Multi-modality method and system for printing three-dimensional structures or parts thereof - Patents.com

Abstract

Provided are multi-modality methods and systems for printing three-dimensional (3D) constructs or portions thereof, the methods including: (i) supporting a flowable precursor with a flowable support at a location that allows cross-linking of the flowable precursor via irradiation with a radiation source, (ii) depositing one or more materials on / within the flowable precursor, and (iii) cross-linking at least a portion of the flowable precursor that is in contact with the one or more materials in the flowable precursor via irradiation with a radiation source to form the 3D construct or portion thereof.

Description

[Technical field]

[0001] The present disclosure relates generally to multi-modality methods and systems for printing three-dimensional (3D) structures or portions thereof. [Background technology]

[0002] Multimodality 3D printing is an area of ​​great interest as most manufactured items are made from multiple materials. Multimodality printing can be used in bioprinting to combine both soft and hard materials to create structures such as bone / ligament interfaces, for example, as well as bioprinted constructs that contain blood vessels. Multimodality 3D printing can also combine other material deposition methods, such as inkjet printing, to establish concentration gradients of materials, such as biologics, within the bioprinted construct.

[0003] However, conventional 3D printers may not be suitable for multi-modality printing because they typically have two solid surfaces: a build plate and a patterning screen / projection window. During use, the build plate is typically brought close to the patterning screen and gradually raised as the material is crosslinked. Such a configuration leaves no space between the build plate and the patterning screen to introduce secondary modalities (e.g., extruders and / or print heads) for depositing other materials.

[0004] Furthermore, the majority of bioprinters employ a syringe extrusion method, where hydrogel bioink, potentially containing cells, is deposited by rastering the syringe to create each layer and then proceeding to the next. In such systems, there is a trade-off between resolution, speed, and cell compatibility, and one of them always has to be sacrificed. For example, the inventors have previously developed a 3D printer capable of printing live cells at speeds of up to 200 mm / hour, at least an order of magnitude faster than previous systems. However, the absence of a vasculature in the printed structures limits the thickness of the structures to a few hundred microns before diffusion limitations affect cell viability.

[0005] Therefore, there is a need for a multi-modality method and system for printing 3D structures or parts thereof that seeks to address or at least ameliorate one of the above problems. Summary of the Invention

[0006] In one aspect, a multi-modality method of printing a three-dimensional (3D) construct or portion thereof is provided, the method comprising the steps of: (i) supporting a flowable precursor with a flowable support at a location where the flowable precursor can be crosslinked via irradiation with a radiation source, (ii) depositing one or more materials on / within the flowable precursor, and (iii) crosslinking at least a portion of the flowable precursor in contact with the one or more materials in the flowable precursor via irradiation with a radiation source to form the 3D construct or portion thereof.

[0007] In one embodiment, the method further comprises (iv) supporting the flowable precursor at a new location where the flowable precursor can be crosslinked via irradiation with a radiation source; (v) optionally further depositing one or more materials on / within the flowable precursor; (vi) optionally further crosslinking at least a portion of the flowable precursor in contact with the one or more materials at the new location via irradiation with a radiation source to form a 3D construct or a portion thereof; and (vii) optionally repeating steps (iv) to (vi) until a desired 3D construct is formed.

[0008] In one embodiment, depositing one or more materials comprises extruding a sacrificial material to form an array of one or more sacrificial structures within the 3D construct.

[0009] In one embodiment, the method further comprises removing sacrificial material from the array of one or more sacrificial structures to form an array of one or more channels in the 3D construct.

[0010] In one embodiment, the one or more channels comprise an open end facilitating fluid communication between the interior environment of the 3D construct and the exterior environment of the 3D construct.

[0011] In one embodiment, the sacrificial material is in a semi-solid or gel state when extruded to form the array of one or more sacrificial structures.

[0012] In an embodiment, removing the sacrificial material comprises liquefying the sacrificial material to facilitate removal of the sacrificial material from the 3D construct or portion thereof.

[0013] In one embodiment, liquefying the sacrificial material comprises cooling the sacrificial material to a temperature that causes the sacrificial material to liquefy.

[0014] In one embodiment, depositing the one or more materials includes propelling droplets containing the one or more materials onto the flowable precursor.

[0015] In one embodiment, propelling droplets containing one or more materials onto the flowable precursor comprises propelling the droplets at variable speeds such that a spatial concentration gradient of the one or more materials is formed within the 3D construct.

[0016] In one embodiment, the step of cross-linking via irradiation with a radiation source is carried out in a bottom-up direction, such that the 3D construct or part thereof is formed layer-by-layer in a bottom-up direction.

[0017] In one embodiment, the step of crosslinking via irradiation with a radiation source is carried out continuously.

[0018] In one embodiment, depositing one or more materials on / within the flowable precursor comprises depositing one or more materials in a top-down direction toward a top surface of the flowable precursor.

[0019] In one embodiment, the flowable support is immiscible with the flowable precursor.

[0020] In one embodiment, the flowable precursor and the one or more materials are biocompatible.

[0021] In one embodiment, the 3D construct has a higher density than the flowable precursor and the flowable support.

[0022] In one embodiment, the flowable precursor and / or the one or more materials include cells.

[0023] In one aspect, a multi-modality system for implementing the multi-modality method disclosed herein is provided, the system comprising: a tank configured to contain a flowable support and a flowable precursor disposed above the flowable support, an irradiation source configured to irradiate the flowable precursor, and one or more dispensing outlets disposed between the tank and the irradiation source and configured to deposit one or more materials on / in the flowable precursor, wherein the irradiation source is configured to crosslink via irradiation at least a portion of the flowable precursor in contact with the one or more materials in the flowable precursor to form a 3D construct or a portion thereof.

[0024] In one embodiment, the one or more dispensing outlets comprise an extruder nozzle for extruding a sacrificial material to form a template for an array of one or more channels in a 3D construct.

[0025] In one embodiment, the one or more dispensing outlets comprise inkjet nozzles for propelling droplets containing the one or more materials onto the flowable precursor.

[0026] definition As used herein, the term "biocompatibility" should be broadly interpreted to refer to the ability of a material to perform its intended function without eliciting significant inflammatory responses, immunogenicity, or cytotoxicity to native cells, tissues, or organs.

[0027] As used herein, unless otherwise specified, the term "cell" refers to an individual cell, a cell line, a primary culture, or a culture derived from such a cell.

[0028] As used herein, the term "substrate" should be interpreted broadly to refer to any supporting structure.

[0029] The term "layer", when used to describe a first material, should be broadly interpreted to refer to a first depth of the first material that is distinct from a second depth of the second material. The layer of the first material may exist as a continuous film, discontinuous structures, or a mixture of both. The layer may also be of substantially uniform depth throughout or of varying depth. Thus, when a layer is formed by discrete structures, each of the discrete structures may have different dimensions. The first and second materials may be the same or different, and the first and second depths may be the same or different.

[0030] The term "continuous," when used to describe the printing of a 3D construct, broadly refers to a method in which the formation of the 3D construct is performed without any significant stops, interruptions, or pauses occurring between successive portions or sections of the formed 3D construct.

[0031] The term "substantially transparent to light," as used herein to describe an object, should be broadly interpreted to mean that 50% or more of incident light perpendicular to the surface of the object can be transmitted through the object. In some examples, an object that is substantially transparent to light can transmit 60% or more, 65% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more of incident light perpendicular to the surface of the object through which it is transmitted. In one example, an object that is substantially transparent to light can transmit more than 70% of incident light perpendicular to the surface of the object.

[0032] As used herein, the term "micro" should be interpreted broadly to include dimensions from about 1 micron to about 1000 microns.

[0033] As used herein, the term "nano" should be interpreted broadly to include dimensions less than about 1000 nm.

[0034] The term "particle" as used herein broadly refers to a discrete object or discrete body. The particles described herein may include organic particles, inorganic particles, or biological particles. The particles described herein may also be macroparticles formed by an aggregate of multiple subparticles or fragments of smaller objects. The particles of the present disclosure may be spherical, substantially spherical, or non-spherical, such as irregularly shaped particles or ellipsoidal particles. The term "size", when used to refer to a particle, broadly refers to the largest dimension of the particle. For example, if the particle is substantially spherical, the term "size" may refer to the diameter of the particle. Alternatively, if the particle is substantially non-spherical, the term "size" may refer to the largest length of the particle.

[0035] The terms "coupled" or "connected," as used in this description, are intended to encompass both direct connection and connection through one or more intermediary means, unless otherwise specified.

[0036] The term "associated" as used herein when referring to two elements refers to a broad relationship between the two elements, including, but not limited to, a physical, chemical, or biological relationship. For example, if element A is associated with element B, then elements A and B are directly or indirectly bound to each other, or element A contains element B, or vice versa.

