Title

A three-dimensional printer comprising a print head and insert and a method of using the same

Field of the Invention

The invention relates to a three-dimensional (3D) printer comprising a print head and an insert. Specifically, the invention relates to 3D printer comprising a print head and an insert for depositing, for example, hydrogels while modifying their properties in a liquid phase or in air during high resolution three-dimensional additive manufacturing.

Background to the Invention

Three-dimensional (3D) bioprinting is an additive process which involves sequential deposition of material in layers to create a 3D object. This additive process can be done through a wide variety of methods, including granular material deposition (initial depositions of granules are fused into a layer, often via laser sintering, that is then lowered and built further upon), photopolymerization (a vat of UV-reactive liquid is exposed to controlled lighting, causing the liquid to harden and form layers that build into a model) and extrusion deposition (extrusion of material through the extruder opening onto a surface). 3D printing is applicable to many industries on scales ranging from the creation of structures of the order of micrometers to meters.

Fused filament fabrication, also known as extrusion deposition 3D printing, involves the production of the model through the deposition of small particles or beads that immediately fuse into a solid substance. In the majority of cases, the printer contains an X, a Y and a Z stage. The X and Y stages, which are controlled independently by stepper motors, position the extruder head on the platform in X and Y axes. The Z stage controls the position of the extruder in the Z axis or the platform on which the structure is being created. Lowering this platform allows for successive layers to be added to the growing structure. Conversely, the extruder can be raised. The printer heats the extruder, with the temperatures depending on the materials being used.

US2015057786 describes a 3D printer device and methods of use thereof, for printing 3D constructs for use in fabricating 3D objects. The printer device comprises a means for applying a wetting agent to one or more of: the printer stage; the receiving surface, the deposition orifice, bio-ink, support material, or the printed construct. The wetting agent can be water, tissue culture media, buffered salt solutions, serum, or a combination thereof. The wetting agent is applied simultaneously or substantially simultaneously with or prior to the bio-ink or supporting material being dispensed by the bioprinter. WO 2014/194180 describes a printing method also, as well as an apparatus for placing cells on a surface comprising: a cell monolayer or biomaterial surface; one or more printing tips; a cartridge for holding said one or more printing tips; and a three-axis motion control system configured to move said cartridge in three dimensions with respect to said cell monolayer or biomaterial surface. A printing platform upon which this is carried out is also described. WO 2014/194180 also appears to describe building a construct using the printer when the printer tips are placed under an aqueous film.

WO 2015/017421 appears to disclose a method for fabricating a structure such as a biological tissue or a tissue engineering scaffold using 3D printing, where the printing method comprises a support bath within which the tissue scaffold is fabricated, and which provides divalent cations for crosslinking the printed material. Further, use of a cross-linker concentration in a method for producing rapid prototyping is discussed in EP1517778B; while DE102012100859A discloses a method for producing and printing a 3D structure containing living cells, which may comprise printing in a high-density liquid.

WO 216/019435 appears to disclose an additive manufacturing apparatus comprising a deposition head to extrude a first material into a reservoir containing a second material, wherein a least a portion of the object being manufactured is submerged in the second material. Further, the second material (a fluid) may be recirculated from the reservoir and back again. The document also appears to disclose that the reservoir is temperature controlled and different fluids may be mixed within the extruder.

WO 2017/081040 appears to disclose a 3D printer based on traditional nozzle-injection with multiple syringe capability in order to print different materials in air or liquid. The 3D printer is a temperature-controlled, fluid-exchange system which provides an unlimited range of printing possibilities as the liquid can be modified at will in real time (e.g., temperature, pH, ions, dyes, cross-linkers, drugs, growth factors, enzymes, extracellular matrix components). The problem(s) associated with the 3D printers and methods described in (i) US2015057786, (ii) WO 2014/194180, (iii) WO 2015/017421 , (iv) EP 1517778 and (v) DE 102012100859 is that it is not possible to influence or control the structure being the fabricated by manipulating the fabrication environment freely. In (i), the use of a wetting agent is to reduce evaporation during the printing process; in (ii) the method is to provide suitable conditions for cell deposition on a surface; for (iii) the support bath is generally removed by chemical treatment and results in printer clogging; (iv) the method discusses creating a polymer and adding a dye to change colour; and (v) the method relates to extrusion into a dense liquid that provides support to the structure, rather than changing the properties of the structure. The problem with the apparatus of WO 2016/019435 is that the method relates to extrusion into a liquid with a density that provides support to the structure being manufactured, rather than changing the properties of the structure itself. The method only uses one solution which is recirculated for reuse and to keep the level of the solution so that the last layer printed is submerged. None of the methods described above can locally modify the properties of the individual hydrogel layers.

The problem associated with the 3D printer described in WO 2017/081040 is that it may be difficult to reliably, repeatedly and accurately position or locate and retain the direction of flow of fluid from the print head to the stage on which a structure is being printed.

US 2017/251713 appears to describe a method for producing objects using a 3D printer, wherein the printer includes a thermoplastic extruder and a liquid mixing extruder (print-head with nozzle and extruder). The thermoplastic extruder is a standard fused filament deposition system employed to create a container to hold the liquid mixed in the second extruder prior to curing this liquid and solving the problem of printing liquid materials which do not hold their shape upon extrusion. The problem with the printer of US 2017/251713 is that beyond the liquid mixing ratio, the characteristics of the print cannot be precisely controlled spatially.

