. 154 and 155, Wu et al., eds., Academic Press Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987; Handbook Of Experimental Immunology, Volumes I-IV, D.M. Weir and C.C. Blackwell, eds., 1986.

[0039] The subject matter of the present disclosure is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data representing data collected at various times during the development and experimentation related to the present invention.

[0040] For clarity, it is understood that certain features of the present invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the present invention described in the context of a single embodiment may also be provided separately, or in any suitable partial combination, or in any other described embodiment of the present invention as being suitable. Specific features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiments would be inoperable without those elements.

[0041] FIG. 1 shows an embodiment of bioprinting a hydrogel rod or tube onto a superhydrophobic surface using a hydrophilic surface (e.g., a thin rod of Pluronic that can be dried on a hydrophobic surface) and maintaining the position and shape in the cross-section of the hydrogel after extrusion (e.g., the contact angle of the rod / tube exceeding 150 degrees). Examples of hydrogels that can be used include, but are not limited to, collagen, fibrin, aqueous solutions (including water, physiological saline), and alginates.

[0042] Figure 3 shows one embodiment of acquiring a biological image, converting the image into a CAD design, and manufacturing the image as a Pluronic F127 printed structure. In this example, the left ventricular Purkinje system is used as an example. Left panel: Biological image of the left ventricular Purkinje system. Central panel: CAD design of the Purkinje system based on the image in the left panel. Right panel: Pluronic F127 structure printed on a hydrophobic surface based on the CAD design in the central panel. Those skilled in the art will be able to easily bioprint other biological systems, such as the heart, kidneys, liver, the systemic and microcirculation of the lungs, the airway system of the lungs, e.g., the trachea to the alveolar system, ligaments and tendons in the orthopedic system, and soft tissue implants used in formation and reconstructive surgery, following the same approach. Another biological system is tissue implants used when correcting the shape of the cornea and lens of the eye. In addition to CAD, other techniques well known in the art, such as computer numerical control or computer-aided manufacturing, can be used.

[0043] Figures 4A - 4D present an example of hydrogel rods printed on a hydrophilic surface. This example shows that the hydrogel does not maintain its shape when printed directly onto the hydrophilic surface. Thus, one objective of the present disclosure is to provide a method for bioprinting a 3D structure containing one or more bio-related materials onto a superhydrophobic surface. In one embodiment, the method includes providing a composition having one or more bio-related materials dispersed within a biocompatible medium. A pattern containing a hydrophilic material is deposited onto a defined area of the superhydrophobic surface, and this pattern is modeled after a biological structure. Next, the composition having one or more bio-related materials is bioprinted onto the hydrophilic surface to form a 3D structure, and the hydrophilic surface maintains the 3D structure in the desired position or shape on the superhydrophobic surface.

[0044] In one embodiment, a method for fabricating a 3D structure containing one or more bio-related materials is provided, the method comprising (i) Depositing a pattern containing a triblock copolymer on a superhydrophobic surface to form a hydrophilic surface on the superhydrophobic surface, wherein the pattern is modeled after a biological structure, and the triblock copolymer has an amphiphilic block structure that imparts hydrophilicity and hydrophobicity to the pattern. In another embodiment, the pattern can also be modeled after a non-biological structure such as a linear branched structure (Figure 2D) or a chaotic structure. (ii) Providing a composition comprising one or more bio-related materials dispersed in a biocompatible medium. (iii) Bioprinting the composition onto the hydrophilic surface to form a 3D structure comprising the one or more bio-related materials, wherein the hydrophilic surface maintains the 3D structure at a desired position or shape on the superhydrophobic surface. In one embodiment, the superhydrophobic surface is printed with rods / tubes that constrain a structure that consistently maintains a contact angle greater than 150 degrees.

[0045] In one embodiment, the biocompatible medium is a hydrogel. In one embodiment, the hydrogel comprises type I collagen.

[0046] In one embodiment, modeling the biological structure as described above includes computer-aided design (CAD), CAM, or CNS.

[0047] In one embodiment, the bio-related material comprises a stromal vascular fraction, microvascular fragments, or stem cells. In one embodiment, the stem cells are embryonic stem cells, adult stem cells, or pluripotent stem cells. In another embodiment, the bio-related material comprises one or more cells suitable for the repair, reconstruction, or reconstitution of a tissue or organ. Examples of cells suitable for the repair, reconstruction, or reconstitution of a tissue or organ include, but are not limited to, neurons, cardiomyocytes, myocytes, vascular cells, or gastrointestinal smooth muscle cells, chondrocytes, pancreatic acinar cells, islets of Langerhans, pancreatic beta cells, osteocytes, hepatocytes, Kupffer cells, fibroblasts, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, or bile duct epithelial cells.

[0048] In one embodiment, the above method further includes the step of incubating the 3D structure at physiological temperature for a suitable period after bioprinting the 3D structure. In another embodiment, the above method further includes the step of culturing the 3D structure in a cell culture medium after bioprinting the 3D structure.

