Bioprinter for fabricating 3D cellular constructs

Abstract

A bioprinter for producing three-dimensional (3D) cellular constructs, comprising: one or more holding reservoirs for holding fluid samples; a print stage for holding sample containers and supporting a substrate on which the 3D cellular constructs are printed; a sample loading system in fluid communication with the one or more holding reservoirs, the sample loading system configured to load the sample from the sample container into the one or more holding reservoirs; a pump in fluid communication with the sample loading system, the pump configured to draw the sample from the sample container and deliver the sample to the one or more holding reservoirs; and a droplet dispensing system in fluid communication with the one or more reservoirs, the droplet dispensing system configured to print sample droplets from the one or more reservoirs onto a substrate supported by the print stage.

Description

[Technical field]

[0001] The present technology relates to bioprinters for producing three-dimensional (3D) cellular constructs, processes for bioprinting 3D cellular constructs, and bioprinted 3D cellular constructs.

[0002] Related Applications This application claims priority to Australian Provisional Patent Application No. 2017904946, filed December 8, 2017, the contents of which are incorporated by reference in their entirety. [Background technology]

[0003] The workhorse of in vitro cell biology is cell culture, where primary or immortalized cells are simply plated onto a plastic or glass surface. Many cellular properties, such as in cell proliferation, differentiation and response to external stimuli, are fundamentally different for cells in two-dimensional (2D) and 3D environments found in vivo. Particularly for drug development and precision medicine programs, cell culture conditions that better reflect the 3D animal environment and thus limit the number of failed animal experiments would be highly advantageous.

[0004] For example, in cancer cell biology, 3D models exhibit more in vivo tumor-like features compared to 2D cell culture models, including hypoxic regions, gradient distribution of chemical and biological factors, and expression of pro-angiogenic and multidrug resistance proteins.

[0005] For this reason, 3D multicellular models are generally considered better models of in vivo systems than the more common 2D cell cultures.

[0006] Moreover, most cellular structures in multicellular biology are organized in three dimensions. Numerous studies have reported the printing of cells using 3D bioprinting techniques (reviewed in (Murphy and Atala, 2014)).

[0007] There are many commercially available 3D bioprinters, for example, 3D-Bioplotter® by EnvisionTEC, BioScaffolder by GeSiM, Bio X by Cellink, BioFactory® by RegenHU, BioBot 2 by BioBots, etc. Commercially available 3D bioprinters are most commonly based on microextrusion, thermal inkjet or piezoelectric inkjet technology. Commercially available 3D bioprinters most commonly utilize cartridges (e.g., Nordson Optimum® Syringe Barrels) to load materials into the printer. Each of these cartridges is often coupled to a single printhead. Maintaining sterility is difficult during cartridge filling, handling, installation and removal.

[0008] The design of 3D models of organs or tissue structures for 3D bioprinting applications is mainly based on: 1) non-invasive medical imaging techniques for data collection (e.g., computed tomography (CT) and magnetic resonance imaging (MRI)); and 2) Computer-aided design and computer-aided manufacturing (CAD-CAM) tools and mathematical modelling for information digitisation, generation of 3D rendering models and generation of 2D cross-sectional images (Murphy and Atala, 2014; Horn and Harrysson, 2012).

[0009] There is a need for tools and technologies that facilitate the application of 3D cell culture models in drug discovery, personalized medicine and general cell biology in a scalable, reproducible and cost-effective manner.

[0010] The present inventors have developed devices, systems and methods for producing in vitro 3D cell culture assays and arrays thereof. Summary of the Invention

[0011] overview In a first aspect, the present invention provides a bioprinter for producing three-dimensional (3D) cellular constructs, said bioprinter comprising: one or more holding reservoirs for holding the fluid sample; a print stage for holding the sample container and supporting the substrate on which the 3D cell constructs will be printed; a sample loading system in fluid communication with the one or more holding reservoirs, the sample loading system configured to load a sample from a sample container into the one or more holding reservoirs; a pump in fluid communication with the sample loading system configured to draw sample from a sample container and pump the sample into the one or more holding reservoirs; and and a droplet dispensing system in fluid communication with the one or more reservoirs, the droplet dispensing system configured to print sample droplets from the one or more reservoirs onto a substrate supported by the print stage.