WO 2003/017745 appears to describe a 3D printer with a multifunctional print-head capable of dispensing material onto a surface, and a vacuum for extraction. The nozzle in the printing head can also be heated to locally heat the surface. The problem with the printer of WO 2003/017745 is that aside from heating, there is no provision for the local modification of print characteristics. For example, there is no possibility to locally deposit adhesion proteins while printing.

CN 109397686 appears to describe a 3D printer with a print-head having a mixing extruder that allows the heating and mixing of materials using a heater and a helical blade to improve the uniformity of the material extruded at the nozzle. The problem with the printer of CN 109397686 is that it is designed to solve a uniformity ( e.g ., in colour) problem related to fused glass pellet manufacturing wherein agitation aids in mixing. It is not designed for the precise local modification of the print characteristics.

CN 109130175 appears to describe a 3D printing device comprising a print-head with a nozzle and an extruder. The extruder acts to agitate material - various pigments - by stirring and to melt by heating prior to extrusion of printed material through a nozzle. The colour can be controlled by adjusting the ratios of the pigments. The problem with the printer of CN 109130175 is that it is designed to solve shortcomings of monochrome single material printers. It is not designed for the precise local modification of the print characteristics; the large mixing chamber provides a large reservoir that would need to be emptied in order to precisely modify the colour of adjacent 3D printed voxels.

It is an object of the present invention to overcome at least one of the above-mentioned problems.

Summary of the Invention

According to the present invention there is provided, as set out in the appended claims, a 3D printer or bioprinter central inflow print head (CIPH) for use in, the CIPH comprising: an extruder for printing at least one polymer, at least one inflow port for delivering a fluid to a platform; at least one outflow port for removing a fluid from the platform; and at least one reservoir to supply a fluid to the platform to create the liquid phase.

In one aspect, there is provided, a 3D printer comprising a central inflow print head (1 ) and an insert (100), the central inflow 3D printer print head (1 ) comprising a nozzle (2) in fluid communication with an extruder head (3), wherein the nozzle (2) has an inflow port (8) at a proximal end (12) and an outlet port (9) at a distal end; and wherein the extruder head (3) comprises an inlet port (4) at a distal end (10) in fluid communication with an outflow port (5) at a proximal end (1 1 ) through a channel (7), and a plurality of tubing (160) separate to the nozzle (2) that delivers a fluid to, or removes a fluid from, a printing platform; and the insert (100) comprising at least one chamber (101 ) having an inner wall (102) and an outer wall (103) and an internal baffle wall (104a,b) cooperating with the internal wall (102) to form an internal space (106); an upper surface (108) and a lip (109).

In one aspect, there is provided, a central inflow print head (1 ) for use with a 3D printer, the central inflow print head (1 ) comprising a nozzle (2) in fluid communication with an extruder head (3), wherein the nozzle (2) has an inflow port (8) at a proximal end (12) and an outlet port (9) at a distal end; and wherein the extruder head (3) comprises an inlet port (4) at a distal end (10) in fluid communication with an outflow port (5) at a proximal end (1 1 ) through a channel (7), and a plurality of tubing (160) adapted to accommodate a fluid for delivery to or removal from a printing platform.

In one aspect, the nozzle (2) has a substantially conical shape, narrowing in diameter in the direction of the proximal end (12) to the distal end (14).

In one aspect, the inflow port (8) is adapted deliver a fluid (such as a crosslinker, a protein in suspension, a cell, an additive, and the like) to a structure being printed in the 3D bioprinter.

In one aspect, the nozzle (2) of the central inflow print head (1 ) further comprises a hollow central portion adapted to accommodate a syringe or needle (180) which his configured to deliver a hydrogel to the printing platform to be modified.

In one aspect, there is provided an insert (100) for use with the central inflow print head (1 ) and 3D printer as described above, the insert comprising at least one chamber

(101 ) having an inner wall (102) and an outer wall (103) and an internal baffle wall (104a,b) cooperating with the internal wall (102) to form an internal space (106); an upper surface (108) and a lip (109). Preferably, the internal baffle wall (104a, 104b) traverses the width of the internal space (106) so that it connects with the internal wall

(102) on two sides forming a tube compartment (1 10) configured to accommodate the tubing (160). Preferably, the width of the tube compartment (1 10) is greater than 75% of the diameter of the tubing (160) and no more than 1 10% of the diameter of the tubing (160). Preferably, the tube compartment (1 10) further comprises a tapered lip (1 12) on the inner wall (102). Ideally, the tapered lip (1 12) further comprises a notch (1 14) at the point furthest away from the internal baffle wall (104b). The notch (1 14) is typically at an incline (1 16) of between about 15° and about 75° relative to the lip (108). Ideally, the incline (1 16) is at an angle of about 45°. In one aspect, the tapered lip (1 12) further comprises a rounded profile.

