Additive manufacturing system and method for three-dimensional structures

Abstract

To provide a system and method for additive manufacturing of three-dimensional structures, including three-dimensional cellular structures.SOLUTION: The system has at least one print head for receiving and dispensing materials. The materials comprise a sheath fluid and a hydrogel. The print head has an orifice for dispensing the materials, microfluidic channels for receiving and directing the materials, and fluidic switches corresponding to one of the microfluidic channels in the print head and configured to allow or disallow a fluid flow in the microfluidic channels. The system further comprises a receiving surface for receiving a first layer of the materials dispensed from the orifice, a positioning unit for positioning the orifice of the print head in a three-dimensional space, and dispensing means for dispensing the materials from the orifice of the print head.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention generally relates to the three-dimensional (3D) printing and fabrication of three-dimensional biological structures from digital files. In particular, the present invention relates to systems, devices, and methods for fabricating 3D cell-containing hydrogel structures.

[0002]

Description of Related Applications

Background Art

[0003] 3D printing (3D printing), which is a form of additive manufacturing (AM), is a process of directly fabricating three-dimensional objects from digital files. Software is used to slice a computer-aided design (CAD) model or 3D scan of an object into a number of thin cross-sectional layers. This collection of layers is sent to an AM system, which constructs the three-dimensional object layer by layer. Each layer is deposited on top of the previous layer, and eventually, the object is completely constructed. Support materials may be used to support overhanging and complex features of the object. There are various AM processes that can construct parts from plastic, metal, ceramic, and / or biological materials.

[0004] Additive manufacturing can be utilized in biological systems. For example, until recently, most cell culture studies have been conducted on two-dimensional (2D) surfaces, such as microwell plates and Petri dishes. However, 2D culture systems do not mimic the 3D environment in which cells exist in vivo. Researchers have discovered that 3D cell culture behaves at least partially more like native biological tissue than 2D cell culture because the 3D arrangement of cells in native tissue affects cell-cell interactions, thereby affecting cell growth and physiological properties.

[0005] Additive manufacturing apparatuses and systems for producing cellular constructs are known. For example, the known fused fiber deposition method has been applied to biological materials. In fused fiber deposition, a high-viscosity liquid is dispensed in small amounts through a relatively narrow orifice and then rapidly solidified by various means. Biocompatible plastics, thermogelled hydrogels, UV-crosslinkable polymers, and high-concentration alginates are used as scaffolds for 3D cellular structures, and cells are added to the scaffold after it has solidified. The drawback of these techniques is that these methods require adding cells to the scaffold after printing, making it difficult to control cell placement. Furthermore, the composition of the scaffold substrate may not be suitable for facilitating cell proliferation and growth.

[0006] Printing systems for 3D structures, including the direct printing of cellular materials, are known and desired, for at least one reason being that such systems allow cells to be deposited within a 3D scaffold. For example, inkjet printing is used to print biological materials. However, the shear forces involved in the propulsion of fluid droplets onto the substrate can damage cells dispersed in the fluid. Furthermore, inkjet printing is a time-consuming process that presents challenges in adapting to biological materials that require specific environmental conditions for manufacturing.

[0007] Other systems for directly printing cells into 3D structures include the system described in U.S. Patent No. 8,639,484, which relates to the use of a CAD model and a 3D positioning unit for fabricating a 3D object by depositing cellular material layer by layer through a number of nozzles. By using a number of nozzles, a wide variety of materials can be incorporated into the 3D object. U.S. Patent Application Publication 2012 / 0089238 discloses a multi-cartridge printing system for fabricating composite organic 3D structures, thereby constructing the structure using at least two syringes, one for a structural support polymer and the other for a living cell composition, and these syringes repeatedly deposit the structural support polymer and living cell composition onto the surface. U.S. Patent Application Publication 2014 / 0012407 discloses a device having one or more print heads, each configured to accept and hold one or more cartridges. Each cartridge has a bioink containing a fluid, such as cells or a supporting material, and an orifice through which the fluid can be dispensed from the cartridge. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent No. 8,639,484 [Patent Document 2] U.S. Patent Application Publication No. 2012 / 0089238 [Patent Document 3] U.S. Patent Application Publication No. 2014 / 0012407 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Prior art methods generally require a large number of nozzles and / or cartridge orifices to facilitate printing of a wide variety of materials (i.e., one material is dispensed by one nozzle or cartridge orifice). Using multiple nozzles to dispense different materials requires positioning appropriate nozzles or cartridge orifices in a controlled sequence, thereby increasing the movement of the printing system to dispense a series of different materials. This increase in movement reduces the printing speed and efficiency.

[0010] It is desirable to eliminate or mitigate one or more of the above-mentioned drawbacks. [Means for solving the problem]

[0011] In a first aspect, a system for additive manufacturing of three-dimensional structures is provided. The system includes at least one print head for receiving and dispensing material, the material including a sheath fluid and a hydrogel. In one embodiment, the print head has an orifice for dispensing material and a microfluidic channel including one or more first channels for receiving and directing the sheath fluid and one or more second channels for receiving and directing the hydrogel, the second channels intersecting each other at a first intersection with the first channels, the second channels and the first channels connecting each other at the first intersection to form a dispensing channel extending to the orifice, and the print head further includes a fluidic switch, each corresponding to one of the microfluidic channels in the print head and configured to allow or deny the flow of fluid in the microfluidic channels of the print head when activated. In one embodiment, the system further includes a receiving surface for receiving a first layer of material dispensed from an orifice, and a positioning unit for positioning the orifice of a print head in three-dimensional space, the positioning unit being operatively coupled to the print head, and the system further includes dispensing means for dispensing material from the orifice of the print head.

[0012] In one embodiment of the first aspect, the system includes a programmable control processor that controls a positioning unit and controls the dispensing of material from the print head onto the receiving surface.

[0013] In one embodiment of the first aspect, one or more first channels include at least two channels, and one or more first channels are configured to be adjacent to each second channel at a first intersection.

[0014] In one embodiment of the first aspect, the sheath fluid includes a crosslinking agent that causes the hydrogel to solidify at the intersection and / or within the dispensing channel upon contact with the sheath fluid.

[0015] In one embodiment of the first aspect, each second channel has a smaller diameter than the diameter of the first channel and the dispensing channel, and the flow from the first channel forms a coaxial sheath around the hydrogel within the dispensing channel.

[0016] In one embodiment of the first aspect, the hydrogel contains living cells.