[0037] The term "adjacent" as used herein when referring to two elements refers to the proximity of one element to another, which may be elements that are in contact with one another, but is not limited to such elements, and may further include elements that are separated by one or more additional elements.

[0038] The term "and / or", e.g., "X and / or Y", is understood to mean either "X and Y" or "X or Y" and should be interpreted as explicitly endorsing both meanings or either meaning.

[0039] Further, in the present description, the term "substantially", whenever used, is understood to include, but is not limited to, "entirely", or "completely", etc. Furthermore, terms such as "comprising", "including", and the like, whenever used, are intended to be open-ended descriptive language in that they broadly include the elements / components described after such terms, in addition to other components not expressly described. For example, when using "comprising", a reference to "a" feature is also intended to be a reference to "at least one" of that feature. Terms such as "consisting of", "comprising", and the like, may be considered as subsets of terms such as "comprising", "comprises", and the like, in the appropriate context. Thus, in embodiments disclosed herein using terms such as "comprising", "comprising", and the like, it will be understood that such embodiments teach for corresponding embodiments using terms such as "consisting of", "comprising", and the like. Furthermore, terms such as "about", "approximately", and the like, whenever used, typically refer to reasonable variations, such as + / - 5% variation of the disclosed value, 4% variation of the disclosed value, 3% variation of the disclosed value, 2% variation of the disclosed value, or 1% variation of the disclosed value.

[0040] In addition, the description herein may disclose specific values ​​within a range. The endpoints of the range are intended to indicate the preferred range. Whenever a range is described, it is intended that the range encompasses and teaches all possible subranges as well as individual numerical values ​​within the range. That is, the endpoints of the range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to specifically disclose values ​​within the range of 1%, 2%, 3%, 4%, 5%, etc., individually, as well as subranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc. Individual numerical values ​​within the range should be understood to include integers, fractions, and decimals as well. In addition, whenever a range is described, it is intended that the range encompasses and teaches values ​​from the numerical endpoints described to two or two additional decimal places or significant digits (as appropriate) after the decimal point. For example, a description of a range of 1% to 5% is intended to specifically disclose 1.00% to 5.00%, as well as the range of 1.0% to 5.0% and all intermediate values ​​therein (e.g., 1.01%, 1.02%...4.98%, 4.99%, 5.00% and 1.1%, 1.2%...4.8%, 4.9%, 5.0%). The above specific disclosure intent is applicable to any depth / breadth of range.

[0041] Furthermore, when describing some embodiments, the present disclosure may disclose a method and / or process as a particular sequence of steps. However, it will be understood that unless otherwise required, the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps are possible. The particular order of steps disclosed herein should not be construed as unduly limiting. Unless otherwise required, the method and / or process disclosed herein should not be limited to steps performed in the order described. The order of steps may be altered and still fall within the scope of the present disclosure.

[0042] Furthermore, while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, it will be understood that one or more of such features / characteristics may also be waived in other alternative embodiments, and the present disclosure provides support for such waivers and such related alternative embodiments.

[0043] Description of the embodiments Non-limiting embodiments of a multi-modality method for printing a 3D construct or a portion thereof and a multi-modality system for implementing the multi-modality method for printing a 3D construct or a portion thereof are disclosed below.

[0044] In various embodiments, a multi-modality method of printing a three-dimensional (3D) construct or portion thereof is provided, which includes the steps of: (i) supporting a flowable precursor with a flowable support in a position that allows for cross-linking of the flowable precursor via irradiation with a radiation source, (ii) depositing one or more materials on / within the flowable precursor, and (iii) cross-linking at least a portion of the flowable precursor in contact with the one or more materials in the flowable precursor via irradiation with a radiation source to form the 3D construct or portion thereof.

[0045] In various embodiments, the one or more materials are deposited using one or more modalities different from the modality for crosslinking the flowable precursor. In various embodiments, crosslinking the flowable precursor via irradiation by an irradiation source is performed by a first printing modality. The first printing modality may be, for example, a Fluid-Supporting Liquid Interfacial Polymerization (FLIP) printer according to various embodiments disclosed herein. In various embodiments, depositing the one or more materials on / in the flowable precursor is performed using a second or additional printing modality, such as inkjet or extrusion printing, different from the first printing modality (e.g., Fluid-Supporting Liquid Interfacial Polymerization). In various embodiments, when more than two printing modalities are used in the method, the second and additional (e.g., third) printing modalities may be the same or different from each other, but both the second and third printing modalities are different from the first printing modality.

[0046] In various embodiments, the one or more materials may be the same or different from the crosslinked flowable precursor. In various embodiments, the one or more materials may differ from the crosslinked flowable precursor in terms of physical / mechanical characteristics / properties (e.g., shape, size, mass, volume, density, melting point, ductility, stiffness, etc.) and / or chemical characteristics / properties (e.g., chemical composition, pH, reactivity, reaction rate, ionic strength, bond strength, etc.). In various embodiments, when multiple different materials are deposited on / in the flowable precursor, the modalities used for depositing the different materials may be different from each other. In various embodiments, the one or more materials may be incorporated into the 3D construct, e.g., the final structure of the 3D construct.

[0047] In various embodiments, the method further comprises (iv) supporting the flowable precursor at a new location that allows for crosslinking of the flowable precursor via irradiation with a radiation source, (v) optionally further depositing one or more materials on / within the flowable precursor, (vi) optionally further crosslinking at least a portion of the flowable precursor that contacts the one or more materials at the new location via irradiation with a radiation source to form a 3D construct or a portion thereof, and (vii) optionally repeating steps (iv) through (vi) until a desired 3D construct is formed. In various embodiments, step (v) of further depositing one or more materials on / within the flowable precursor may be optional for some repetitions of steps (iv) through (vi), such that the one or more materials are present only in selected portions or regions of the 3D construct.

[0048] Advantageously, the multi-modality method may be used to print 3D constructs that include two or more materials, i.e., multi-material printing. Various embodiments of the method may be used in bioprinting applications to print living 3D constructs (i.e., constructs that include cells), such as bioprinted constructs including bone / ligament interfaces and blood vessels, constructs with complex geometries related to anatomical structures, etc.

[0049] In various embodiments, the fluid precursor supported by the fluid support includes a top surface that interacts with or is exposed to the fluid medium. In various embodiments, the fluid medium may be air or an inert gas or liquid (e.g., mineral oil or silicone oil) that does not react with the precursor and / or does not react (e.g., crosslink) in the presence of irradiation by the irradiation source. In various embodiments, the fluid medium may also be one that allows the radiation to pass through. In various embodiments, the method does not have a screen or panel (e.g., a glass screen) that contacts the top surface of the fluid precursor. In various embodiments, the pattern is projected directly onto a thin layer (e.g., about 3 mm thick) of the fluid-supported precursor (e.g., a hydrogel precursor) that acts as a floating liquid projection screen. This advantageously avoids inadvertent adhesion to the screen or panel (e.g., a glass screen) that affects typical resin-based 3D printers, allowing for high-speed continuous printing.

[0050] In various embodiments, the top surface of the flowable precursor provides a relatively unobstructed free surface at the precursor-fluid medium interface. Advantageously, such a configuration of the first printing modality provides great flexibility in introducing / integrating other different printing modalities due to the degree of spatial access provided by the free surface. The free surface of the flowable precursor may advantageously enable or facilitate the addition of one or more materials onto the flowable precursor. For example, in a 3D printer setting for carrying out the methods disclosed herein (e.g., a fluid-supported liquid interfacial polymerization (FLIP) printer), the free surface may advantageously allow other printing modalities, such as extrusion or piezoelectric deposition of one or more different materials, to be combined or integrated into the 3D printer setting. Some advantages of using different modalities may include the ability to increase throughput (e.g., some other modalities may allow for faster printing of certain finer and more complex structures within the 3D construct), the ease of introducing different types of materials into the 3D construct, and the ability to intermittently cool / deactivate / recharge different modalities while still allowing for uninterrupted continuous printing when swapping between different modalities during operation, improving part lifespan and increasing efficiency.

[0051] In various embodiments, the flowable precursor supported by the flowable support includes a bottom surface that interacts with or is exposed to the flowable support. In other words, the flowable precursor is disposed (e.g., suspended) above the flowable support. In various embodiments, the flowable precursor may directly interact with the flowable support or indirectly interact with one or more intervening intermediate layers (e.g., another flowable liquid or a separate substrate / solid) disposed between the flowable precursor and the flowable support. In various embodiments, the flowable precursor is immiscible with the flowable support and forms a distinct phase separate from each other. Advantageously, the flowable support provides the necessary buoyancy to support the flowable precursor, such that the flowable precursor is properly presented / supported toward the irradiation source for crosslinking. In various embodiments, the buoyancy of the flowable support is sufficient to support the flowable precursor but insufficient to float the 3D construct in / on the flowable support. Thus, in various embodiments, the 3D construct is still supported by the build plate. In various embodiments, the flowable support has a density higher than that of the flowable precursor and the fluid medium has a density lower than that of the flowable precursor. In various embodiments, the 3D construct has a density greater than the density of the flowable precursor, the flowable support, and the fluid medium.