In one aspect, there is provided, a method for fabricating a structure by means of 3D printing using 3D printer, the central inflow print head, and the insert described herein, the method comprising the steps of extruding at least one polymer in a liquid phase or in air to form the structure on a platform, wherein the liquid phase is configured to modify the physical, chemical, mechanical and biofunctional properties of the structure being fabricated by controlling a fluid exchange in the liquid phase in real time during fabrication, characterised in that central inflow print head (1 ) is adapted to apply a plurality of difference crosslinking fluids simultaneously when printing the structure on the platform.

In one aspect, the liquid phase is contained within the platform area upon which the structure is fabricated.

In one aspect, the liquid phase is formulated to comprise at least one component selected from a buffer, a cell culture media, a cross-linking solution, and aqueous solutions containing ions, proteins, drugs, a prokaryotic cell or a eukaryotic cell.

In one aspect, at least one property of the liquid phase can be modified in real-time by actively replacing or adding a component to the liquid phase to modify the structure being fabricated. Preferably, the property is selected from temperature, pH, and ion concentration, or a combination thereof. Preferably, the component is selected from a dye, a cross-linking agent, a drug, a growth factor, an enzyme, an extracellular matrix component, a cell, a biomaterial, an organic material, a composite material, a nanomaterial, an encapsulated material, a drug eluting material, a dye, a fluorescent label, a quantum dot, a cell, and a diatom, or a combination thereof.

In one aspect, the fabricated structure is selected from a hydrogel, a biofunctional tissue, a microtissue, a hierarchical organotypic tissue, a scaffold, and a drug delivery particle. In one aspect, the platform is a petri-dish, a cell culture dish, a multi-well plate, a glass slide, a mesh, or any vessel capable being adapted for use in a fluid exchange system with inlets and outlets. In one aspect, the polymer is one or more selected from a monomer, a copolymer, a homopolymer, a multipolymer, a natural or synthetic multipolymer such as a hydrogel, alginate, collagen, chitosan, fibrin, polyethylene glycol), a synthetic hydrogel, a peptide, hyaluronic acid, and block copolymers. In one aspect, the extruder further comprises a solution selected from a buffer, cell culture media, a cross-linking solution, and aqueous solutions containing ions, proteins and drugs.

In one aspect, the extruder is a syringe or a syringe pump. Preferably, the extruder is a syringe with a plunger, or other suitable pump device, such as a cartridge or a tube. Preferably, the extruder is a syringe having a needle gauge selected from 1 , 2, 3, 4, 5,

6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29,

30. In one aspect, the shape of the opening of the extruder can control the shape of the print from the extruder. Preferably, the shape of the opening of the extruder is configured to print a structure selected from a solid tube, a hollow tube, a star-shaped extrusion, a square a circle, a polygon. In one aspect, there is provided a 3D bioprinter comprising the central inflow print head described herein and the insert described herein.

The CIPH is compatible with a 3D printer and allows, e.g., the cross-linking solution to be changed while a structure is being printed, thus enabling, for example, the user to tailor the physical, chemical and biofunctional properties of the structure, and locally introduce physical, chemical and biofunctional property gradients into the 3D object. Tailoring the chemical properties can be achieved by influencing the structure being fabricated as it responds to changes in pH, the ionic strength of the buffer or media, the solvent composition of the same and the molecular species being used. The method and fluid exchange system described herein allows a user to print stable structures onto a platform when the pumps for the liquid phase and extruder are running.

The advantages of the 3D printer comprising the CIPH described herein are that: The CIPH can be used for printing in air. In this mode, the biocompatible gel is extruded under pneumatic pressure as usual or with a physical syringe plunger but it is extruded in the presence of air rather than submerged in crosslinking fluid. Central inflow can be simply not used and, due to its design, the print is unaffected. Additionally, crosslinker may be applied to the print to partially alter the gels properties. While although it is not submerged this is an advantage over competitor print-in-air machines. This function offers the opportunity to tailor the gel around its circumference as it is extruded. As five or more (each of the supply tubes could have split connections going to multiple sources and controlled via valves/switches) crosslinking fluids can be applied simultaneously or in any combination around the circumference of the extruder tip, the gel may be altered in 72-degree arc segments (360/5) for even greater fidelity of crosslinking. This is advantageous over other approaches described for other extruder heads and 3D printers, which are time consuming (retracing print with second crosslinking syringe, spraying to crosslink, etc.).

Printing with a support utilises changing the fluid so that the gel is printed into a denser fluid, which is utilised to support the printed structure. The density of the support fluid/gel is such that it can support the weight of printed structures while also allowing the syringe to pass through it freely. In this case, the well plate or similar apparatus is filled with support material prior to the print starting. Future iterations of the device may automate this process. The print then is carried out, as material is extruded is pushes the support material out of the way. In essence, the support material surrounds the printed structure meaning greater overhangs and larger bridges can be printed. The CIPH allows the crosslinking or molecule loading to still take place at the nozzle extrusion as previously described.

Using the CIPH it is possible to invert the materials extruded. The crosslinking fluid is placed in the syringe and pneumatically extruded into the biocompatible gel. In this instance, the well is filled with the non-crosslinked biocompatible gel prior to printing. The crosslinker is extruded into the gel in a similar fashion to the support material. This mode is restricted to biocompatible gels of the correct viscosity. This mode offers the opportunity to combine the benefits of printing in support and printing submerged. The remaining non-crosslinked gel could then be rinsed away, leaving the printed structure intact. It is possible to print on typical substrates used in cell culture such as glass, plastic, metals, and also on other hydrogels, biopolymers, biomaterials, membranes, and mono- or multi-cell layers.