[0017] In one embodiment of the first aspect, the system further includes a fluid removal feature to remove excess sheath fluid dispensed from the print head.

[0018] In one embodiment of the first aspect, the receiving surface includes a porous membrane having pores sized to allow excess sheath fluid to flow through.

[0019] In one embodiment of the first aspect, the fluid removal feature includes an absorbent material or vacuum that draws excess sheath fluid away from the receiving surface.

[0020] In one embodiment of the first aspect, the absorbent material or vacuum is applied below the porous membrane. In one embodiment of the first aspect, the vacuum is applied above the receiving surface.

[0021] In one embodiment of the first aspect, a vacuum is applied through one or more vacuum channels provided in the print head, and the one or more vacuum channels have orifices disposed near the orifices of the print head.

[0022] In one embodiment of the first aspect, the system further includes a reservoir for containing a material, and the reservoir is fluidly coupled to microfluidic channels within the print head, respectively.

[0023] In one embodiment of the first aspect, the print head further has at least two inlets for receiving the material from the reservoir, and each of the inlets is in fluid communication with a corresponding microfluidic channel and a corresponding reservoir, respectively.

[0024] In one embodiment of the first aspect, the metering means includes a pressure control unit.

[0025] In one embodiment of the first aspect, the fluidic switch includes a valve.

[0026] In one embodiment of the first aspect, the print head further has a hollow protrusion configured to extend from the orifice toward the receiving surface.

[0027] In one embodiment of the first aspect, the print head has two second channels, each of the second channels is adapted to carry a corresponding hydrogel, and the two second channels intersect each other at a second intersection and are connected to each other at the second intersection, thereby forming a third channel extending to the first intersection.

[0028] In a second aspect, a system for additive manufacturing of three-dimensional structures is provided, comprising at least one print head for receiving and dispensing material, the material comprising a sheath fluid and a hydrogel. In one embodiment, the print head has an orifice for dispensing material, a microfluidic channel for receiving and directing material into the orifice, and a fluidic switch, each corresponding to one of the microfluidic channels in the print head and configured to allow or deny fluid flow in the microfluidic channels of the print head when activated. In one embodiment, the system further comprises a receiving surface for receiving material dispensed from the orifice, a fluid removal feature for removing excess sheath fluid dispensed from the orifice, and a positioning unit for positioning the orifice of the print head in three-dimensional space, the positioning unit being operatively coupled to the print head, and the system further comprises dispensing means for dispensing material from the orifice of the print head.

[0029] In one embodiment of the second aspect, the fluid removal feature includes a vacuum to draw excess sheath fluid from the hydrogel that is dispensed from or through the receiving surface and / or onto the receiving surface.

[0030] In one embodiment of the second aspect, the receiving surface includes a porous membrane having pores sized to allow excess sheath fluid to flow through.

[0031] In one embodiment of the second aspect, the vacuum is applied below the porous membrane. In one embodiment of the second aspect, the vacuum is applied above the receiving surface.

[0032] In one embodiment of the second aspect, the vacuum is applied through one or more vacuum channels provided in the print head, and one or more vacuum channels have orifices located near the print head orifice.

[0033] In one embodiment of the second aspect, the fluid removal feature includes an absorbent material that absorbs excess sheath fluid from the receiving surface.

[0034] In one embodiment of the second aspect, the system further includes a programmable control processor that controls a positioning unit and controls the dispensing of material from the print head onto the receiving surface.

[0035] In one embodiment of the second aspect, the print head further includes a hollow projection configured to extend from the orifice toward the receiving surface.

[0036] In one embodiment of the second aspect, the print head has one or more first channels that receive and direct a sheath fluid and one or more second channels that receive and direct a hydrogel, the second channels intersect each other at a first intersection with the first channels, and the second channels and the first channels are connected at the first intersection to form a dispensing channel that extends to an orifice.

[0037] In one embodiment of the second aspect, the print head has two second channels, each of which is configured to transport its respective hydrogel, and the two second channels intersect and connect with each other at a second intersection, thereby forming a third channel that extends to a first intersection.

[0038] In a third aspect, a method is provided for printing a three-dimensional (3D) structure, the method comprising the step of preparing a 3D printer, the printer having at least one print head having an orifice for dispensing material, a receiving surface for receiving a first layer of material dispensed from the orifice of the print head, and a positioning unit operatively coupled to the print head for positioning the print head in three-dimensional space. In one embodiment, the method further includes the step of preparing a material to be dispensed, the material to be dispensed comprising a sheath fluid and one or more types of hydrogels, the method further includes the steps of encoding a printer with a 3D structure to be printed, dispensing the material to be dispensed from a print head orifice, adhering a first layer of the dispensed material onto a receiving surface, repeating the adhering step by adhering the next dispensed material onto the first layer and onto any subsequent layers of dispensed material, thereby adhering the layers onto the layers of dispensed material in a geometric arrangement according to the 3D structure, and removing excess sheath fluid dispensed from the print head orifice at one or more points during or between the adhering steps.

[0039] In one embodiment of the third aspect, the sheath fluid contains a crosslinking agent suitable for crosslinking and solidifying the hydrogel upon contact with the sheath fluid, thereby producing hydrogel fibers.

[0040] In one embodiment of the third aspect, the sheath fluid and the hydrogel are dispensed in a coaxial arrangement, with the sheath fluid surrounding the hydrogel.

[0041] In one embodiment of the third aspect, the adhesion step and the removal step are performed sequentially, thereby continuously removing excess sheath fluid as layers of dispensing material are adhered.

[0042] In one embodiment of the third aspect, the removal step is performed intermittently and / or simultaneously with the deposition steps, thereby intermittently removing excess sheath fluid while the material layers are being deposited.

[0043] In one embodiment of the third aspect, one or more hydrogels are configured to support the growth and / or proliferation of living cells dispersed in such hydrogels.

[0044] The features of the present invention will become more apparent in the following detailed description, with reference to the accompanying drawings. [Brief explanation of the drawing]

[0045] [Figure 1] This is a perspective view of one embodiment of the printing system of the present invention. [Figure 2] This is a perspective view of a software design object and a corresponding object printed using one embodiment of the printing system of the present invention. [Figure 3] This is a perspective view of one embodiment of the print head of the present invention. [Figure 4] Figure 3 is a cross-sectional view of a valve located in the print head, showing the state in which the valve membrane flexes when the valve is activated. [Figure 5] Figure 3 is a cross-sectional view of another modified embodiment of the print head. [Figure 6] Figure 3 is a plan view of a modified embodiment of the print head. [Figure 7] This is a disassembled and assembled perspective view of one embodiment of the printing bed assembly of the present invention. [Figure 8] Figure 7 is a cross-sectional view of the printing bed in its assembled state. [Figure 9] Figure 7 is a cross-sectional view of a modified embodiment of the printing bed. [Figure 10] This is a perspective view of one embodiment of the print head of the present invention. [Modes for carrying out the invention]

[0046] Definitions of certain terms used herein are provided below. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as generally understood by those skilled in the art relating to the present invention.