[0052] In various embodiments, the flowable precursor is disposed between the flowable support and the fluid medium (e.g., air), e.g., the flowable precursor interacts with the flowable support as well as the fluid medium. In various embodiments, the flowable precursor (the entire flowable precursor) may directly interact with the flowable support and the fluid medium. That is, there are no other intervening layers. In other words, the flowable precursor may be in direct contact with the fluid medium and the flowable support, e.g., the top surface of the flowable precursor forms an interface with the fluid medium and the bottom surface of the flowable precursor forms an interface with the flowable support. In various embodiments, the flowable precursor interacts with the flowable support as well as the fluid medium in a flowable support-flowable precursor-fluid medium configuration. In other words, the flowable precursor may be sandwiched between the flowable support and the fluid medium without the presence of an intermediate solid screen / panel (e.g., glass screen).

[0053] In various embodiments, supporting the fluid precursor at a new position includes adjusting / changing the position / level of the fluid precursor. In various embodiments, adjusting / changing the position / level of the fluid precursor includes adjusting / changing the position / level of the fluid support. For example, adjusting the position of the top surface of the cross-linked fluid precursor includes adjusting the position of the top interface of the fluid support (e.g., the top surface of the fluid support that interacts with another medium, such as a precursor). The adjusting step may then further include adjusting the position of an intervening intermediate layer. In various embodiments, adjusting the position of the top interface of the fluid support will change or adjust in conjunction with the position / level of any intervening intermediate layer and / or the position of said top surface of the fluid precursor. In various embodiments, as the position / level of the fluid precursor increases, a pattern corresponding to the cross-section of the 3D construct to be printed is sequentially transferred onto the fluid precursor by the irradiation source, so that the fluid precursor is cross-linked according to this pattern.

[0054] In various embodiments, adjusting / changing the position / level of the fluid support includes raising the level of the fluid support (e.g., by increasing the volume of the fluid support and / or moving the level of a base (which may be a solid substrate or an intermediate liquid layer) on which the fluid support is placed facing upwards without changing the volume of the fluid support. In various embodiments, increasing the volume of the fluid support may include maintaining a laminar flow (e.g., having a Reynolds number of about 3) of the fluid support into a vessel / tank in which the fluid precursor is held to minimize or prevent interruption of the crosslinking process of the fluid precursor. In various embodiments, since the fluid precursor interacts with the fluid support, adjusting may additionally / alternatively include adjusting the position of the interface between the fluid precursor and the fluid support.

[0055] In various embodiments, supporting the fluid precursor at a new position includes adjusting / changing the position / level of the fluid precursor relative to the 3D construct or part thereof. In various embodiments, adjusting / changing the relative position / level of the fluid precursor may include adjusting / changing the position / level of the 3D construct or part thereof. For example, adjusting / changing the position / level of the fluid precursor relative to the 3D construct or part thereof may include keeping the volume of the fluid support constant and changing the position of the 3D construct or part thereof relative to the fluid precursor. This may be achieved by moving the print bed supporting the 3D construct or part thereof downwards such that the relative position of the fluid precursor and the 3D construct or part thereof changes.

[0056] In various embodiments, the flowable support may be an immiscible liquid (with the precursor) and may be selected according to the flowable precursor. For example, in the case of an aqueous precursor, the flowable support may be a perfluoro oil such as FC-40, HFE7500, or an immiscible aqueous solution (i.e., to form an aqueous two-phase system). In the case of a common resin (when used as a flowable precursor), the support fluid (or flowable support) may be water. In various embodiments, one criterion for selecting the flowable support is that the flowable support is denser than the flowable precursor.

[0057] In various embodiments, the fluid precursor comprises at least one polymerizable monomer / crosslinkable polymer and at least one photoinitiator. In various embodiments, the fluid precursor is a hydrogel precursor that can be crosslinked to form a hydrogel. The hydrogel can be formed using either monomers (e.g., acrylic acid, methacrylic acid) or macromonomers (e.g., gelatin methacrylate, PEG-diacrylate). Non-limiting examples of polymers or copolymers suitable for forming hydrogels include polyacrylic acid, polymethacrylic acid, polyacrylamide, polymethacrylamide, polyvinylpyrrolidone and copolymers thereof. Other examples include polyethers, polyurethanes and polyethylene glycols that can be functionalized with crosslinking groups or used in combination with compatible crosslinkers. In various embodiments, the polymer chains can be modified with reactive groups such as acrylates or methacrylates to make the polymer chains photocrosslinkable. In various embodiments, the hydrogel may include polymers including, but not limited to, modified polycaprolactone, gelatin, gelatin methacrylate, alginate, alginate methacrylate, modified chitosan, chitosan methacrylate, glycol chitosan, glycol chitosan methacrylate, modified hyaluronic acid (HA), HA methacrylate, and other non-crosslinked natural or synthetic polymer chains. In various embodiments, the polymers may be modified to render the polymer crosslinkable. For example, acrylates, methacrylates, or other types of conjugation chemistries may be used to form covalent crosslinks. In various embodiments, the flowable precursor may include gelatin methacrylate (GelMA) and / or alginate methacrylate (ALMA).

[0058] In various embodiments, the photoinitiator may be a free radical photoinitiator that generates free radicals upon exposure to radiation, such as ultraviolet or visible light, thereby initiating the polymerization reaction. Non-limiting examples of suitable photoinitiators include benzophenones (aromatic ketones), such as benzophenone and methylbenzophenone, acylphosphine oxide type photoinitiators, such as diphenyl(2,4,6 trimethylbenzoyl)phosphine oxide, benzoins and benzoin alkyl ethers, such as benzoin, benzoin methyl ether, benzoin isobutyl ether, and the like.

[0059] In various embodiments, the flowable precursor starting material may include a monomer (e.g., acrylic acid) or a macromonomer (e.g., poly(ethylene glycol) diacrylate (PEGDA), a polymer chain modified with two acrylate groups). The macromonomer may have different chain lengths (e.g., from about 100 Da to about 10,000 kDa, from about 200 Da to about 9,500 kDa, from about 300 Da to about 9,000 kDa, from about 400 Da to about 8,500 kDa, from about 500 Da to about 8,000 kDa).

[0060] In various embodiments, the concentration of the monomer or macromonomer is in the range of about 1% to about 100% by weight of the total flowable precursor, depending on the type of material used to make the 3D construct. For 3D hydrogel constructs, the concentration of the monomer or macromonomer may be in a range having a start point and an end point selected from the following numerical groups: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, and 50% by weight of the total flowable precursor. For 3D plastic resin structures, the concentration of the monomer or macromonomer may be in a range having a start point and an end point selected from the following numerical groups: 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100% by weight of the total flowable precursor.

[0061] In various embodiments, the concentration of the photoinitiator is in the range of about 0.05% to about 10% by weight of the total flowable precursor. The concentration of the photoinitiator may range, for example, from about 0.05% to about 10%, from about 0.065% to about 9%, from about 0.08% to about 8%, from about 0.095% to about 7%, from about 0.11% to about 6%, or from about 0.125% to about 5%.

[0062] In various embodiments, the one or more materials in step (ii) are different from the flowable precursor. The one or more materials may differ from the flowable precursor in terms of physical and / or chemical characteristics / properties. In various embodiments, the one or more materials may be deposited using a different modality, for example, inkjet printing or extrusion printing.

[0063] In various embodiments, depositing the one or more materials includes extruding the sacrificial material to form an array of one or more sacrificial structures in the 3D construct. In various embodiments, the sacrificial material may be extruded from a dispensing outlet, for example, an extruder nozzle disposed between the irradiation source and the flowable precursor. The array of one or more sacrificial structures may include an array of one or more sacrificial fibers that serve as a mold / template for the array of one or more channels of the 3D construct. The sacrificial fibers may be straight or curved. The array of one or more sacrificial fibers may be arranged in a regular pattern or an irregular pattern. The one or more sacrificial structures may be shaped and sized according to a predetermined design and may serve as a mold / template for various structures in the 3D construct, such as overhanging structures, free-standing hollow structures, complex shapes related to anatomical structures, etc. The sacrificial structures themselves may also be three-dimensional and span different planes of the 3D construct.