Both ion concentration and temperature-mediated cross-linking, can be implemented through software or manual control during the extrusion via control of extrusion rate and printing speed. The shape of the extruded material further depends on the physical parameters of the syringe tip (gauge, flat or bevelled end, opening shape, etc.). All parameters of printing and fluid exchange are controllable by software, script or manually during the printing process. The invention relates to an integrated fluid exchange 3D printer.

The Cl PH and insert described herein may be retrofitted to commercially available printers.

One advantage of the fluid exchange system of the 3D printer described herein is that optimization of alginate-gelatin and oxidized alginate-gelatin scaffolds, for example, can be achieved by controlling crosslinking, ratios, print speed, extrusion speed, nozzle size, viscosity, etc. during the printing process. This optimization can be done with very high precision, affecting the object being printed locally instead of layer per layer.

The advantage of pre-conditioning the platform by printing a sacrificial priming skirt thereon is that it improves adhesion and stability of the structure being printed. One could also use surface roughening and a mesh to promote attachment of the first layers.

In the specification, the term“3D printer” or“3D bioprinter” should be understood to mean a 3D printing system that is capable of printing structures that have organic and/or inorganic components and properties. The terms can be used interchangeably.

In the specification, the term“priming skirt” should be understood to mean a layer of polymer or hydrogel deposited or printed on the platform surface and which is an outline of the structure being fabricated. The skirt also helps to ensure that the printed polymer or hydrogel is securely attached to the surface of the platform or layer or base and also stabilises the resulting structure. The skirt primes the extruder by beginning the flow of polymer or gel through the extruder. It also allows time for the polymer or hydrogel to adhere to the platform, which can often take several seconds, before the printing of the structure, construct or scaffold begins. The priming skirt can be printed in air or solution and the structure being fabricated is then printed in solution. The priming skirt facilitates the attachment and stabilisation of the fabricated structures, which allows the structures themselves to be moved or removed from the platform (or from within the printing apparatus, or a layer or base or petri dish, or whatever it is printed on) or from one location to another on the printing apparatus. For example, one can fabricate individual structures on a support platform primed with a sacrificial skirt and move the structures to a 96-well plate, and test molecules in each of the wells on the separate structures. This allows one to move the structures to a plate rather than moving the platform in the printing apparatus to test the molecules. Alternatively, one could print directly into a multi-well plate with (or without, depending on the polymer and conditions) a priming skirt, and then move the multi-well plate from the platform and place it, for example, in an incubator. It allows the user to pick up the soft object (the printed structure) and move it directly from the platform or layer or base without damaging the printed structure.

In the specification, the term“liquid phase” should be understood to mean extrusion of the polymer into solution.

In the specification, the term“modified in real-time” should be understood to mean that parameters can be modified during the extrusion process, such as the feedback loop between the fluid exchange and the extrusion step. The modifications can be controlled by a computer system either via a pre-set program or manually by the user during the printing process. For example, the flow rate of the extruder or the rate of buffer flow into the platform, the temperature of the extruder or the liquid within the platform, the speed and direction of the extruder in X, Y and Z planes, the addition of agents (physical, chemical, biofunctional) to the extruder or platform ( e.g . liquid phase).

In the specification, the term“formulated to modify the structure” should be understood to mean modification of the physical, chemical, and biofunctional properties of the structure through fluid exchange. For example, any solution aqueous or otherwise which can modify the physical chemical or biofunctional properties or the extruded material or composite material including but not limited to buffer, cell culture media, a cross-linking solution, serum, aqueous solutions containing ions, proteins, drugs, etc. or a combination thereof or serve as a support structure. The addition of a liquid phase with high steroid concentration will lead to a print layer loaded with steroid that can be later used by cells to grow or migrate faster at this given position, e.g. the increase in the crosslinking concentration in the fluid phase will increase the cross-linked state of the extruded polymer, leading to a higher density print at the following layers and can give migration cues to cells or nerve endings. The increase in the crosslinking concentration in the fluid phase will increase the cross-linked state of the extruded polymer leading to a higher density print at the following layers and can give migration cues to cells or nerve endings or create a local density that can improve the print (stiffness).

In the specification, the term“modified structure” should be understood to mean and include modification of the composition of the structure, modification of the, or a, physical parameter of the structure (such as, for example, by controlling the rate of crosslinking, the mechanical properties, topography, roughness of the structure), chemical modification (such as, for example, adding functional groups to the polymers making up the structure; controlling the cross-linking and surface chemistry of the structure), and biofunctional modifications (such as, for example, the additional of proteins, drugs, growth factors etc. to the structure).

In the specification, the term“microcontact lithography” should be understood to mean a form of soft lithography that uses the relief patterns on a master polydimethylsiloxane (PDMS) stamp to form patterns of self-assembled monolayers (SAMs) of ink or proteins on the surface of a substrate through conformal contact, as in the case of micro contact or nanotransfer printing. Its applications are wide-ranging, including microelectronics, surface chemistry and cell biology.