[0012] In one embodiment, the bioprinter further comprises a housing that encloses one or more holding reservoirs, a print stage, a holder, a sample loading system, a pump, and a droplet dispensing system.

[0013] In one embodiment, the bioprinter further comprises an airflow system disposed within the housing, the airflow system configured to induce laminar airflow within the housing.

[0014] In one embodiment, the airflow system includes a fan that induces laminar airflow within the housing.

[0015] In one embodiment, the air flow system includes at least one air filter.

[0016] In one embodiment, the sample loading system includes a needle for insertion into the sample vessel, and the pump is configured to draw fluid through the needle when the needle is inserted into the sample vessel.

[0017] In one embodiment, the bioprinter further comprises a first positioning unit coupled to the needle, the first positioning unit configured to insert the needle into the sample container and to withdraw the needle from the sample container.

[0018] In one embodiment, the bioprinter further comprises a second positioning unit having a track, the second positioning unit coupled to the needle and the droplet dispensing system and configured to move the needle and the droplet dispensing system along the track of the second positioning unit.

[0019] According to one embodiment, the bioprinter further includes a third positioning unit having a track, the third positioning unit coupled to the print stage and configured to move the print stage along the track of the third positioning unit.

[0020] According to one embodiment, the track of the second positioning unit extends substantially perpendicular to the track of the third positioning unit.

[0021] In one embodiment, the bioprinter further comprises a plurality of holding reservoirs, and the sample loading system is configured to load a sample from a sample container into any one of the plurality of reservoirs.

[0022] In one embodiment, the sample container is a tray having a plurality of sample wells, the sample wells configured to receive a sample, and the sample loading system configured to load the sample from any one of the sample wells into any one of the retention reservoirs.

[0023] In one embodiment, the bioprinter further comprises a waste container configured to receive waste material from the sample loading system.

[0024] In one embodiment, the waste container is provided on a tray.

[0025] In one embodiment, a pump is configured to draw the sample from one of the holding reservoirs and pump the sample out of the sample loading system.

[0026] In one embodiment, the bioprinter further comprises a pressure regulator coupled in fluid communication to each retention reservoir for regulating the pressure within each retention reservoir.

[0027] In one embodiment, the bioprinter further comprises a selector valve in fluid communication with the pump, the sample loading system, each holding reservoir, and the pressure regulator, the selector valve configured to selectively couple the pump in fluid communication with the sample loading system and each holding reservoir.

[0028] According to one embodiment, the pressure regulator is removably coupled in fluid communication with the compressed air source.

[0029] In a second aspect, the present invention provides a method of producing a three-dimensional cellular construct comprising depositing droplets of one or more samples using a bioprinter of the first aspect.

[0030] In a third aspect, the present invention provides a method for producing a three-dimensional cell construct, the method comprising: providing a bioprinter of a first aspect; Presenting the substrate to a print stage; providing a sample container to the print stage, the sample container containing a sample; loading the sample into one of the holding reservoirs by a sample loading system; and Printing the sample from the holding reservoir onto a substrate using a droplet dispensing system to form a three-dimensional cellular construct.

[0031] Also disclosed is a bioprinter for producing a three-dimensional cell construct, the bioprinter comprising: a sample loading system for loading the sample from the sample container into the holding reservoir; a selector valve in fluid communication with the holding reservoir for directing the sample to the holding reservoir; a droplet dispensing system in fluid communication with the holding reservoir, the droplet dispensing system adapted to print sample droplets from the holding reservoir onto a substrate; a control system for controlling the operation of the sample loading system, the selection valve, and the droplet dispensing system; a laminar airflow system; and a housing containing the sample loading system, the selector valve, the droplet dispensing system, and the laminar airflow system.

[0032] In one embodiment, a sample loading system includes a plurality of sample containers and a plurality of holding reservoirs for holding samples from each container.

[0033] In one embodiment, multiple sample vessels are housed in a removable sample tray.

[0034] In one embodiment, a removable sample tray includes 10 positions for holding sample vessels in an array.

[0035] In one embodiment, the sample container is a vial having a cap and a septum.

[0036] In one embodiment, the removable sample tray further includes a waste container for receiving waste from flushing the sample loading system.