[0064] In various embodiments, the method further comprises removing sacrificial material from the array of one or more sacrificial structures to form an array of one or more channels in the 3D construct. In various embodiments, the one or more channels comprise open ends that facilitate fluid communication between an internal environment of the 3D construct and an external environment of the 3D construct. In various embodiments, the method further comprises removing sacrificial material from the array of one or more sacrificial structures to form various structures in the 3D construct, such as overhanging structures, free-standing hollow structures, complex shapes associated with anatomical structures.

[0065] In various embodiments, the sacrificial material is in a semi-solid or gel state when it is extruded to form the sacrificial structure. Advantageously, the gel state helps the sacrificial structure maintain its shape and form in the liquid flowable precursor, such that the sacrificial material does not mix with the flowable precursor and does not substantially lose its shape and form during formation of the 3D construct.

[0066] In various embodiments, removing the sacrificial material may include liquefying the sacrificial material to facilitate its removal from the 3D construct or portion thereof. In various embodiments, liquefying the sacrificial material depends on the type of sacrificial material used. For example, the sacrificial material may be liquefied using physical methods (e.g., by changing the temperature of the sacrificial material), chemical methods (e.g., by modifying / changing the chemical composition of the sacrificial material), or a combination thereof.

[0067] In various embodiments, liquefying the sacrificial material may include cooling the sacrificial material to a temperature that liquefies the sacrificial material. Cooling the sacrificial material may include immersing the 3D construct or a portion thereof in a water bath having a temperature that liquefies the sacrificial material. In various embodiments, the sacrificial material may be a heat-sensitive material that changes its physical and / or chemical properties in response to a change in temperature. The temperature at which the physical and / or chemical properties change depends on the type of sacrificial material used.

[0068] In one example, the sacrificial material may include a fugitive ink (e.g., poloxamer 407, also known by the trade name Pluronic® F127). F127 is configured to switch between a liquid state and a semi-solid or gel state depending on the concentration. For example, at a concentration of 20% by weight, the F127 material is liquid below 20° C., gels from about 20° C. to about 70° C., and liquefies again above 70° C. The sacrificial ink is configured to maintain a gel state at the printing temperature and melt away when the temperature increases or decreases. In other words, other materials such as agarose can also be used if the printing application allows the temperature to be increased to 40° C. to 50° C. For bioprinting applications, high temperatures above 40° C. are not suitable for living cells as they may affect cell viability and even cause cell death. For this reason, the sacrificial material needs to be selected such that the printing temperature is suitable for the cells. For other printing applications not involving living cells, the choice of sacrificial material may be more flexible as cell viability may not be a considered factor in such printing applications.

[0069] In various embodiments, liquefying the sacrificial material may include changing / modifying the chemical composition of the sacrificial material to liquefy the sacrificial material. For example, changing the chemical composition of the sacrificial material may include removing a reversible crosslinker used to maintain the sacrificial material in a solid or semi-solid form. In various embodiments, liquefying the sacrificial material may include dissolving the sacrificial material. In various embodiments, the sacrificial material may include alginate. The alginate molecules form a gel in the presence of a crosslinking solution containing multivalent cations such as calcium. In various embodiments, the gelled alginate fibers may be extruded to form an array of one or more sacrificial structures in the 3D construct. In various embodiments, liquefying the sacrificial material made from alginate may include immersing the 3D construct in a chelating agent-containing buffer (e.g., citrate, EDTA, EGTA), which removes the cations and dissolves the alginate fibers.

[0070] In various embodiments, removing the sacrificial material is primarily accomplished by melting the sacrificial material (e.g., sacrificial ink) under appropriate temperature or chemical conditions. In various embodiments, removing the sacrificial material may further include actively removing or diluting the liquefied sacrificial material from the 3D construct, for example, by rinsing / washing with a fluid, such as water, a suitable buffer (e.g., a chelating agent-containing buffer), or cell culture medium, to allow the liquefied sacrificial material to drain from the 3D construct. In various embodiments, removing the sacrificial material may further include passively removing or diluting the liquefied sacrificial material from the 3D construct, for example, by immersing or submerging the 3D construct in a fluid, such as water, a suitable buffer (e.g., a chelating agent-containing buffer), or cell culture medium for a period of time, to allow the liquefied sacrificial material to drain from the 3D construct. In various embodiments, the temperature of the fluid (e.g., medium) is used to induce a phase change in the sacrificial material (e.g., sacrificial ink). In various embodiments, removing the sacrificial material may include both actively and passively removing the liquefied sacrificial material from the 3D construct.

[0071] In various embodiments, the 3D constructs comprising an array of one or more channels may be applied to bioprinting of 3D constructs for culturing cells. The presence of channels in the 3D constructs may advantageously address or ameliorate a fundamental challenge in bioprinting, which is to provide nutrients to cells in thick structures beyond the diffusion limit. The size, density and arrangement of the channels may be optimized to ensure a sufficient supply of nutrients to cells distributed throughout the 3D construct.

[0072] In various embodiments, depositing the one or more materials includes propelling droplets containing the one or more materials onto the fluid precursor. In various embodiments, the one or more materials may be propelled from a droplet dispensing outlet, e.g., an inkjet nozzle, an acoustic levitation nozzle, disposed between the irradiation source and the fluid precursor.

[0073] In various embodiments, propelling the droplets including the one or more materials onto the flowable precursor includes propelling the droplets at a substantially constant velocity such that the one or more materials are substantially uniformly distributed throughout the 3D construct. The one or more materials may be substantially uniformly distributed in selected regions of the 3D construct or throughout the 3D construct.

[0074] In various embodiments, propelling droplets containing one or more materials onto the flowable precursor includes propelling the droplets at variable speeds to form a spatial concentration gradient of the one or more materials onto the 3D construct, e.g., a spatial pattern of molecules within the 3D construct. The one or more materials may be dispersed in a spatial concentration gradient in selected regions of the 3D construct or throughout the 3D construct. A "spatial concentration gradient" is a concentration gradient in which the concentration may vary along one or more spatial dimensions.

[0075] Non-limiting examples of the one or more materials contained in the droplets include biological response modifiers, cytokines, growth factors, antigens, drugs, hormones, etc. In one example, the method may be used to print controlled spatial gradients of angiogenic factors to promote a desired vascularization outcome within a 3D construct for tissue engineering applications. In another example, the method may be used to print a 3D construct having a first region containing a first growth factor for promoting a first type of cell (e.g., a growth factor for promoting the growth of chondrocytes in the first region) and a second region containing a second growth factor for promoting a second type of cell (e.g., a growth factor for promoting the growth of bone cells in the second region).

[0076] In various embodiments, depositing the one or more materials may include extruding a sacrificial material to form an array of one or more sacrificial structures in the 3D construct only. In various embodiments, depositing the one or more materials may include propelling droplets containing the one or more materials onto the flowable precursor only. In various embodiments, depositing the one or more materials may include both extruding a sacrificial material to form an array of one or more sacrificial structures in the 3D construct and propelling droplets containing the one or more materials onto the flowable precursor.

[0077] In various embodiments, crosslinking of the fluid precursor occurs at, near, adjacent, or proximate to the interface between the fluid precursor and the fluid medium. Advantageously, in various embodiments, only a small amount or a thin layer of the fluid precursor needs to be used at any one time during crosslinking. In various embodiments, minimizing the thickness can help reduce the amount of expensive precursor used and also improve resolution by avoiding excessive photocrosslinking. This reduces waste of valuable resources while allowing better control of the formation of structures at high resolution and high speed. However, the amount of precursor material needs to be sufficient to print the desired structure (e.g., 10 mL of precursor volume is insufficient to print a 20 mL volume of structure). The actual amount of fluid precursor required depends on, for example, the precursor properties (e.g., viscosity, surface tension, etc.), but in various embodiments, as an example, a layer of fluid precursor about 3 mm thick may be used / sufficient to 3D print a structure. The amount of fluid precursor should ideally be sufficient to maintain a continuous layer of precursor. A lower surface tension (e.g., by use of a surfactant) can help the precursor spread across the flowable support, but may not be desirable if it is toxic to cells. In various embodiments, the layer of flowable precursor may have a thickness / height ranging from about 0.5 mm to about 5 mm. The layer of flowable precursor may be in a range having a start and end point selected from the following groups of values: 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, and 5 mm.

[0078] In various embodiments, cross-linking at least a portion of the precursor by irradiation includes irradiating at least a portion of the precursor to be cross-linked with electromagnetic radiation from an electromagnetic radiation source (i.e., irradiation source). In various embodiments, the flowable precursor is capable of cross-linking upon application of electromagnetic radiation. Electromagnetic radiation includes, but is not limited to, electromagnetic radiation having wavelengths in the infrared, ultraviolet, or visible light spectrum. In various embodiments, the flowable support is not cross-linkable by application of the same electromagnetic radiation that cross-links the flowable precursor. Similarly, in various embodiments, the fluid medium that interacts with the top layer of the flowable precursor is not cross-linkable by application of the same electromagnetic radiation that cross-links the flowable precursor.