In the specification, the term“polymer” should be understood to mean any natural or synthetic polymer commonly used in any combination and also as composite materials incorporating particles, nanomaterials, etc. polyethylene glycol; synthetic hydrogel, hyaluronic acid or any material and scaffolds that are extrudable, biocompatible, with limited by-products and stable.

In the specification, the term “hydrogel” or“hydrogels” can be interchangeable with “polymer” and should be understood to mean a network of natural, synthetic or hybrid polymer chains that are hydrophilic and/or hydrophobic. A hydrogel can be a homopolymer (a single polymer chain), a copolymer (two polymer chains), or a multipolymer (a plurality of different polymer chains). The polymers may be selected from alginate, collagen, fibrin, silk, lysozyme, synthetic hydrogel, polyethylene glycol), Matrigel® (a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced and marketed by Corning Life Sciences), calmodulin, elastin-like polypeptides; polysaccharides such as hyaluronic acid (HA), agarose, dextran, chitosan; protein/polysaccharide hybrids such as collagen/HA, laminin/cellulose, gelatin/chitosan and fibrin/alginate; deoxyribonucleic acid (DNA); degradable and non-degradable synthetic polymers such as the block copolymers polylactide-b/oc/(-poly(ethylene glycol)-b/oc/(-polylactide (PLA-PEG-PLA) and polyethylene glycol)-b/oc/(-polylactide-b/oc/(-poly(ethylene glycol) (PEG-PLA-PEG) diacrylates, disulfide-containing polyethylene glycol diacrylates (PEG(SS)DA), (hydroxyethyl)methacrylate (HEMA), acrylamide (AAm), acrylic acid (AAc), (N- isopropylacrylamide) (NIPAm), Poly(N-isopropylacrylamide) (PNIPAm) and polyethylene glycol) methacrylate (mPEGMA); natural/synthetic hybrids such as PEG- modified heparin, dextran, HA, fibrinogen, albumin; PNIPAm-modified collagen, chitosan and alginate; other synthetic peptide-modified proteins or polysaccharides; poly(vinyl alcohol) (PVA) modified natural polymers.

In the specification, the term“fabricated structure” should be understood to mean a structure that can be fabricated using the 3D printer, such as, for example, a hydrogel, a biofunctional tissue (for example, a printed tissue designed to elicit a particular cellular response, and extracellular matrix mimic, etc.), a microtissue (for example, a micron sized cell-tissue construct), a hierarchical organotypic tissue (for example, complex tissues with multiple levels of structure that are typical of particular organs, e.g., kidney, liver, lung, etc.), a scaffold, a biomaterial (for example, a collagenous object), a drug delivery particle, and the like.

In the specification, the term“baffle wall” should be understood to mean a physical barrier composed of a grid, a mesh or a pegboard having different mesh, pore or hole sizes. The number and size of the pores used would depend on the speed (pump pressure) of the flow or density of the fluid. For example, if a fluid is delivered from the inlet port at high pressure, it would be necessary to use a flow barrier to break the flow of fluid, release the pressure and ensure an even distribution of the fluid on the platform. This“baffle wall” is similar to a baffle system which is used to reduce turbulent flow in a fluid. For example, to achieve a higher pressure the baffle wall may have a reduced pore size so as to reduce maximum velocity. To achieve a low-pressure flow, the baffle wall may have larger pores. Alternatively, the baffle wall may be removed altogether. In baffle systems, the height of the wall, the thickness of wall and the porosity can be changed. The baffle wall may be made from any material that is suitable to safely support ( e.g . inert, stable) the fluid being used in the liquid phase of the printing process. For example, the baffle wall may be composed of poly(methyl methacrylate) (PMMA), or polymers having similar physical properties when set. In the system described herein, the function of the baffle wall is to control the inflow and the outflow of the fluid from the inlet port to the liquid phase and from the liquid phase to the outflow port, while avoiding any fluid-based disturbance of the printing process (e.g. drag, drift, lateral displacement). The advantage of the baffle wall is that it allows for a smooth exchange of fluid into and out of the liquid phase while printing, in between layer prints, or any other related printing steps.

In the specification the term“central inflow print head” should be understood to mean the combination of a printing nozzle to extrude a hydrogel, or other suitable material, jacketed by a multi-entry flow system that run alongside the extruded material to modify or act upon the extruded material properties. The combination can be, but is not limited to, one fluid for one hydrogel type while the fluid element can be modified physically (e.g. temperature), chemically (e.g. pH), or biologically (e.g. protein addition).

Materials and Methods

Printer Equipment

Any ‘do it yourself kit for assembling a bioprinter is readily available, such as the Ultimaker Original™. 3D printers generally consist of a platform with an adjustable bed, X, Y and Z axes run by stepper motors, an extruder head and an extruder which pushes the filament through a heated nozzle. The extruder head sits on two metal bars attached to the X and Y axes which control the movement around the bed. The bed platform sits on a threaded bar (the Z axis) which controls the Z positioning during printing. One embodiment of a printer used herein was constructed using these parts and the extruder modified for use with a syringe. It should be understood that the system of the invention can be used in other printers, such as RepRap (replicating rapid prototype) printers and 2D printers. The RepRap printers are 3D printers that are an open design, released under a free software license (the GNU General Public License), and use an additive manufacturing technique called fused filament fabrication (FFF) to lay down material in layers. It will also be possible to inject a polymer with a syringe in a photopolymerisation-based 3D printer and exchange the resin bath with different polymers ( e.g . to change colours, add reagents) to affect the printed structure.