[0047] The term “approximately” as used herein will be understood by those skilled in the art, and there may be some variation depending on the context in which it is used. Where there is a use of terminology that is not obvious to those skilled in the art, and where the context in which it is used is given, “approximately” means within ±10% of the listed values.

[0048] As used herein, the term "hydrogel" means a composition comprising water and a network or lattice structure of hydrophilic polymer chains. Examples of natural hydrogels include, for example, gels primarily composed of alginates, agarose, collagen, fibrinogen, gelatin, chitosan, hyaluronic acid, or any combination thereof. Various synthetic hydrogels are known and can be used in embodiments of the systems and methods provided herein. For example, in embodiments of the systems and methods provided herein, one or more hydrogels form a structural base for a printed three-dimensional structure. In some embodiments, the hydrogel has the capacity to support the growth and / or proliferation of one or more cell types, such cell types may be dispersed within the hydrogel or added to the hydrogel after it has been printed in a three-dimensional form. In some embodiments, the hydrogel is crosslinkable with a chemical crosslinking agent. For example, hydrogels containing alginate are preferably crosslinkable in the presence of divalent cations, hydrogels containing fibrinogen are preferably crosslinkable in the presence of thrombin, and hydrogels containing collagen or chitosan are preferably crosslinkable in the presence of heat or a basic solution. Crosslinking of hydrogels increases their hardness and, in some embodiments, enables the formation of hydrogels that behave like solids.

[0049] As used herein, the term “sheath fluid” means, at least in part, a liquid used to surround or “sheath” a material to be dispensed, such as a hydrogel. In some embodiments, the sheath fluid is an aqueous solvent, such as water or glycerol, and a chemical crosslinking agent, such as a divalent cation (e.g., Ca 2+ Ba 2+ Sr 2+ It contains one or more of the following: thrombin, or pH-adjusting chemicals, such as sodium bicarbonate.

[0050] As used herein, the term “excess sheath fluid” means a portion of sheath fluid that is dispensed from the printhead orifice and does not form part of a three-dimensional structure printed using one or more embodiments of the system or method provided herein. For example, excess sheath fluid can be useful in facilitating the passage of hydrogel through the dispensing channels and printhead orifice provided within the printhead. Once dispensed from the printhead orifice, excess sheath fluid flows away from the surface of the dispensed hydrogel layer and onto a receiving surface, where it can accumulate or pool.

[0051] As used herein, the term “receiving surface” means the surface to which the first layer of material dispensed from the printhead orifice is deposited. The receiving surface also receives excess sheath fluid dispensed from the printhead orifice, which flows away from one or more layers of material dispensed from the printhead orifice. In some embodiments, the receiving surface is made of a solid material. In some embodiments, the receiving surface is made of a porous material. For example, in some embodiments, the porosity of the porous material is sufficient to allow the sheath fluid to pass through. In some embodiments, the receiving surface is substantially flat, thereby providing a flat surface to which the first layer of dispensed material can be deposited. In some embodiments, the receiving surface has a topography corresponding to the three-dimensional structure to be printed, thereby facilitating the printing of a three-dimensional structure having a non-flat first layer.

[0052] In one view, the present invention generally relates to apparatus, systems, and methods for the additive manufacturing of three-dimensional (3D) biological structures.

[0053] Overview of the printing system

[0054] In one view, the present invention provides a system for additive manufacturing of three-dimensional structures (hereinafter also referred to herein as “printer,” “3D printer,” “printing system,” or “system”). The system includes a microfluidic print head, which is a microfluidic liquid handling device having one or more microfluidic channels that receive and direct material to be dispensed, a fluidic switch corresponding to the microfluidic channels and adjusting the flow rate of material to be printed, and a single orifice for dispensing the material to be dispensed.

[0055] The materials to be dispensed include a sheath fluid and at least one type of hydrogel. In a preferred embodiment, the sheath fluid includes a chemical crosslinking agent suitable for solidifying the hydrogel upon contact with the sheath fluid. In a preferred embodiment, the sheath fluid acts as a lubricant for the solidified hydrogel.

[0056] Microfluidic channels act as conduits that direct the material to be dispensed and combine such material in a controlled manner. Microfluidic channels are arranged within the print head such that one or more first channels that receive and direct the sheath fluid and second channels that receive and direct the hydrogel intersect and connect with each other at a first intersection, thereby forming a dispensing channel that extends to the print head orifice. In a preferred embodiment, the first channels are configured to be alongside the second channels at the first intersection. In this way, the sheath fluid is directed to flow along each side of the hydrogel within the dispensing channel.

[0057] In a preferred embodiment, the material in the dispensing channel is coaxially oriented, the hydrogel is concentrated in the center of the dispensing channel, and the sheath fluid surrounds the hydrogel fluid, thereby forming a sheath around the hydrogel. In a preferred embodiment further comprising a chemical crosslinking agent suitable for crosslinking the hydrogel in the sheath fluid, solidified hydrogel fibers are formed in the dispensing channel and then dispensed from the orifice of the print head.

[0058] From one perspective, the system further includes a receiving surface that receives a first layer of material dispensed from an orifice, and a positioning unit that positions the orifice of the print head in three-dimensional space, the positioning unit being operationally coupled to the print head. For example, the print head may be coupled to a commercially available motorized positioning system with three degrees of freedom of motion, thereby positioning the print head above the receiving surface and oriented it so that the dispensed material is directed downward toward the receiving surface.

[0059] From one perspective, the system may include means for dispensing material in small amounts from the print head orifice, and may further include and / or be in data communication with such programmable control processor for adjusting the positioning of the print head orifice. The programmable control processor may also be used to control the dispensing of material to be dispensed in small amounts from the print head orifice.

[0060] Figure 1 is a schematic perspective view of one embodiment of a 3D printing system provided herein.