[0079] In various embodiments, the electromagnetic radiation is in the form of a projection from an electromagnetic source (e.g., an illumination or irradiation source) onto the flowable precursor. In various embodiments, the projection onto the flowable precursor corresponds to a cross-sectional area of ​​the 3D construct formed by cross-linking of the precursor. In various embodiments, the projection (corresponding to a cross-sectional area of ​​the 3D construct formed) changes continuously as the location / level of said flowable precursor contacting or interacting with a portion of the 3D construct formed changes and / or cross-linking occurs.

[0080] In various embodiments, the flowable precursor is irradiated in a direction substantially perpendicular to a surface of the flowable precursor (e.g., the plane of the flowable precursor-fluid medium interface). In various embodiments, crosslinking via irradiation by an irradiation source is performed in a bottom-up direction such that the 3D construct or a portion thereof is formed layer-by-layer in a bottom-up direction. In various embodiments, depositing one or more materials on / in the flowable precursor includes depositing one or more materials in a top-down direction toward a top surface of the flowable precursor.

[0081] In various embodiments, the step of crosslinking at least a portion of the precursor via irradiation is performed simultaneously with the step of depositing one or more materials on / inside the flowable precursor. For example, a sacrificial material may be extruded to form an array of one or more sacrificial structures in the 3D construct while crosslinking occurs in the flowable precursor. Although the extrusion of the sacrificial material may require temporary positioning of an extruder nozzle between the projector and the flowable precursor, the extrusion of the sacrificial material may occur faster than the crosslinking of the flowable precursor. Thus, the extrusion of the sacrificial material may not significantly affect the crosslinking process. In alternative embodiments, the step of crosslinking at least a portion of the precursor via irradiation and the step of depositing one or more materials on / inside the flowable precursor may be performed sequentially. For example, one or more materials may be first deposited on / inside the flowable precursor, and then the flowable precursor is crosslinked by the irradiation source.

[0082] In various embodiments, the step of adjusting / changing the position / level of the flowable precursor is performed at a rate that substantially matches or substantially parallels (or is substantially proportional to) the crosslinking rate of the flowable precursor. For example, the step of adjusting the position of the top surface of the flowable precursor is performed at a rate that substantially matches the rate of crosslinking the flowable precursor. In various embodiments, the rate of increasing the volume of the flowable support and / or the rate of adjusting / changing the position / level of the flowable precursor matches or even parallels (or is substantially proportional to) the crosslinking rate of the flowable precursor. In various embodiments, the method is a continuous printing method and / or a high-speed printing method. In various embodiments, the method is a continuous printing method, even if, for example, different modalities for depositing one or more materials are used. In various embodiments, the step of crosslinking via irradiation by the irradiation source is performed continuously without pause. In various embodiments, continuous printing advantageously enables printing of 3D constructs that are substantially free of layering artifacts that may occur as a result of non-continuous printing processes.

[0083] In various embodiments, the method may further include removing the fluidic support from the 3D construct. During printing, the already printed 3D construct or a portion thereof is placed on the build plate and immersed in the fluidic support (i.e., the supporting fluid). After printing, the supporting fluid is removed from the printed 3D construct, for example, by rinsing with another fluid, such as water or a suitable buffer solution.

[0084] In various embodiments, the method may have a printing speed of about 0.05 mm / min (or about 3 mm / hr) to about 5 mm / min (or about 300 mm / hr) along a vertical axis perpendicular to the surface plane of the flowable precursor. For ease of understanding, the X and Y axes are defined as parallel to the surface plane of the flowable precursor, and the Z axis is defined as perpendicular to the surface plane of the flowable precursor. The print speed may be within a range having a beginning and an end point selected from the following group of values: 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 and 5 mm / min. In various embodiments, the printing speed may depend on factors such as the formulation of the flowable precursor, the reaction rate of the flowable precursor, and the behavior of the flowable precursor when crosslinked rapidly (e.g., whether warping of the structure occurs). Depending on the formulation of the flowable precursor, 3D constructs (e.g., soft hydrogel structures with complex shapes) may be printed using the methods disclosed herein in a matter of minutes to hours, depending on the materials used and the size of the construct. For example, for a 3 cm tall structure, the printing time may be as little as 5-10 minutes for fast curing materials, or several hours for very slow curing materials. In one embodiment, the method can print 3D hydrogel constructs at 3.3 mm / min, which is faster than many DLP (digital light processing) printers, which typically operate at about 0.1 mm / min to 1 mm / min.

[0085] In various embodiments, the 3D construct may include dimensions having a scale on the order of centimeters. In various embodiments, the channels formed within the 3D construct may include dimensions having a scale on the order of millimeters or sub-millimeters. In various embodiments, the 3D construct may be a 3D hydrogel construct having a stiffness ranging from about 7 kPa to about 4 MPa. The stiffness of the 3D hydrogel construct may be within the following group of values: 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, The stiffness may be within a range having a beginning and an end selected from 0, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, and 4000 kPa. In various embodiments, the 3D hydrogel construct may have a stiffness of greater than 4 MPa. In various embodiments, the 3D construct may be a 3D plastic resin construct having a stiffness of greater than about 0.1 GPa. In various embodiments, the 3D construct may have a stiffness of about 1 kPa or less depending on the material and structure printed.

[0086] In various embodiments, the method may be used for bioprinting of cell-containing 3D constructs. In various embodiments, the flowable precursor and one or more materials are biocompatible. In various embodiments, the flowable precursor and / or one or more materials include cells. In various embodiments, the method may advantageously achieve high speed printing of soft materials without exposure to high shear, extreme temperatures or other harsh conditions, and may be compatible with cell printing. Typically, the printing conditions are different (in terms of osmolarity, temperature, pH, etc.) from the final culture state of the printed construct, so the printing speed allows cells to be rapidly returned to optimal culture conditions. In various embodiments, the speed, ease of use, and versatility allow the method to be used for a wide range of manufacturing and biomedical applications.

[0087] In various embodiments, a system for carrying out the methods disclosed herein is provided, comprising a tank configured to contain a flowable support and a flowable precursor disposed above the flowable support, an irradiation source configured to irradiate the flowable precursor, and one or more dispensing outlets disposed between the tank and the irradiation source and configured to deposit one or more materials on / in the flowable precursor, wherein the irradiation source is configured to crosslink via irradiation at least a portion of the flowable precursor in contact with the one or more materials in the flowable precursor to form a 3D construct or a portion thereof.

[0088] In various embodiments, the system components (i.e., tank, fluid support, irradiation source) that cooperate to effect crosslinking of the fluid precursor are based on a first printing modality. The first printing modality may be, for example, a fluid-supported liquid interfacial polymerization (FLIP) printer according to various embodiments disclosed herein. In various embodiments, the one or more dispensing outlets are based on a second or additional printing modality, such as inkjet or extrusion printing, that is different from the first printing modality (e.g., fluid-supported liquid interfacial polymerization).

[0089] In various embodiments, the first printing modality of the system provides a relatively unobstructed free surface on the top surface of the flowable precursor. The free surface of the flowable precursor may advantageously provide high flexibility in introducing / integrating other different modalities due to the degree of spatial access provided by the free surface, thereby enabling or facilitating the addition of one or more materials on / inside the flowable precursor. Advantageously, various embodiments of the system overcome the limitations of conventional 3D printers that lack space between the build plate and the patterning screen to introduce / integrate other different printing modalities. Some advantages of using different modalities may include the ability to increase throughput (e.g., some other modalities may print certain finer and more complex structures in the 3D construct faster), ease of introducing different types of materials to the 3D construct, and improved part life and efficiency due to the ability to continuously print without interruption while allowing intermittent cooling / non-operation / recharging when switching between different modalities during operation.

[0090] In various embodiments, the one or more dispensing outlets comprise an extruder nozzle for extruding the sacrificial material to form a template for an array of one or more channels of the 3D construct. In various embodiments, the one or more dispensing outlets comprise a propelling nozzle (e.g., inkjet nozzle, acoustic levitation nozzle) for propelling droplets containing the one or more materials onto the flowable precursor. In various embodiments, the one or more dispensing outlets (e.g., extruder nozzle, inkjet nozzle, acoustic levitation nozzle) are coupled to respective reservoirs containing the one or more materials (e.g., sacrificial material, additives).