Brief Description of the Drawings

The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which

Figure 1 illustrates (A) a perspective view of a cross-section of the CIPH, (B) a perspective view of the CIPH as a whole, (C) a perspective view of a cross-section of the lower portion of the CIPH, and (D) a plan view of a CIPH of the claimed invention.

Figure 2 illustrates (A) a perspective view of an insert of the claimed invention, (B) is a side view of the insert, and (C) and (D) are plan views of one chamber of the insert of the claimed invention, where (D) shows the insert in use with a plate and tubing.

Figure 3 illustrates the print head and insert of the claimed invention in use with connected inflow tubes in the insert (100).

Figure 4 illustrates the print head and insert of the claimed invention in use with connected inflow tubes in the printing head.

Detailed Description of the Drawings

A solution may be extruded into a liquid bath that can be modified at any given time. The temperature or crosslinking concentration of the bath can be modified by removing one liquid and adding another via a pumping system with incident control and grid systems to reduce flow turbulence. To address the issues of the current 3D printers, the Applicant has developed a 3D printer with a print head which enables the user to load a combination of location-specific crosslinking and biochemical factors to different locations of a hydrogel during the biofabrication of a 3D structure. The Applicant has also developed an insert for use with the 3D printer comprising the print head which facilitates the ingress and egress of fluids during the biofabrication process by reliably, repeatedly and accurately position or locate and retain tubing from the print head in a well plate. The edges of the insert act as a guide for the (flexible) tubing coming from the print head. The use of the print head and the insert increases the precision of the application of the crosslinking fluid as it can be applied on a point per point basis during the print rather than a layer by layer application. The approach is based on traditional nozzle-injection with multiple syringe capability in order to print different materials, including cells and tubes for, for example, vascularization, in air or liquid. The primary advantage of the approach described herein is that printing in a liquid environment gives a user the possibility to tune the properties of the material - to tailor the physical, chemical and biofunctional properties of the print in real time with micrometre resolution - in order to print reproducible microtissues onto a variety of platforms, including petri dishes or cell culture dishes, and multiple well-plates (12, 24, 36, 48, 72, 96 wells, etc.) already compatible with many biomedical characterisation tools. Furthermore, the central inflow printer head of the invention provides an unlimited range of printing possibilities as the liquid can be modified at will in real time ( e.g ., pH, ions, dyes, cross-linkers, drugs, growth factors, enzymes, extracellular matrix components).

Turning now to Figure 1 , there is illustrated a 3D printer central inflow print head (CIPH) of the present invention. Specifically, Figure 1 illustrates (A) a cross-section of the CIPH, (B) a plan perspective view of the CIPH as a whole, (C) a perspective view of a cross-section of the lower portion of the CIPH, and (D) a plan view of the CIPH of the claimed invention and is generally referred to by reference numeral 1 . The CIPH 1 comprises an extruder head 3 (also known as a CIPH body (both terms are interchangeable)) and a nozzle 2 (also known as a CIPH head (both terms are interchangeable)). The extruder head 3 generally having at least one inlet port 4 at a distal end 10 and at least one outflow port 5 at a proximal end 1 1 . The inlet port 4 and outflow port 5 are in fluid communication (in line) with each other through a channel 7. The channel 7 runs the length of the extruder head 3, from the distal end 10 to the proximal end 1 1 . The extruder head 3 further comprises an outer wall 20 and an inner wall 21 which together wrap around to form a hollow centre 15.

The nozzle 2 generally comprises at least one inflow port 8 and an outlet port 9. The inflow port 8 is at a proximal end 12 of the nozzle 2, while the outlet port 9 is at a distal end 14 of the nozzle 2. The inflow port 8 at the proximal end 12 is in fluid communication with the outflow port 5 of the extruder head 3. The inflow port 8 is also in fluid communication with the outlet port 9. The outlet port 9 being at the distal end 14 of the nozzle 2. Thus, the extruder head 3 and the nozzle 2 are in fluid communication with each other from the inlet port 4 to the outlet port 9. The nozzle 2 also comprises an outer wall 22 and an inner wall 23, which wrap around to form a hollow centre 16. The nozzle 2 narrows in diameter in the direction of the proximal end 12 to the distal end 14, forming a substantially conical shape.

The extruder head 3 is connected to the nozzle 2 via connectors 30,32, respectively. The connector 30 is found on the distal end 1 1 of the extruder 3, while the connector 32 is found on the proximal end 12 of the nozzle 2. The connectors 30,32 are affixed to each other by a connection means known to the skilled person, such as a male-female arrangement (fastener, nut and bolt, etc.), a snap-fit connection, a cable, a hook, a pin, a rivet, a seam, an adhesive, a switch, a collar, a tightening nut, a screw, a tie (for example, a piece of string, twine, a shoe lace etc.) and the like.