[0049] In another embodiment, another example of a rod structure is described in U.S. Patent No. 10,889,799 (see FIG. 12 therein). This example shows the printing of a spheroid having an inner core of a cell product and an outer core of microvascular fragments, but the same delivery pen can be used to print a rod containing the same material. Tubes can also be printed on hydrophilic / hydrophobic surfaces. In one embodiment, the bioprinting of spheroids / rods can be performed in a manner that enables the production of pre-angiogenic hydrogel spheroids. For example, in some implementations, a method of making a pre-angiogenic hydrogelated spheroid includes providing a first suspension containing one or more relevant cells dispersed in a biocompatible medium, and providing a second suspension containing one or more microvascular fragments dispersed in a biocompatible medium. A bioprinter having a first delivery pen surrounded by a second delivery pen (e.g., the B.A.T. assembly described above herein) is then provided, the first suspension is placed within the first delivery pen, and the second suspension is placed within the second delivery pen. The first suspension and the second suspension are then extruded substantially simultaneously from the first delivery pen and the second delivery pen, respectively, such that droplets are formed using the second suspension to coat the first suspension. In other words, by co-extruding the first suspension and the second suspension substantially simultaneously from the first delivery pen and the second delivery pen, a biocompatible medium containing one or more microvascular fragments surrounds a core containing a biocompatible medium containing one or more stromal vascular fraction cells, stem cells, and / or one or more relevant cells, and droplets are formed. In some embodiments, once the droplets are formed, the droplets are then contacted with the surface of a salt solution to form pre-angiogenic spheroids.

[0050] In this specification, certain features of the present invention have been illustrated and described, but now many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the present invention.

Example

[0051] Example 1 Using a 3D bioprinter to form hydrophilic regions on a superhydrophobic surface Fabrication of a superhydrophobic surface. In one embodiment, a two-step aerosol coating of NEVERWET™ (Rust Oleum, Vernon Hills, Ill.) was used to form a superhydrophobic surface on a polystyrene 48-well plate (Corning, Corning, N.Y.) and a 35 mm Petri dish. The first step was the application of a binder as a base coat to the surface, and this base coat was air-dried at room temperature for at least 1 hour. Subsequently, an upper sheet containing polydimethylsiloxane modified with hexamethyldisilazane was applied to form a superhydrophobic layer. The thickness of the superhydrophobic layer was measured to be 0.07 mm. Thereafter, the upper sheet was air-dried at room temperature for an additional 1 hour. NEVERWET™ has a reported contact angle of 165°, and a surface with a contact angle exceeding 150° was considered a superhydrophobic surface. The contact angles of both water and non-polymerized collagen in solution were measured via side photographs and subsequent contact angle measurements in ImageJ.

[0052] Creating hydrophilic regions. In one embodiment, hydrophilic regions on a superhydrophobic surface were created using a 3D bioprinter (Bio-Assembly Tool (BAT) 3D printer, nScrypt, Inc., Orlando, Fla.) and Pluronic F-127 (Sigma, St. Louis, Mo.) was extruded. In one embodiment, for each hydrophilic spot or line, the BAT extruded 3.8% (weight / weight) Pluronic F-127 with a target volume of 2.5 μL (for spots) and 10 μL / cm (for lines) in 1× phosphate buffered saline (PBS). In the BAT time-pressure extrusion system, in this case, an exposure time of 100 ms (for spots) and a continuous time (for lines) with 2.5 PSI had to be applied via a 25G needle to generate the appropriate extrusion force for dispensing the target volume. These spots were then air-dried for 30 minutes before use.

Claims

1. A method for fabricating a 3D structure comprising one or more bio-related materials, comprising: depositing a pattern comprising a triblock copolymer on a superhydrophobic surface to form a hydrophilic surface on the superhydrophobic surface, wherein the pattern is modeled after a biological structure and the triblock copolymer has an amphiphilic block structure that imparts hydrophilicity and hydrophobicity to the pattern; providing a composition comprising one or more bio-related materials dispersed in a biocompatible medium; bioprinting the composition onto the hydrophilic surface to form a 3D structure comprising the one or more bio-related materials, wherein the hydrophilic surface maintains the 3D structure at a desired position or shape on the superhydrophobic surface.

2. The method according to claim 1, wherein the biocompatible medium is a hydrogel.

3. The method according to claim 2, wherein the hydrogel comprises type I collagen.

4. The method according to claim 1, wherein modeling the biological structure after the model comprises computer-aided design (CAD).

5. The method according to claim 1, wherein the bio-related materials comprise stromal vascular fraction, microvascular fragments, or stem cells.

6. The method according to claim 5, wherein the stem cells are embryonic stem cells, adult stem cells, or pluripotent stem cells.

7. The method according to claim 1, wherein the bio-related materials comprise one or more cells suitable for tissue or organ repair, reconstruction, or regrowth.

8. The method according to claim 7, wherein the one or more cells suitable for tissue or organ repair, reconstruction, or regrowth comprise neurons, cardiomyocytes, muscle cells, vascular cells, or gastrointestinal smooth muscle cells, chondrocytes, pancreatic acinar cells, islets of Langerhans, islet beta cells, osteocytes, hepatocytes, Kupffer cells, fibroblasts, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, or bile duct epithelial cells.

9. The method according to claim 1, further comprising incubating the 3D structure at physiological temperature for a suitable period of time after bioprinting the 3D structure.

10. The method according to claim 1, further comprising culturing the 3D structure in a cell culture medium after bioprinting the 3D structure.

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