[0091] In various embodiments, the propulsion nozzle may be configured to propel droplets containing the one or more materials at a substantially uniform velocity onto the flowable precursor such that the one or more materials are substantially uniformly distributed throughout the 3D construct. The propulsion nozzle may be configured to distribute the one or more materials substantially uniformly over selected regions of the 3D construct or throughout the 3D construct.

[0092] In various embodiments, the propulsion nozzle may be configured to propel droplets containing one or more materials at variable speeds onto the flowable precursor such that a spatial concentration gradient of the one or more materials is formed in the 3D construct, e.g., in a spatial pattern of molecules within the 3D construct. The propulsion nozzle may be configured to disperse the one or more materials in a spatial concentration gradient in selected regions of the 3D construct or throughout the 3D construct.

[0093] In various embodiments, one or more dispensing outlets (e.g., extruder nozzle, inkjet nozzle, acoustic levitation nozzle) are configured to be movable. One or more dispensing outlets may be configured to be movable along one or more axes. For example, one or more dispensing outlets may be configured to be movable in a horizontal direction substantially parallel to a surface of the flowable precursor. One or more dispensing outlets may be configured to be movable in a vertical direction substantially perpendicular to a surface of the flowable precursor. The movability of one or more dispensing outlets may advantageously facilitate deposition of one or more materials on / within the flowable precursor.

[0094] In various embodiments, the system may include a combination of an extruder nozzle for extruding a sacrificial material to form an array of one or more sacrificial structures in a 3D construct, and a propelling nozzle for propelling droplets containing one or more materials onto a flowable precursor. Advantageously, various embodiments of the system may combine extrusion-based printing techniques with continuous 3D printing methods such as DLP (Digital Light Processing) to create a multi-modality printer that can be used to create bioprinting structures with channels that can supply cells with nutrients necessary for their survival. In addition to a fiber extruder for creating sacrificial structures of channels, various embodiments of the system may also be used in combination with a propelling nozzle, such as an inkjet nozzle and / or an acoustic levitation nozzle, that can establish a concentration gradient of additives, such as cytokines, growth factors, etc., to create a heterogeneous matrix.

[0095] In various embodiments, the vessel / tank includes an inlet (i.e., at least one inlet) that allows for the flow of the fluid support at a predetermined rate. The system may further include an actuator (e.g., a pump, a lift arm) for facilitating the flow of the fluid support through the inlet of the vessel / tank. In various embodiments, the vessel / tank may include one or more inlets, and in such embodiments, the actuator may accordingly facilitate the flow of the fluid support through the one or more inlets of the vessel / tank.

[0096] In various embodiments, the system further comprises a processing module configured to adjust the position of said top surface of the flowable precursor at a rate substantially corresponding to the rate at which the flowable precursor is cross-linked by the irradiation source. In various embodiments, the processing module may be configured to control, for example, the flow rate of the flowable support to the vessel / tank, the intensity of the irradiation by the irradiation source, and the position of the irradiation source to synchronize these parameters that may affect the rate at which the position of the top surface of the flowable precursor is adjusted and / or the rate at which the flowable precursor is cross-linked by the irradiation source. The processing module may also control the type and amount of one or more materials deposited on / within the flowable precursor, as well as the timing and rate of deposition. The processing module may also control and determine the type or shape of the projection irradiated on the flowable precursor as the relative level of the flowable precursor (e.g., relative to a portion of an already formed 3D construct) along a vertical axis perpendicular to the top surface of the flowable precursor is changed or altered.

[0097] In various embodiments, the system further comprises a reservoir for holding a volume of the fluidic support outside the container / tank. In various embodiments, the reservoir is in fluid communication with the container / tank (e.g., via an inlet). In various embodiments, the fluidic support is transferred from the reservoir to the container / tank using a passive pumping system, e.g., a gravity pumping system, by raising the position of the reservoir relative to the container / tank (e.g., by using a lift arm), thereby causing the fluidic support to flow into the container / tank. Advantageously, this ensures a laminar flow of the fluidic support into the container / tank, minimizing or preventing interruption of the crosslinking process of the fluidic precursor.

[0098] In various embodiments, the system further comprises a print bed / build plate disposed on or near a base of the vessel / tank. The print bed can support the 3D structure or a portion thereof. In various embodiments, the position of the build plate can also be adjustable (e.g., by the processing module) along a vertical axis substantially perpendicular to a top surface of the flowable precursor such that the position of the 3D structure or a portion thereof relative to the flowable precursor is altered.

[0099] In various embodiments, the illumination sources are positioned over the tank (e.g., a top-down projection system), beneath the tank / object (e.g., a bottom-up projection system), to the side of the tank / object (e.g., a side projection system), and / or around the tank / object (e.g., a circumferential projection system such as a radial / ring projection system).

[0100] In various embodiments, the container / tank is substantially transparent to the radiation from the radiation source. For example, the container / tank allows electromagnetic waves emitted from the radiation source to be transmitted to the flowable precursor. In various embodiments, such a container / tank may be particularly useful when using a projection system other than a top-down projection system, i.e., when using a bottom-up projection system, a side projection system, and / or a circumferential projection system. In various embodiments, the flowable precursor, the flowable support, and the 3D construct or a portion thereof are disposed in the tank.

[0101] In various embodiments, a multi-modality 3D printer is provided that may be used to create cell-laden hydrogel structures, utilizing a 3D printer with a free surface of a flowable precursor. In various embodiments, the multi-modality 3D printer is combined with other printing modalities, such as extrusion or piezoelectric deposition of different materials. In various embodiments, the multi-modality 3D printer uses specific inks for creating cell-laden structures (e.g., gelatin methacrylate, alginate methacrylate, etc.), as well as fugitive inks (e.g., Pluronic® F127) that liquefy at a low but cell-compatible temperature to create channels in the cell-laden structures as a means of supplying nutrients. In various embodiments, the multi-modality 3D printer combines free surface printing with inkjet deposition of molecules (e.g., biologics) to generate a concentration gradient of the molecules within the hydrogel structures. [Brief description of the drawings]

[0102] [Figure 1] FIG. 1 is a schematic diagram illustrating a side view of a system for printing a 3D construct or a portion thereof (e.g., a Fluid-Supported Liquid Interfacial Polymerization (FLIP) printer) according to various embodiments disclosed herein. [Diagram 2] FIG. 2 is a schematic diagram showing a side view of a conventional 3D printing system. [Figure 3A] FIG. 3A is a schematic diagram illustrating a method of printing a 3D construction or a portion thereof using a FLIP printer, according to various embodiments disclosed herein. [Figure 3B] FIG. 3B is a schematic diagram illustrating a method of printing a 3D construction or a portion thereof using a FLIP printer according to various embodiments disclosed herein. [Figure 3C] FIG. 3C is a schematic diagram illustrating a method of printing a 3D construction or a portion thereof using a FLIP printer according to various embodiments disclosed herein. [Figure 3D]FIG. 3D is a schematic diagram illustrating a method of printing a 3D construction or a portion thereof using a FLIP printer according to various embodiments disclosed herein. [Figure 4A] FIG. 4A is a screenshot from a video showing the process of printing a 3D construct, according to various embodiments disclosed herein. [Figure 4B] FIG. 4B is a screenshot from a video showing the process of printing a 3D construct, according to various embodiments disclosed herein. [Figure 4C] FIG. 4C is a screenshot from a video showing the process of printing a 3D construct, according to various embodiments disclosed herein. [Figure 4D] FIG. 4D is an image of a cross section of the 3D construct showing the removed channels within the 3D construct. [Figure 5A] 5A is a micrograph showing a cross-section of a hydrogel construct according to various embodiments disclosed herein. Scale bar = 200 microns. [Figure 5B] 5B is a micrograph showing a cross-section of a hydrogel construct according to various embodiments disclosed herein. Scale bar = 200 microns. [Figure 6A] 6A is a micrograph showing a cross-section of another hydrogel construct according to various embodiments disclosed herein. Scale bar = 200 microns. [Figure 6B] 6B is a micrograph showing a cross-section of another hydrogel construct according to various embodiments disclosed herein. Scale bar = 200 microns. [Figure 6C] 6C is a micrograph showing a cross-section of another hydrogel construct according to various embodiments disclosed herein. Scale bar = 200 microns. [Figure 7] FIG. 7 is a state diagram for Pluronic® F127. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0103] Exemplary embodiments of the present disclosure will be better understood and readily apparent to those skilled in the art from the following discussion, when appropriate, in conjunction with the drawings. It should be understood that other modifications related to structural changes may be implemented without departing from the scope of the present invention. Exemplary embodiments are not necessarily mutually exclusive, since some embodiments may be combined with one or more embodiments to form new exemplary embodiments. Exemplary embodiments should not be interpreted as limiting the scope of the present disclosure.

[0104] In the following example, a strategy for printing 3D constructs is described.