[0037] In one embodiment, the removable sample tray further includes a wash reservoir for cleaning the sample loading system, the selector valve, and the droplet dispensing system.

[0038] In one embodiment, the sample loading system comprises a needle for insertion into the sample vessel and a pump operably coupled to the needle for transporting the sample in the sample vessel to the holding reservoir.

[0039] According to one embodiment, the pump is a positive displacement pump.

[0040] In one embodiment, the pump is a peristaltic pump, a diaphragm pump, or a syringe pump.

[0041] In one embodiment, the pump is reversible and operable for resuspension of the sample within the container.

[0042] In one embodiment, the sample loading system further includes a first positioning unit operably coupled to the needle, the first positioning unit positions the needle into and out of piercing engagement with the sample vessel.

[0043] In one embodiment, the sample in the sample container can be a cell suspension, water, ethanol, bio-ink, an activator, a washing solution, a flushing fluid, a cell culture medium, or a drug dispersed in a solution.

[0044] In one embodiment, the sample in the sample container is sterile.

[0045] According to one embodiment, the sample loading system further comprises a second positioning unit operably coupled to the needle, the second positioning unit positioning the needle in two-dimensional space.

[0046] In one embodiment, the retention reservoir is an elongated tube.

[0047] In one embodiment, an elongated tube is coiled and contained within the chamber.

[0048] In one embodiment, the retention reservoir is formed from a spool of flexible tubing.

[0049] In one embodiment, the flexible tubing comprises a polytetrafluoroethylene (PTFE) tubing.

[0050] In one embodiment, a droplet dispensing system comprises at least one print head operably coupled to a plurality of holding reservoirs and adapted to dispense sample droplets from each holding reservoir onto a substrate.

[0051] In one embodiment, at least one printhead is an array of valves.

[0052] According to one embodiment, each valve is a micro-solenoid valve.

[0053] In one embodiment, the sample is stored in a holding reservoir upstream of the micro-solenoid valve.

[0054] In one embodiment, each holding reservoir has its own printhead.

[0055] In one embodiment, each holding reservoir is coupled to a pressure regulator.

[0056] According to one embodiment, a compressed air supply is connected to a regulator manifold.

[0057] According to one embodiment, each micro solenoid valve is coupled to a respective pressure regulator.

[0058] According to one embodiment, a droplet dispensing system includes a plurality of pressure regulators in a regulator manifold, at least one check valve, and a compressed air supply operatively coupled to each pressure regulator in the regulator manifold.

[0059] According to one embodiment, the pressure regulator is coupled to the selector valve.

[0060] In one embodiment, the sample is drawn from a sample container into the holding reservoir using a sample loading system, and from the holding reservoir into the print head through operation of the droplet dispensing system, with a pressure regulator of the droplet dispensing system and a selector valve of the sample loading system operating to move the sample from the holding reservoir to the print head.

[0061] In one embodiment, the droplet dispensing system further comprises a print stage for supporting the substrate.

[0062] In one embodiment, the substrate is a multi-well plate.

[0063] According to one embodiment, the droplet dispensing system further includes a third positioning unit operably coupled to the print stage, the third positioning unit positioning the print stage in two-dimensional space.

[0064] In one embodiment, the control system records the identity of the sample in the sample container from a user input.

[0065] In one embodiment, the control system comprises a non-transitory computer readable medium containing programmed instructions for operating the bioprinter.

[0066] In one embodiment, the non-transitory computer readable medium is located separately from the bioprinter and operably connectable to the bioprinter.

[0067] In one embodiment, the laminar flow system includes a fan for drawing air into the housing, an air inlet for admitting air, a filter, and an air outlet.

[0068] In one embodiment, the fan is a centrifugal fan.

[0069] In one embodiment, a fan draws air from below the print stage, to the front of the housing, around the sample loading system, through one or more filters, and out of the housing.

[0070] According to one embodiment, a fan draws air through the front access door of the bioprinter housing.

[0071] In one embodiment, the laminar flow system includes two filters, one to exhaust air and one to receive air toward the print stage.

[0072] According to one embodiment, each filter is a high efficiency particulate air (HEPA) filter.

[0073] In one embodiment, each filter receives approximately 50% of the airflow.

[0074] In one embodiment, the housing includes a hinged door to allow a user access to the interior of the bioprinter.