[0061] Referring to Figure 1, the system includes a microfluidic printhead

[0100] , which has a printhead orifice

[0114] and at least one inlet for receiving material to be dispensed from the printhead

[0100] . The material to be dispensed is stored in a material reservoir

[0110] and is then fed to the printhead through corresponding first connecting pipes

[0122] , which enable fluid communication between the printhead and the material reservoir. In the illustrated embodiment, the means for dispensing the material to be dispensed from the printhead orifice is a pressure control unit

[0112] , which is fluidly coupled to the material reservoir

[0110] by corresponding second connecting pipes

[0120] . The pressure control unit is a means for providing force to dispense the material to be dispensed. The pressure control unit supplies air pressure to the material to be printed reservoir

[0110] via the corresponding second connecting pipes

[0120] . The pressure applied to the material to be printed reservoir pushes the fluid out of the reservoir and into the print head via the respective first connecting pipes

[0122] . Other means for dispensing the material to be dispensed may be used in the illustrated embodiment. For example, a series of electronically controlled syringe pumps can be used to provide force for dispensing the material to be dispensed from the print head orifice.

[0062] Referring to Figure 1, the microfluidic printhead

[0100] is coupled to a 3D motorized stage which includes three arms [102, 103, 104] for positioning the printhead

[0100] and the printhead orifice

[0114] in three-dimensional space above the print bed

[0108] , the print bed

[0108] having a surface

[0109] for receiving the material to be printed. In one embodiment, the 3D motorized stage (i.e., the positioning unit) is preferably controlled to position a vertical arm

[0104] which extends along the z-axis of the 3D motorized stage so that the printhead orifice

[0114] is oriented downward. A first horizontal arm

[0102] extending along the x-axis of the motorized stage is fixed to a stationary base platform

[0116] . A second horizontal arm

[0103] extending along the y-axis of the motorized stage is movably coupled to the upper surface of the first horizontal arm

[0102] , with the longitudinal directions of the first and second horizontal arms [102,103] perpendicular to each other. To be understood, the terms “perpendicular” and “horizontal” used above with respect to the arms describe the way the print head is moved and do not necessarily limit the physical orientation of the arms themselves.

[0063] In the embodiment shown in Figure 1, the print bed

[0108] is positioned on the top of the platform

[0118] , and the platform is coupled to the upper surface of the second horizontal arm

[0103] . In this embodiment, the 3D motorized stage arms [102, 103, 104] are each driven by three corresponding motors [105, 106, 107] and controlled by a programmable control processor, such as a computer (not shown). In a preferred embodiment, the print head

[0100] and the print bed

[0108] can move together along all three major axes of the Cartesian coordinate system by the 3D motorized stage, the motion of which is determined using computer software.

[0064] As should be understood, the present invention is not limited to the positioning system described, and other positioning systems are known in the art.

[0065] In the embodiment shown in Figure 1, as the material is dispensed from the print head orifice

[0114] , the positioning unit is moved in a software-controlled pattern, thereby forming a first layer of dispensed material on the receiving surface

[0109] . Additional layers of dispensed material are stacked on top of each other such that the final 3D geometric shape of the dispensed material layers as a whole is a replica of a 3D geometric design provided by the software. The 3D design is created using typical 3D CAD (computer-aided design) software or generated from a digital image, as is known in the art. Furthermore, if the geometric shape generated by the software includes information about the specific material to be used, according to one embodiment of the present invention, it is possible to assign specific types of material to different geometric locations. For example, Figure 2 shows three 3D structures printed using one embodiment of the system provided herein, e.g., a cube

[0128] , a hollow cylinder

[0129] , and a hollow coaxial cylinder

[0130] . The software is used to generate cube, hollow cylinder, and hollow coaxial cylinder design examples (

[0125] ,

[0126] , and

[0127] , respectively), each design example including two different types of materials (dyed alginates) dyed in different colors to provide visual clarity for the materials used to generate the printed cube and hollow cylinder.

[0066] Any software, application, or module used herein is executable using computer-readable / executable instructions, and such instructions are preferably stored in or otherwise held on such computer-readable media.

[0067] Print head

[0068] Figure 3 is a schematic perspective view of one embodiment of a microfluidic printhead

[0100] used in the provided system.

[0069] Referring to Figure 3, the illustrated embodiment shows a microfluidic printhead

[0100] having microfluidic channels for carrying various fluids. In the illustrated embodiment, the microfluidic channels have a cylindrical shape. However, channel shapes other than cylindrical are also available for the printheads provided herein. Channel

[0200] is a conduit for a crosslinking agent, and channel

[0202] is a conduit for water. In the illustrated embodiment, the crosslinking agent and water, separately or together, serve as a “sheath fluid”. Channel

[0204] is a conduit for a first hydrogel composition (hereinafter referred to as “Hydrogel A”), and channel

[0206] is a conduit for a second hydrogel composition (hereinafter referred to as “Hydrogel B”). In a preferred embodiment, one or more types of living cells are compatible with Hydrogel A and / or Hydrogel B and are optionally dispersed in Hydrogel A and / or Hydrogel B. In the illustrated embodiment, each microfluidic channel has a fluid inlet [208a, 208b, 208c, 208d] that allows fluid contained in the connecting tube

[0122] to enter the respective channel of the print head

[0100] . Downstream of the fluid inlets [208a, 208b, 208c, 208d] are valves [210, 212, 214, 216] corresponding to each channel. In the illustrated embodiment, these valves serve as "fluidic switches" that can be operated to allow or deny the flow of fluid through the channel, and each valve has a corresponding inlet [218, 218a, 218b, 218c, 218d] that facilitates the operation and deactivation of the valve. In one embodiment, the valves [210, 212, 214, 216] can be electronically actuated. In another embodiment, the valves [210, 212, 214, 216] can be actuated, for example, by a solenoid piston, by a change in the pressure applied to them. The electronic or pressure-driven operation of the different valves facilitates rapid changes in the amount of material dispensed, thereby allowing the dispensed material to consist of different materials in a controlled sequence.

[0070] Referring further to Figure 3, in the illustrated embodiment, the crosslinking agent channel

[0200] and the water channel

[0202] intersect at the intersection point

[0203] in a "Y" shape, thereby connecting them to form a channel called a "sheath flow channel"

[0224] immediately downstream of the crosslinking agent and water channels [200,202]. The hydrogel A channel

[0204] and the hydrogel B channel

[0206] intersect at the intersection point

[0207] in a "Y" shape, thereby connecting them to form a channel called a "focusing channel"

[0226] immediately downstream of the two hydrogel channels. The sheath flow channel

[0224] and the focusing channel

[0226] intersect at the intersection

[0228] in a "trifugal" or three-branch configuration with respect to the embodiments described above, in which case the sheath flow channel

[0224] is attached alongside the focusing channel

[0226] , and these channels are connected to each other to form a channel referred to herein as the dispensing channel

[0220] . The dispensing channel

[0220] terminates at the dispensing orifice

[0222] . In the preferred embodiment shown in Figure 1, the dispensing channel protrudes from the print head

[0100] and terminates at the dispensing orifice

[0114] .