The extruder head 3 stores a polymer or a polymer mix in a syringe 180 connected to a needle/nozzle 182 (see Figures 3 and 4) prior to extruding the polymer or polymer mix through the outlet port 9 and the distal port 14 of the nozzle 2 and into a liquid phase or dry phase (printing area) held on a platform. The syringe 180 can be inserted within or be wrapped up with a heating element to improve the polymer flow into the nozzle/needle 182. Liquid flows through distal port 9 onto the nozzle/needle 182 and is guided towards the extruded polymer and can chemically, physically or biologically interact with it. The polymer or polymer mix stored in the syringe 180 connected to the needle/nozzle 182, which is connected to the extruder head 3, is generally a hydrogel forming polymer as described herein.

Turning now to Figure 2, there is illustrated a perspective view of a 3D printer insert of the claimed invention and is generally referred to by reference numeral 100. The insert 100 generally comprises at least one chamber 101 or a number of chambers 101 that correlate with the number of wells of the platform that the CIPH 1 is printing into. For example, the insert 100 could comprise a single chamber 101 to a multi-chamber 101 insert 100, for example, a 6-chamber insert. The insert 100 is designed such that the central area of the platform is sufficiently large to allow structures of a specified print volume/size to printed, while allowing for the free movement of the CIPH 1 , so as to not limit the structures that can be printed.

For example, the internal dimensions of the chamber 101 can be calculated as follows:

• Minimum size = [(print volume) + (2O.D. of syringe tip)] mm in x and y direction (1 )

• Functional size = [(2 ^* (edge length of print volume)]mm in x and y direction (2) • Maximum size = [(vessel edge length) - (2OD of tubing) - (4 ^* wall thickness of insert)] mm in x and y (3)

The chamber 101 comprises an inner wall 102, an outer wall 103, an internal baffle wall 104a, 104b, an upper surface 108 and a lip 109. The internal baffle wall 104a and internal baffle wall 104b face across from each other, while being attached to or are continuous with the inner wall 102. The inner wall 102 and outer wall 103, together with baffle walls 104a, 104b, cooperate with each other to form an internal space 106 located inside the inner wall 102 and between the baffle walls 104a, 104b. The location of the internal baffle wall 104b is determined by the internal size of the chamber 101 as defined above. For example, if the internal chamber size was 20 mm x 20 mm, it would mean that the inner baffle wall 104b would be 10 mm from the centre of the platform. The baffle wall 104b tends to be in contact with the surface of the platform within which the insert 100 is placed.

The baffle wall 104a, 104b can be made of materials that are inert and stable in the fluids being exchanged, including cellulose, ceramic, plastic, nylon, polycarbonate, polytetrafluorethylene (PTFE), polyamide or any other filtering-type material known in the art, but also can be in the form of grids made from materials such as, for example, metals (stainless steel, titanium, aluminium, etc.), polyvinyl chloride (PVC), polylactic acid (PLA, polylactide), Poly(methyl methacrylate) (PMMA), or other materials known in the art.

The insert 100, when in contact with the platform, must meet the following criteria:

1 . The geometry of the inner wall 102 of the insert 100 should match the wall geometry of the platform e.g., if the platform is circular then the inner wall 102 of the insert 100 should also be circular. While other geometries would work, it means that space is being used inefficiently and makes guiding the tubing into place more difficult.

2. The outer wall 103 of the insert 100 should be at most either, 98% of the edge dimensions of the platform or 1 mm less than the edge dimension. The lower limit is determined by the size of tube coming from the CIPH 1 and the space that it requires.

3. The height of the wall ideally should be between 90% and 97%, but not less than 0.5mm, of the depth of the total depth of the vessel (platform). Other heights can be used depending on the needs of the specific application. The space between the inner wall 102 and the baffle wall 104a, 104b defines a tube compartment 1 10, which accommodates a tubing coming from the CIPH 1 . The space between the inner wall 102 and the baffle wall 104a, 104b also defines the shape and size of the tube compartment 1 10 which allows the tubing to be positioned therein, constrains the tubing and allows for fluid to flow into the print area. The maximum width of the tube compartment 1 10 needs to be greater than 75% of the diameter of the tube, as smaller widths will start to pinch the tube; and no more than 1 10% of the diameter of the tube, as larger widths will not retain the tubing. These figures are based on standard flexible silicone tubing.

In an aspect, the tube compartment 1 10 located at the outer periphery of the insert 100 is formed by the internal baffle wall 104a and the structure of the platform within which the insert 100 is placed. The outer wall 103 of the insert 1 has a cut-out section 150 to accommodate the placement of the insert 100 into the wells of the platform. The wall of the well of the platform creates one side of the tube compartment 1 10 while the internal baffle wall 104a forms the other side.

Additionally, the shape of the tube compartment 1 10 is further defined by a tapered lip 1 12 of the inner wall 102 which forms one side of the tube compartment 1 10. The tapered lip 1 12 helps guide the tubing into the tube compartment 1 10. The tapered lip 1 12 can optionally have a notch 1 14 at the point furthest away from the baffle wall 104. The notch 1 14 can help in directing the tubing into the compartment 1 10. While the insert 100 would be functional without the notch 114, it was found that if a notch having the same length as the edge length of the chosen build volume, or a width correlating with the outer diameter of the tubing, was removed from the insert, it improved the positioning or freedom of the tube to move during the printing or insertion process while still retaining the tube and allowing for lateral movement. This ensures that the CIPH 1 is not restricted during the printing process.