[0105] (System for printing 3D structures) FIG. 1 is a schematic diagram illustrating a side view of a system 100 (e.g., a fluid-supported liquid interfacial polymerization (FLIP) printer) for printing a 3D construct 102 or a portion thereof, according to various embodiments disclosed herein. The system 100 includes a tank 104 configured to contain a flowable support 106 (e.g., a supporting fluid) and a flowable precursor 108 (e.g., a hydrogel precursor) disposed above the flowable support 106. The system 100 further includes an irradiation source 110 (e.g., a patterning light source or projector) configured to irradiate the flowable precursor 108. The system 100 further includes a dispensing outlet 112 disposed between the tank 104 and the irradiation source 110 and configured to deposit a material 114 on / in the flowable precursor 108. The system 100 may include two or more dispensing outlets 112 configured to deposit two or more materials 114 on / in the flowable precursor 108. The irradiation source 110 is configured to crosslink at least a portion of the flowable precursor 108 in contact with the material 114 via irradiation to form the 3D construct 102 or a portion thereof. The formed 3D construct 102 or a portion thereof is disposed on a build plate 116.

[0106] In the system 100 shown in FIG. 1, a pattern from an irradiation source 110 is projected directly onto the flowable precursor 108, and photocrosslinking occurs at the surface of the flowable precursor 108, which is suspended above the flowable support 106. This configuration frees up the top surface of the flowable precursor 108 and provides a free surface to allow a dispensing outlet 112 (e.g., an extruder) to be introduced into the gap between the irradiation source 110 and the flowable precursor 108 to deposit a different material (e.g., a second material different from the flowable precursor 108) into the flowable precursor 108 and onto the initial printing of the 3D construct 102 or a portion thereof. Such a configuration may address the problem faced with conventional 3D printers, which lack the space to introduce additional printing modalities different from the FLIP printing modality.

[0107] For example, FIG. 2 is a schematic diagram showing a side view of a conventional 3D printing system 200, highlighting the problems faced by conventional 3D printers. The 3D printing system 200 includes a build plate 202, a patterning screen 204 (e.g., an oxygen-permeable window), and an imaging unit 206 configured to project a patterned light image onto a mirror 208, which in turn projects the patterned light image onto the patterning screen 204. The conventional 3D printing system 200 includes two solid surfaces: the build plate 202 and the patterning screen 204. In use, the build plate 202 is brought close to the patterning screen 204 and gradually lifts as it crosslinks the liquid resin 210 to form a 3D construct 212, or a portion thereof, that hangs from the underside of the build plate 202. In the conventional 3D printing system 200, a blind spot 214 is formed between the build plate 202 and the patterning screen 204 as the 3D construct 212, or a portion thereof, is formed. This leaves no space between the build plate 202 and the patterning window 204 to introduce other printing modalities for depositing other materials.

[0108] (How to print 3D structures) 3A-3D are a series of schematic diagrams illustrating a method of printing a 3D construction or a portion thereof using a FLIP printer, according to various embodiments disclosed herein.

[0109] As shown in Figure 3A, a flowable precursor 308 (compare 108 in Figure 1) is supported by a flowable support 306 (compare 106 in Figure 1) in a position that allows the flowable precursor 308 to be crosslinked via irradiation by an irradiation source (not shown) (compare 110 in Figure 1). The flowable precursor 308 is positioned to contact the top surface of a portion of an already printed cylindrical construct 302 (e.g., hydrogel construct) (compare 102 in Figure 1). The portion of the cylindrical construct 302 is placed on a build plate 316 (compare 116 in Figure 1).

[0110] In FLIP, photocrosslinking occurs within the floating precursor layer 308. The space above the fluid precursor 308 is relatively unobstructed, which provides a free surface so that an extruder 312 (compare 112 in FIG. 1) based on a different printing modality can be used to deposit fugitive ink fibers (e.g., Pluronic® F127 fibers) onto the print in the early stages while printing is still in progress. As shown in FIG. 3A, fibers 314 (compare 114 in FIG. 1) are formed on the top surface of the cylindrical construct 302 as the fugitive ink extruded from the extruder 312 translates horizontally across the fluid precursor 308, as indicated by block arrow 318. Extrusion of the fugitive ink onto the free surface of the fluid precursor 308 during printing creates a mold / template of the nutrient delivery channels. As printing of the cylindrical construct 302 continues, the increasing volume of the fluid support 306 causes the position / level of the fluid precursor 308 to rise upwards, as indicated by block arrow 320.

[0111] As shown in Figure 3B, the flowable precursor 308 continues to flow over the fibers and upon cross-linking embeds the fibers within the cylindrical construct 302. The cylindrical construct 302 is then immersed in an ice bath at 4°C to liquefy the fibers 314. Figure 3C shows a schematic perspective view of the cylindrical construct 302 with channels 322 formed where the fibers 314 were originally embedded. As shown in Figure 3D, the cylindrical construct 302 with channels 322 is then immersed in culture medium 324 such that the medium fills the channels 322.

[0112] 4A, 4B, and 4C are screenshots from a video showing the process of printing a 3D construct according to various embodiments disclosed herein, and FIG 4D is an image of a cross section of the 3D construct showing a removed channel in the 3D construct.

[0113] FLIP printing creates a free surface at the precursor-air interface. This allows for direct deposition of material, for example using an extruder, to deposit Pluronic® F127 fibers. As shown in FIG. 4A, a GelMA (gelatin methacryloyl) cylinder 402 (compare 102 in FIG. 1) is formed from its precursor 408 (compare 108 in FIG. 1). A Pluronic® F127 gel fiber 414 (compare 114 in FIG. 1) is incorporated during the formation of GelMA by extrusion from a syringe 412 (compare 112 in FIG. 1). As shown in FIG. 4B, when a supporting fluid (not visible in the figure, compare 106 in FIG. 1) flows in, the precursor 408, which is floating on the supporting fluid, is lifted upward and flows over the deposited fiber 414. As the photocrosslinking continues, the fiber 414 becomes encapsulated within the gel, as shown in FIG. 4C. The location of the extruder 412 between the projector (not visible in the figure, compare 110 in FIG. 1) and the precursor 408 may interfere with the projected pattern, but does not significantly affect the crosslinking process, since the extrusion of Pluronic® F127 occurs in a few seconds, much faster than photocrosslinking. As shown in FIG. 4D, when the entire cylinder is immersed in a cold water / ice bath after photocrosslinking is complete, F127 liquefies and becomes transparent, forming channels in the GelMA cylinder. As shown in the phase change diagram in FIG. 7, F127 is a heat-sensitive material that changes its physical state in response to changes in temperature. For example, at a concentration of 20% by weight, the F127 material is liquid below 20° C., gels from about 20° C. to about 70° C., and liquefies again above 70° C. The sacrificial ink remains in a gel state at the printing temperature, but melts when the temperature is increased or decreased.

[0114] Bioprinting of cells in hydrogels containing nutrient delivery channels The ability to print soft hydrogel structures has clear applications in 3D bioprinting, but two additional goals must be achieved to successfully print living constructs.

[0115] First, the 3D printing process itself must not be cytotoxic. To demonstrate the bioprinting potential of the FLIP printer disclosed here, green fluorescent protein (GFP)-expressing fibroblasts were suspended in gelatin methacrylate (GelMA) and printed into a gelatin methacrylate (GelMA) matrix. The hydrogel constructs were cultured in cell culture medium under static conditions (i.e., without active flow of medium). After 7 days, the hydrogel constructs were cut in half and stained with propidium iodide (PI) to identify dead cells.

[0116] As a result of the gentle printing conditions (no high shear, high temperature, excessive irradiation) and rapid printing, the cells were still viable even one week after printing. However, as shown in FIG. 5A, without the printed channels, GFP-expressing cells were only found around the media-facing surface 500 within a few hundred microns. Beyond a few hundred microns from the surface, cell viability drops off sharply due to limited nutrient diffusion, and the core region 502 of such gels is virtually devoid of live cells. FIG. 5B is a micrograph of the core region / necrotic core 502 showing only dead P1 stained cells.

[0117] This highlights the second condition necessary for bioprinting, namely that the printed structure must supply sufficient nutrients to support the metabolism of the cells, for example, through one or more channels located within the printed structure. Although it is possible to directly print channels, the tendency for over-crosslinking often results in very slow printing (using excess quenching dye to control the process) or partially blocking the channels. The method disclosed here utilizes a temporary ink strategy by depositing F127 fibers directly onto the partially printed construct during the printing process (see Figure 3A to Figure 3D). This was possible because FLIP employs a non-interactive strategy, where the fibers can be deposited on a free surface using an extruder while the photocrosslinking proceeds without pause. After printing, immersion of the construct in a coolant caused the fibers to liquefy and form channels through which the cells were supported (see 322 in Figure 3C). The advantage of such channels is evident around the channel lumen where GFP-expressing cells are clearly seen, despite being located near the necrotic core of the gel.