[0071] Referring further to Figure 3, in the illustrated embodiment, the sheath flow channel

[0224] and the dispensing channel

[0220] have larger diameters than the focusing channel

[0226] . When hydraulic pressure is applied to the sheath flow channel

[0224] and the focusing channel

[0226] , the liquid in the focusing channel

[0226] is compressed laterally and "focused" into a narrow flow along the central axis of the focusing channel

[0226] . When the fluid from the large-diameter sheath flow channel

[0224] intersects with the focusing channel

[0226] at intersection

[0228] , it surrounds and encloses the narrow focused flow of hydrogel dispensed from the focusing channel

[0226] .

[0072] In a preferred embodiment, the liquid in the sheath flow channel

[0224] contains a chemical crosslinking agent, and the liquid in the focusing channel

[0226] contains one or more types of chemically crosslinkable hydrogels containing one or more types of living cells. When one or more types of chemically crosslinkable hydrogels are focused into a narrow flow in the focusing channel

[0226] and then encapsulated by the crosslinking agent in the dispensing channel

[0220] , at least the outer surfaces of one or more types of chemically crosslinkable hydrogels solidify in the dispensing channel

[0220] , thereby creating crosslinked or "solid" hydrogel fibers. The hydrogel fibers are then dispensed in a controlled manner from the dispensing orifice

[0222] onto the receiving surface, thereby constructing a 3D structure layer by layer.

[0073] In a particularly preferred embodiment, the sheath flow surrounding the hydrogel fibers can act to facilitate the passage of the hydrogel fibers through the dispensing channel

[0220] , until the hydrogel fibers are eventually dispensed from the print head orifice

[0222] .

[0074] In one embodiment, the sheath fluid comprises a chemical crosslinking agent, water, or a combination thereof. In embodiments where the sheath flow does not contain a chemical crosslinking agent, the hydrogel will not solidify but will be dispensed as a liquid. It is preferable to operate the crosslinking agent channel valve

[0210] and the water channel valve

[0212] to adjust the composition of the sheath fluid to initiate and / or stop the solidification of the hydrogel. It is conceivable that dispensing a liquid instead of a solid hydrogel, or dispensing only the sheath fluid, may be desirable to constitute several viewpoints of various three-dimensional objects.

[0075] In one embodiment, the print head

[0100] may be configured to receive and dispense only one type of hydrogel material. In one embodiment, the print head may be configured to receive and dispense two or more types of hydrogel materials. For example, in an embodiment in which the print head

[0100] is configured to receive two types of hydrogel materials, each containing, for example, different types of cells, the system provided herein is preferably programmed to dispense heterogeneous cell structures, in which case the first and second types of cells can be arranged individually and / or in combination with each other in controlled patterns within and between layers. The boundary between the two types of materials is controlled, for example, by software, and a programmable control processor is used to command a fluidic switch (e.g., one or more of valves

[0210] ,

[0212] ,

[0214] ,

[0216] ) to change the flow of material in one or more microfluidic channels, thereby changing the contents of the material being dispensed from the print head orifice. The number of types of hydrogel materials that can be received and dispensed by the printheads provided herein is limited only by the size of the printhead that the user deems practical.

[0076] Referring to Figure 4, in one embodiment, the fluidic switch is a valve having a membrane

[0332] positioned to cover a bowl-shaped feature

[0318] formed within a microfluidic channel

[0308] . When air pressure (indicated by arrows in Figure 4) is applied to the exposed surface of the valve membrane

[0332] , the valve membrane

[0332] flexes into the bowl-shaped feature

[0318] , thereby blocking the passage of fluid through the microfluidic channel

[0308] . In a preferred embodiment, the thickness of the valve membrane

[0332] is approximately 150 μm. In embodiments where the thickness of the valve membrane is increased, the applied valve operating air pressure must be increased accordingly, as will be understood by those skilled in the art. Similarly, a valve membrane formed of a less elastic material will require a higher operating pressure. Those skilled in the art will understand how to adjust the operating pressure to suit the specific material of the valve membrane.

[0077] In one embodiment, the print head has a separate fluidic switch that adjusts the amount of material to be dispensed from the print head orifice. For example, the flow rate of material in the microfluidic channels may be adjusted by a mechanism that controls the pressure applied to each channel, without using valves.

[0078] In one embodiment, the print head further includes an extension tip having an orifice for dispensing material from the print head. Such an extension tip facilitates precise dispensing and deposition of material into limited or narrow areas, such as into the wells or petri dishes of multi-well plates (e.g., standard microtiter plates, microwell plates, or microplates having 6, 24, 96, etc. wells). Referring to the embodiment shown in Figure 5, a portion

[0500] of the dispensing channel

[0220] located closest to the dispensing orifice

[0222] has a larger diameter than the upstream portion of the dispensing channel

[0220] . The extension tip

[0502] has a tube (e.g., made of plastic, glass, or metal) with an exterior configured to fit into the larger diameter portion

[0500] of the dispensing channel and an interior configured to align with the dispensing channel

[0220] (defining the hollow space within the tube). The extension tip

[0502] is preferably inserted into the larger diameter portion

[0500] of the dispensing channel, thereby extending the length of the dispensing channel

[0220] , which facilitates the accumulation of material dispensed from the orifice

[0503] of the extension tip

[0502] into a narrow space, such as a well plate insert

[0504] or a petri dish (not shown).

[0079] Referring to the embodiment shown in Figure 1, the extension tip

[0130] is a projection extending from the print head

[0100] , and the extension tip

[0130] terminates at the print head orifice

[0114] . In this embodiment, the extension tip

[0130] is integrated with the print head.