The geometry of the upper surface 108, combined with an inclined inner surface 1 12, is important when it comes to guiding the tubing into position in the tube compartment 1 10 and to facilitate the removal of the tubing. There are two main features of the geometry of the upper surface 108: the inclined surface 1 12 that is applied to the notch 1 14 of the tube compartment 1 10 and the roundness of the edges of the notch 1 14 that are created by the incline 1 12. The incline 1 12 can be any angle between about 15° and about 75°, and preferably between about 40°and about 50°. Ideally, the angle is about 45°. The depth of the incline 1 12 applied is ideally as deep as the geometry will allow for a given application, as this provides the most lead-in for the tubing. The incline 1 12 also reduces the force required to pull the tubing out of the insert 100.

The incline 1 12 also has a rounded profile, primarily for, but not limited to, the following reasons:

1 . To eliminate sharp edges in the compartment 1 10 that could snag the tubing, preventing it from moving easily.

2. To increase the surface area on the upper surface 108 that the tubing can make contact with, which improves the ability of the insert 100 to guide the tubing into position.

3. To reduce friction as the tubing is moved. The rounder this can be, the better the insert performs.

Turning now to Figures 3 and 4, there is illustrated the CIPH 1 and the insert 100 in use in a 3D printer. The CIPH 1 and the insert 100 are designed to act in unison when in use in the 3D printer or bioprinter. The insert 100 is designed to act as guide/retention device for tubing 160 as part of a fluid exchange system. One or more tubes 160 are mounted to the edge of the CIPH 1 . A syringe 180 comprising a syringe tip 182 is accommodated within the hollow centres 15,16 of the CIPH 1 . To locate the tubing 160 in the right location in the printing platform, the following procedure is used.

1 . Raise the print head 1 , such that the syringe tip 182 is above the lip of the platform.

2. Advance the print head 1 to the next section of the platform. The tubing 160 will lag behind the print head 1 .

3. The print head 1 is moved ahead of the starting point of the next print. This allows for the tubing 160 to be located into the tubing compartment 1 10 of the insert 100.

4. The print head 1 is moved back to the start position of the print. This forces the tubing 160 to align with the widest section of the tubing compartment 1 10, thus restraining it while printing. During use, for example when 3D bioprinting with a specific hydrogel (either in air or in a liquid) to fabricate a thread-like medical device or a 3D hydrogel structure, the crosslinker or any specific fluid can be entered in the central inflow print head 1 by two distinct entry ports. First the fluid can be exchanged within a well through the plate insert 100 (see arrow A for fluid in and arrow B for fluid out in Figure 4), this method allows for complete modification of the hydrogel being printed at the time. Secondly a crosslinker can be locally exchanged while 3D printing the hydrogel using the CIPH 1 and the insert 100. In that case, the fluid containing the crosslinker is dripped along the needle 182 containing the hydrogel (see Figure 4) such that it enters the print chamber along the same axis of travel as the hydrogel polymer, which modifies the printed structure locally as the hydrogel is extruded rather than globally over the entire printed structure. This means that the CIPH 1 combined with the insert 100 allows for both general and local modification of a 3D printed hydrogel using any fluid configured to introduce a particular reagent, active, or other component to the printed 3D structure.

The fluid(s) are pumped under computer-controlled pressures of flow rates into the central inflow print head 1 through channels 7,8. The print head 1 is adapted to accommodate the syringe 180, or capillary tubes, pipettes, single/multi-hole/coaxial tube spinnerets or other delivery devices required for extrusion of the polymer within the hollow centres 15,16.

The extruder head 3 is generally controlled to move in X, Y and Z stages, the same as any other extruder head in a 3D printer and the movement mechanism can be taken from those printers known in the art. In general, the mechanism consists of a rail system, belts, stepper motors, and is generally referred to by those skilled in the art as a drive train system. This is described here: http://reprap.org/wiki/Cateaorv: DriveTrains. Basically, voltage is applied to a stepper motor that causes rotation, which is translated into independent linear motion along the X, Y and Z stages. The mechanism of movement is the same as that of a Computer Numerical Control (CNC) router, which is a computer controlled cutting machine related to the hand-held router used for cutting various hard materials, such as wood, composites, aluminium, steel, plastics, and foams, and is familiar to those skilled in the art of 3D printing.

The fluid exchange process is a software controlled and feedback circuit controlled by a computer during the extrusion process. The computer is able to adapt the fluid exchange and the extrusion steps as needed. A fluid exchange platform can be linked to more than one polymer reservoir to allow for mixing of components prior to injection in the printing area, allowing for multiple combinations and changes during one single print. In the specification the terms "comprise, comprises, comprised and comprising" or any variation thereof and the terms include, includes, included and including" or any variation thereof are considered to be totally interchangeable and they should all be afforded the widest possible interpretation and vice versa. The invention is not limited to the embodiments hereinbefore described but may be varied in both construction and detail.