[0118] As shown in Figures 6A-6C, green fluorescent protein (GFP) expressing mouse fibroblast cells were printed directly into a gelatin methacrylate (GelMA) matrix containing a channel 600 printed in a similar location corresponding to the necrotic core 502 in Figure 5A. While viability declines 100-200 microns back from the lumen (see Figure 6B), GFP expressing cells were clearly seen around the channel lumen (see Figure 6A). Figure 6C is an overlay image of Figures 6A and 6B showing the gradual transition to low viability.

[0119] Cell viability may be improved if medium exchange is performed by active flow, rather than passive diffusion, as in this experiment. With appropriate channel design, it is possible to allow active flow of medium through the channel, rather than passive diffusion, as in this experiment, which would further improve cell viability.

[0120] This experiment demonstrates the feasibility of rapid and versatile bioprinting using the methods and systems disclosed herein, as high speed printing of cell-laden GelMA (at speeds greater than 70 mm / hr) can be achieved, while channel networks of any design can be easily incorporated to support cell viability.

[0121] (Consideration) The presently disclosed FLIP method and system embodiments provide a continuous 3D printing platform that enables printing of soft structures using a fluid support strategy. The presently disclosed FLIP method and system embodiments may expand the role of 3D printing in biomedical applications such as tissue bioprinting. To this end, FLIP has attempted to address some of the technical challenges associated with bioprinting, including the low mechanical stiffness of hydrogels, the complex geometry associated with anatomical structures, the slow printing speed of most existing bioprinters, and the need for gentle, cell-compatible printing conditions.

[0122] One of the main components of bioinks typically includes hydrogels due to their compatibility with cell physiology. Although hydrogels are more flexible than traditional engineering materials such as metals, plastics, and even many elastomers, it is important to note that hydrogels ostensibly made from the same material (e.g., PEG-diacrylate) can have significantly different mechanical properties depending on the formulation. Megapascal hydrogels can be printed with a variety of printers, including FLIP, for bioprinting purposes, but typically the Young's modulus of the material is no more than a few hundred kilopascals. This necessitates the use of supports for the overhanging structures, a necessity further exacerbated by the fact that many anatomical structures (e.g., blood vessels) are thin and may contain voids (e.g., cardiac chambers).

[0123] To address this issue, possible support strategies include printing with cryogenic supports, printing directly on gels that melt at high temperatures, and printing on granular gels. Although each of these strategies has its advantages, they all employ syringe extrusion techniques with associated limitations. Additionally, the cryogenic supports also make direct cell printing difficult due to the low temperatures required being incompatible with cells. In embodiments of the method disclosed herein, FLIP is employed, using an inert and stable fluid to provide buoyancy to support complex structures so that they do not collapse during the printing process. The use of perfluorinated supporting fluids may result in immiscibility with aqueous precursors and may also minimize interactions with organic components of the precursors, such as monomers, crosslinkers, and initiators. As a result, the supporting fluid of the disclosed method embodiments does not cause any swelling of the hydrogel structures and may be easily removed by rinsing with running water. In these examples, apart from printing overhanging structures, it has been shown that a FLIP printer can be used to estimate the conditions required to successfully print free-standing hollow structures at the sub-millimeter scale, which has been difficult with many resin-based 3D printers.

[0124] It is worth emphasizing that high printing speed is not just a matter of convenience. Rather, speed allows cells to return to optimal culture conditions quickly, since printing conditions are often different (in terms of osmolarity, temperature, pH, etc.) from the final incubation conditions of the printed structures. FLIP has been shown to achieve high speed printing of soft materials without subjecting them to high shear, extreme temperatures, or other harsh conditions, and is compatible with cell printing (see Figures 5 and 6). By introducing channels into the printed structures, embodiments of the presently disclosed methods and systems have also demonstrated the ability to address a fundamental challenge of bioprinting: providing nutrients to cells in thick structures beyond the diffusion limit. The versatility of the presently disclosed system and method embodiments allows the size, density, and arrangement of channels to be optimized to ensure that they are adequately supplied to cells dispersed throughout the structure. In addition to fiber extruders, embodiments of the presently disclosed methods may also be used in combination with acoustic levitation nozzles that can establish concentration gradients of cytokines, growth factors, or other additives to generate heterogeneous matrices. The speed, ease of use, and versatility that FLIP now offers makes it applicable to a variety of manufacturing and biomedical applications.

[0125] Those skilled in the art will appreciate that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the present disclosure as broadly described. For example, features of different exemplary embodiments may be mixed, combined, substituted, incorporated, adopted, modified, subsumed, etc. across different exemplary embodiments as described herein. Thus, the present embodiments should be considered in all respects as illustrative and not restrictive.

Claims

1. 1. A multi-modality method for printing a three-dimensional (3D) construct or a part thereof, the method comprising: (i) supporting a flowable precursor with a flowable support at a location where the flowable precursor can be crosslinked via irradiation with a radiation source; (ii) depositing one or more materials on / within the flowable precursor; (iii) cross-linking at least a portion of the flowable precursor in contact with the one or more materials in the flowable precursor via irradiation by the irradiation source to form the 3D construct or a portion thereof.

2. (iv) supporting the flowable precursor at a new location where the flowable precursor can be crosslinked via irradiation by the irradiation source; (v) optionally further depositing said one or more materials on / within said flowable precursor; (vi) optionally further cross-linking at least a portion of said flowable precursor in contact with said one or more materials at said new location via irradiation by said irradiation source to form said 3D construct or a portion thereof; 13. The method of claim 1, further comprising: (vii) optionally repeating steps (iv) to (vi) until a desired 3D construct is formed.

3. The method of claim 1 or 2, wherein depositing one or more materials comprises extruding a sacrificial material to form an array of one or more sacrificial structures within the 3D construct.

4. 4. The method of claim 3, further comprising removing the sacrificial material from an array of one or more sacrificial structures to form an array of one or more channels in the 3D construct.

5. 5. The method of claim 4, wherein the one or more channels comprise an open end facilitating fluid communication between an internal environment of the 3D construct and an external environment of the 3D construct.

6. The method of claim 3 , wherein the sacrificial material is in a semi-solid or gel state when extruded to form the array of one or more sacrificial structures.

7. The method of claim 4 , wherein removing the sacrificial material comprises liquefying the sacrificial material to facilitate removal of the sacrificial material from the 3D construct or portion thereof.

8. The method of claim 7 , wherein liquefying the sacrificial material comprises cooling the sacrificial material to a temperature that causes the sacrificial material to liquefy.

9. The method of claim 1 , wherein depositing one or more materials comprises propelling droplets containing the one or more materials onto the flowable precursor.

10. 10. The method of claim 9, wherein propelling droplets containing the one or more materials onto the flowable precursor comprises propelling the droplets at variable speeds such that a spatial concentration gradient of the one or more materials is formed within the 3D construct.

11. 2. The method of claim 1, wherein the step of cross-linking via irradiation with a radiation source is performed in a bottom-up direction such that the 3D construct or part thereof is formed layer-by-layer in a bottom-up direction.

12. The method of claim 1 , wherein the step of crosslinking via irradiation with the radiation source is performed continuously.

13. 2. The method of claim 1, wherein depositing the one or more materials on / within the flowable precursor comprises depositing the one or more materials in a top-down direction toward an upper surface of the flowable precursor.

14. The method of claim 1 , wherein the flowable support is immiscible with the flowable precursor.

15. The method of claim 1 , wherein the flowable precursor and the one or more materials are biocompatible.

16. 10. The method of claim 1, wherein the 3D construct has a higher density than the flowable precursor and the flowable support.

17. The method of claim 1 , wherein the flowable precursor and / or the one or more materials comprise cells.

18. A multi-modality system for implementing the multi-modality method of claim 1, comprising: a tank configured to contain a flowable support and a flowable precursor disposed above the flowable support; an irradiation source configured to irradiate the flowable precursor; one or more dispensing outlets disposed between the tank and the irradiation source and configured to deposit one or more materials on / within the flowable precursor; The system, wherein the irradiation source is configured to crosslink via irradiation at least a portion of the flowable precursor that is in contact with the one or more materials in the flowable precursor to form the 3D construct or a portion thereof.

19. 20. The system of claim 18, wherein the one or more dispensing outlets comprise an extruder nozzle that extrudes a sacrificial material to form a template for an array of one or more channels in the 3D construct.

20. 20. The system of claim 18 or 19, wherein the one or more dispensing outlets comprise inkjet nozzles for propelling droplets containing the one or more materials onto the flowable precursor.

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