[0080] In one embodiment, two or more types of hydrogel materials are preferably arranged coaxially in hydrogel fibers dispensed from a system provided herein. Referring to Figure 6, in the illustrated embodiment, the print head

[0100] has microfluidic channels arranged to create coaxial hydrogel fibers containing a hydrogel core material and a hydrogel shell material. In the illustrated embodiment, the shell material carried in channel

[0508] is a rapidly gelling hydrogel, such as an alginate, and the core material carried in channel

[0506] is a different hydrogel selected by the user (e.g., collagen or fibrinogen). Channels

[0508] and

[0506] intersect, for example, in a "Y" shape at a hydrogel convergence intersection

[0510] (similar to intersection

[0528] shown in Figure 3), thereby connecting with each other to form a convergence channel

[0226] downstream of channels

[0506] and

[0508] . At the hydrogel convergence intersection

[0510] , the shell material converges coaxially with the core material, and as a result, the shell material forms a sheath around the core material. In a preferred embodiment, channels

[0508] and

[0226] have a larger diameter than channel

[0506] to facilitate coaxial convergence of the core material and the shell material. In a preferred embodiment, the purpose of the shell material is to provide a physical structural support to the core material, thereby enabling the core material to be formed into a 3D geometric shape. The core is preferably solidified after the material is deposited, and the solidification method, strictly speaking, is specific to each different core material. For example, the core may contain a material that solidifies very slowly. In another embodiment, the core and shell material contain the same type of material. In yet another embodiment, the shell material contains a rapidly solidifying hydrogel, and the core material contains a non-gelling material, thereby facilitating the creation of hollow fibers.

[0081] In one embodiment, the print head

[0100] shown in Figure 6 may further have additional core material channels, each core material channel having a corresponding fluidic switch, such as a valve, for adjusting the flow rate of material in the channel. The fluidic switch facilitates rapid and frequent adjustments to the composition of the core material in the fibers being dispensed, for example, by commands provided by a programmable control processor.

[0082] In one embodiment, several print heads may be arranged, for example, in parallel, to enable simultaneous printing of multiple structures. This increases throughput production.

[0083] In some embodiments, the print head is disposable. The use of disposable print heads can reduce the risk of contamination of materials used in different printing operations.

[0084] The print head is preferably manufactured using, for example, a known microfluidic molding technique (e.g., casting, imprinting, or injection molding) and one or more moldable polymers, such as polydimethylsiloxane (PDMS). As a variation, the print head may be manufactured using commercially available 3D printing technology.

[0085] Fluidic Removal Features

[0086] In one view, the present invention provides an additive manufacturing system for three-dimensional structures, characterized by removing excess sheath fluid from the receiving surface to which a first layer of material dispensed from the print head orifice is deposited, and optionally from the surface of the dispensed hydrogel. During printing, excess sheath fluid can accumulate or “pool” on the receiving surface or the surface of the dispensed hydrogel. Such pooling may interfere with the deposition of the dispensed hydrogel from the print head orifice onto the receiving surface and / or onto one or more layers of the dispensed hydrogel. For example, the pooling of sheath fluid may cause the dispensed hydrogel fibers to slip from their intended position in the 3D structure during printing. Therefore, in embodiments of the system, the removal of excess sheath fluid from the receiving surface and optionally from the surface of the dispensed hydrogel by the fluidic removal feature can improve the additive manufacturing of three-dimensional structures.

[0087] To remove excess sheath fluid from the receiving surface or from the surface of one or more layers of the dispensed hydrogel, it is preferable to absorb the fluid from these surfaces or to enable or facilitate the evaporation of the sheath fluid from these surfaces, or, in embodiments where the receiving surface is porous, the excess sheath fluid can be removed by allowing it to be absorbed into the porous surface.

[0088] In a preferred embodiment, the receiving surface comprises a porous material, the pores being sized to facilitate the passage of a sheath fluid through them and to support one or more layers of hydrogel deposited on the pores.

[0089] Referring to Figures 7 and 8, in the illustrated embodiment, the print bed

[0108] has a porous membrane

[0400] which serves as a surface (i.e., receiving surface) that receives a first layer of dispensed material. The porous membrane

[0400] is held in place within the print bed

[0108] between a box component

[0408] and a lid component

[0402] . The box component

[0408] is a container which can be any shape (e.g., square, round) that is suitable for receiving and containing liquid. The space inside the box component

[0408] is called the chamber

[0404] . The box component

[0408] has a top surface

[0409] which has a recessed lip

[0412] that forms an extension around the top surface

[0409] of the box component

[0408] . The top surface

[0409] has a hole defined by one or more walls

[0410] , which is surrounded by a recessed lip

[0412] and extends into the box component

[0408] .

[0090] Referring further to the embodiments shown in Figures 7 and 8, the lid component

[0402] has a top surface

[0403] through which a hole

[0416] is provided, and a side wall

[0418] configured to fit around the recessed lip

[0412] of the box component

[0408] , thereby facilitating the placement of the lid component

[0402] on the top surface

[0409] of the box component

[0408] . When the lid component

[0402] is placed on the box component

[0408] , the holes

[0416] of the box component and the lid component align with each other. During the operation, the porous membrane

[0400] is placed on the top surface

[0409] of the box component

[0408] so that the porous membrane extends to cover the hole on the top surface

[0409] of the box component

[0408] , and then the lid component

[0402] is placed on the top of the box component

[0408] and pushed downward. The downward pressure from the lid component

[0402] stretches the porous membrane

[0400] over the holes in the upper surface

[0409] of the box component

[0408] , thereby holding the porous membrane

[0400] between the box component

[0408] and the lid component

[0402] . In a preferred embodiment, the lid component

[0402] and the box component

[0408] fit together tightly, thereby providing a joint that remains rigid during operation of the system provided herein.

[0091] Referring further to the embodiments shown in Figures 7 and 8, the box component

[0408] comprises a solid base

[0414] , at least one outlet duct

[0406] directing the fluid away from the chamber

[0404] , and a vacuum source (not shown) in fluid communication with the outlet duct

[0406] of the chamber

[0404] . The porous membrane

[0400] has pores sized to facilitate the passage of the sheath fluid. The vacuum source (not shown) coupled to the outlet duct

[0406] is preferably operated to draw excess sheath fluid collected on the porous membrane

[0400] through the porous membrane

[0400] into the chamber

[0404] and then draw it out of the chamber

[0404] through the outlet

[0406] , after which a small amount of hydrogel fibers are left on the top of the porous membrane

[0400] .

[0092] In a preferred embodiment, a feature for removing excess sheath fluid from the receiving surface, and optionally from the surface of the dispensed hydrogel, is preferably incorporated into the system configured to dispense the material into a multi-well plate or Petri dish. For example, referring to Figure 9, in the illustrated embodiment, a commercially available well-plate insert

[0504] is positioned on top of the box component

[0408] . Some well-plate inserts

[0504] are basket-shaped with a base made of porous membrane material

[0512] . In the illustrated embodiment, a gasket

[0514] is positioned between the well-plate insert

[0504] and the box component

[0408] to improve the sealing between the two components [504, 408]. In such embodiments, the porous membrane

[0512] of the well plate type insert

[0504] serves as a "receiving surface," and excess sheath fluid is preferably removed from the porous membrane using a vacuum connected to the outlet duct

[0406] as described above, or using one of the other fluidic removal features described below.

[0093] In one embodiment (not shown), the receiving surface on the print bed includes or is positioned adjacent to an absorbent material, which facilitates the absorption of excess sheath fluid from the receiving surface. For example, a well-plate type insert having a base made of a porous membrane material (e.g., shown in Figure 9) or any other porous membrane substrate may be positioned on or adjacent to the top of the absorbent material, such as a sponge. The absorbent material works to draw excess sheath fluid from the receiving surface. In embodiments where the absorbent material is positioned beneath the porous receiving surface, excess sheath fluid on the receiving surface is drawn into the absorbent material through the porous receiving surface, thereby preventing the excess sheath fluid from accumulating on the receiving surface. In embodiments where the absorbent material is positioned immediately beside or on top of a portion of the receiving surface (e.g., around the perimeter of the receiving surface so as not to interfere with the accumulation of dispensed material), excess sheath fluid is drawn from the receiving surface and absorbed into the absorbent material.

[0094] In one embodiment (not shown), one or more tubes are provided in a region located near the receiving surface and near the print head orifice, without using one of the above-described print beds. The one or more tubes are preferably fluid-coupled to a vacuum source (not shown), which can provide suction to remove excess sheath fluid from the receiving surface and optionally from the surface of the dispensed hydrogel. In such an embodiment, a solid or porous receiving surface may also be used.

[0095] In one embodiment shown in Figure 10, the print head is configured to further have one or more vacuum channels [700a, 700b], each of which has an orifice [702a, 702b] located near the print head orifice

[0222] . Each of the one or more vacuum channels [700a, 700b] has an inlet [704a, 704b] configured to facilitate fluid communication with one or more vacuum sources (not shown). When the print head

[0100] is in fluid communication with a vacuum source, one or more vacuum channels [700a, 700b] direct negative pressure to a region of the receiving surface from which the material is dispensed from the print head orifice

[0222] and / or to a portion of the surface area of ​​the dispensed hydrogel, thereby drawing up excess sheath fluid from the receiving surface and, optionally, from the surface of the dispensed hydrogel, thereby preventing the sheath fluid from accumulating on the receiving surface and / or the surface of the dispensed hydrogel.

[0096] In one embodiment, one or more vacuum tubes are provided as one or more extensions that protrude at least partially from the print head, the extensions protruding in the same overall direction as the extensions having the print head orifice and dispensing channels (see, for example, Figure 10). In such embodiments, the one or more extensions including the vacuum tubes do not extend any further than the extensions including the print head orifice and dispensing channels so as not to interfere with the dispensed and deposited hydrogel.

[0097] In some embodiments, the fluid removal feature may be a feature of the sheath fluid composition itself. For example, the sheath fluid composition may be designed to evaporate after it is dispensed from the printhead orifice, thereby preventing excess sheath fluid from accumulating on the receiving surface or the surface of the dispensed hydrogel. For example, the sheath fluid may have a boiling point that results in evaporation after dispensing, while remaining in a liquid state before dispensing.

[0098] Printing method for three-dimensional structures

[0099] From one perspective, a method for printing three-dimensional (3D) structures is provided.

[0100] This method first includes the step of providing a design for a 3D structure to be printed. This design is preferably created using commercially available CAD software. In one embodiment, this design includes information about specific materials to be assigned to specific geometric locations in the design (e.g., information about heterogeneous structures containing multiple materials).

[0101] This method involves the use of a 3D printer, which has a print head, a receiving surface for receiving material dispensed by the print head, and a positioning unit operatively coupled to the receiving surface, the positioning unit for positioning the print head at a location in three-dimensional space above the receiving surface. For example, various embodiments of the printing system provided herein can be used for methods of printing 3D structures.

[0102] The method includes the step of providing at least two types of materials to be dispensed by the print head, such as a sheath fluid and a hydrogel fluid. In a preferred embodiment, one or more types of cells are compatible with the hydrogel and optionally dispersed within the hydrogel. In a preferred embodiment, the sheath fluid acts as a lubricant to facilitate the movement of the hydrogel in and out of the print head. In a preferred embodiment, the sheath fluid includes a crosslinking agent that solidifies at least a portion of the hydrogel before or while the hydrogel is being dispensed from the print head.

[0103] This method includes the step of transferring the design to a 3D printer. This transfer can be achieved, for example, by a programmable control processor.

[0104] This method includes controlling the relative positioning of the print head and the receiving surface in three-dimensional space, and simultaneously dispensing the sheath fluid and hydrogel individually or in combination from the print head. In a preferred embodiment, the materials to be dispensed from the print head are dispensed coaxially, so that the sheath fluid envelops the hydrogel. This coaxial arrangement causes the crosslinking agent to solidify the hydrogel, resulting in solid hydrogel fibers, which are then dispensed from the print head.

[0105] This method includes the step of adhering a first layer of dispensed material onto a receiving surface, wherein the first layer includes a material arrangement specified by the design, and the method further includes the step of repeating the adhering step to adhering subsequent layers of material onto the first layer and subsequent layers of material, thereby adhering layers onto the dispensed material in a geometric arrangement specified by the design to create a cell-containing 3D structure.

[0106] In a preferred embodiment, other types of materials, such as other types of hydrogels (at least some of which include one or more types of cells), can be deposited in a controlled sequence, thereby allowing the hydrogels and one or more types of cells to be deposited in a controlled arrangement and in a geometric arrangement specified by the design.

[0107] In a preferred embodiment, this method includes the step of removing excess sheath fluid from the receiving surface and, optionally, from the surface of the dispensed hydrogel. For example, the step of removing excess sheath fluid may be performed continuously throughout the entire printing process, thereby removing excess fluid that might otherwise interfere with the layered arrangement of the dispensed material in the geometric arrangement provided by the design. In a variation, the step of removing excess sheath fluid may be performed intermittently throughout the entire printing process, either in sequence with or concurrently with one or more deposition steps. In some embodiments, the removal of excess sheath fluid is achieved by suctioning the fluid from the receiving surface and, optionally, from the surface of the dispensed hydrogel. In another embodiment, the removal of excess sheath fluid is achieved by allowing the excess fluid to be absorbed into the receiving surface, which has pores dimensioned to allow the passage of the sheath fluid. In yet another embodiment, the removal of excess sheath fluid is achieved by providing sheath fluid that evaporates after being dispensed from the print head orifice.

[0108] Exemplary Use of Embodiments of Systems and Methods for Printing Cell-Containing Three-Dimensional Structures

[0109] In some embodiments, structures produced using the systems and methods provided herein may be useful, for example, in the field of drug discovery where the focus is on determining the cellular response to various chemical formulations and compositions. The use of 3D cell cultures produced using embodiments of the systems and methods provided herein can result in experimental conditions that more closely resemble in vivo cell and tissue conditions compared to 2D cell cultures. The 3D arrangement of cells can more closely mimic in vivo cell-cell interactions, and the response to external stimuli and the heterogeneity of 3D structures produced using the provided apparatus and methods enable the study of tissues and potentially organs. 3D cell-containing structures produced using embodiments of the systems and methods provided herein can similarly benefit the cosmetics industry by providing an alternative means of testing cosmetics.

[0110] In some embodiments, the systems and methods provided herein are compatible with standard wellplate technology. Wellplates or wellplate inserts can be used in or as part of the print bed of the methods and systems provided herein. Thus, various embodiments of the systems and methods provided herein are compatible with machinery and practices that utilize wellplates, thereby allowing the machinery and practices to be easily integrated into existing process flows.

[0111] In some embodiments, the microfluidic channel within the printhead is compatible with other microfluidic modules. For example, a known microfluidic module is preferably located upstream of the printhead orifice within the printhead of the system provided herein. Such modules include, for example, cell counting modules, cell sorting modules, cell analysis modules, and / or concentration gradient generation modules.

[0112] In some embodiments, the throughput of 3D printing can be increased by adding additional print heads to the system in parallel. Each print head contains all the elements necessary to print a multi-material structure, and thus, by providing additional print heads in the system, several 3D structures can be printed simultaneously.

[0113] While the present invention has been described with reference to certain embodiments, various modifications of such embodiments will be apparent to those skilled in the art without departing from the object and scope of the invention as set forth in the claims appended herein. The examples provided herein are provided solely for the purpose of illustrating the present invention and do not limit the invention in any sense. The drawings provided herein are provided solely for the purpose of illustrating various aspects of the invention and are not intended to be drawn to scale or to limit the invention in any sense. All prior art described herein is incorporated herein by reference and its disclosures are incorporated herein by reference.

[0114] References

[0115] The following references are provided as examples of known technologies related to the present invention. The following list does not provide a comprehensive list of all related technologies. All technical documents described herein, including the following, are incorporated herein by reference and their entire contents constitute part of this specification. 1. Su-Jung Shin, Ji-Young Park, Jin-Young Lee, Ho Park, Yong-Doo Park, Kyu-Back Lee, Chang-Mo Whang, and Sang-Hoon Lee, "'On the fly' continuous generation of alginate fibers using a microfluidic device," Langmuir, 2007, Vol. 23, pp. 9104-9108. 2. Saif Khalil and Wei Sun, "Bioprinting endothelial cells with alginate for 3D tissue constructs," Journal of Biomechanical Engineering, 2009, No. 131, pp. 111002-1 to 111002-8. 3. Min Hu, Rensheng Deng, Karl M. Schumacher, Motoichi Kurisawa, Hongye Ye, Kristy Purnamawati, and Jackie Y. Ying, "Hydrodynamic spinning of hydrgel fibers," Biomaterials, 2010, Vol. 31, pp. 863-869. 4. Byung Kim, Intae Kim, Wooseok Choi, Sun Won Kim, Joosung Kim, and Geunbae Lim, "Fabrication of cell-encapsulated alginate microfiber scaffold using microfluidic channel," Journal of Manufacturing Science and Engineering, 2008, No. 130, pp. 021016-1 to 021016-6. 5. Edward Kang, Su-Jung Shin, Kwang Ho Lee, and Sang-Hoon Lee, "Novel PDMS cylindrical channels that generate coaxial flow, and application to fabrication of macrofibers and particles," Lab on a Chip, 2010, No. 10, pp. 1856-1861. 6. Hiroaki Onoe, Riho Gojo, Yukiko Tsuda, Daisuke Kiriyaand, and Shoji Takeuchi, "Core-shell gel wires for the construction of large area heterogeneous structures with biomaterials," IEEE MEMS Conference, 2010, pp. 248-251. 7. Setareh Ghorbanian, "Microfluidic probe for direct write of soft cell scaffolds," Master of Engineering Thesis, McGill University, Canada, 2010. 8. Edward Kang, Gi Seok Jeong, Yoon Young Choi, Kwang Ho Lee, Ali Khademhosseini, and Sang-Hoon Lee, "Digitally tunable physicochemical coding of material composition and topography in continuous microfibers," Nature Materials, 2011, No. 10, pp. 877-883. 9. European Patent Application Publication No. 2489779(A1) 10. U.S. Patent Application Publication No. 2006 / 0105011(A1) 11. U.S. Patent Application Publication No. 2011 / 0136162(A1) 12. U.S. Patent Application Publication No. 2012 / 0089238(A1) 13. International Publication No. 2012 / 009363(A1) Pamphlet

Claims

[Claim 1] A system for additive manufacturing of three-dimensional structures, wherein the system is - Includes at least one print head that receives and dispenses material, the material includes a sheath fluid and a hydrogel, and the print head is - An orifice for dispensing the aforementioned material in small quantities, - comprising one or more first channels for receiving and directing the sheath fluid, one or more second channels for receiving and directing the hydrogel, and a dispensing channel, wherein the first and second channels are joined together at a first intersection, and the dispensing channel extends from the first intersection to the orifice, and further, A first fluidic switch is located in the first channel and, when activated, is configured to allow or deny the flow of fluid in the first channel, A second fluidic switch is located in the second channel and, when activated, is configured to allow or deny the flow of fluid in the second channel, - A receiving surface that receives the first layer of the material dispensed in small amounts from the orifice, - A positioning unit for positioning the orifice of the print head in three-dimensional space, comprising a positioning unit operably coupled to the print head, - A system comprising a dispensing means for dispensing the material in small amounts from the orifice of the print head.

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