[0075] In one embodiment, a removable sample tray can be loaded into the bioprinter through a door.

[0076] In one embodiment, the removable sample tray has a lid.

[0077] In one embodiment, the housing has a recess for receiving a lid for a sample tray and a lid for a multi-well plate.

[0078] In one embodiment, the sample containers are loadable into a removable sample tray inside the bioprinter.

[0079] In a second aspect, there is provided a method of producing at least one three-dimensional cellular construct by depositing a plurality of droplets of a sample using a bioprinter according to the first aspect.

[0080] Throughout this specification, unless the context requires otherwise, the term "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of the stated elements, integers or steps, or group of elements, integers or steps, but not the exclusion of other elements, integers or steps, or group of elements, integers or steps.

[0081] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and is not to be construed as an admission that any or all of such matters form part of the prior art base or were common general knowledge in the art relevant to the present invention as they existed prior to the priority date of each claim herein.

[0082] In order that the invention may be more clearly understood, preferred embodiments will now be described with reference to the following drawings and examples. [Brief description of the drawings]

[0083] [Figure 1] FIG. 1 is a rear perspective view of a bioprinter for fabricating 3D cellular constructs and illustrates the sample loading system;

[0084] [Diagram 2] FIG. 2 is a front perspective view of the sample loading system of FIG. 1;

[0085] [Diagram 3] FIG. 3 is a front perspective view of the sample loading system showing a number of sample vessels, each containing a sample;

[0086] [Figure 4] FIG. 4 is a rear perspective view of the sample loading system of FIG. 3;

[0087] [Diagram 5] FIG. 5 is a perspective view of a removable sample tray used in the sample loading system;

[0088] [Figure 6] FIG. 6 is a front perspective view of the bioprinter with the laminar airflow system installed;

[0089] [Figure 7] FIG. 7 is a rear perspective view of the bioprinter showing only the laminar airflow system;

[0090] [Figure 8] FIG. 8 is a rear perspective view of the bioprinter showing only the laminar airflow system of FIG. 7;

[0091] [Figure 9] Figures 9 and 10 show the bioprinter with a transparent panel showing the air flow path of the laminar airflow system; [Figure 10] Figures 9 and 10 show the bioprinter with a transparent panel showing the air flow path of the laminar airflow system;

[0092] [Figure 11] FIG. 11 is a top perspective view of the bioprinter illustrating the positioning units for the droplet dispensing system and the sample loading system;

[0093] [Figure 12] FIG. 12 is a side perspective view of a bioprinter with multiple holding reservoirs and a compressed air supply system;

[0094] [Figure 13] 12 is a front perspective view of the bioprinter of FIG. 11 with the laminar airflow system installed;

[0095] [Figure 14] FIG. 14 is a rear perspective view of the bioprinter of FIG. 13;

[0096] [Figure 15] FIG. 15 is a rear perspective view of a bioprinter having a single holding reservoir;

[0097] [Figure 16] FIG. 16 is a front perspective view of the assembled bioprinter;

[0098] [Figure 17] FIG. 17 is a rear perspective view of the bioprinter of FIG. 16;

[0099] [Figure 18] FIG. 18 is a flow chart diagram of the components of the bioprinter;

[0100] [Figure 19] FIG. 19 is a side view of the components of the bioprinter showing the air flow paths between the components of the laminar airflow system;

[0101] [Figure 20] FIG. 20 is a flow chart diagram of a bioprinter operatively associated with a control system computer;

[0102] [Figure 21] FIG. 21 is an exemplary graphical user interface of the control system software implemented on a computer showing three-dimensional cell construct design;

[0103] [Figure 22] FIG. 22 is an exemplary graphical user interface of the control system software implemented on a computer showing the printing of 3D cellular constructs in a multi-well plate; and

[0104] [Diagram 23] FIG. 23 is a schematic diagram of a flow chart for designing 3D cell constructs integrated with a bioprinter. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0105] Description of the embodiments As shown in the drawings, a bioprinter 10 for producing 3D cellular constructs is disclosed herein. The bioprinter 10 comprises a sample loading system 20 for loading a sample 100 from a sample container 110 into a holding reservoir 120; a selector valve 30 in fluid communication with the holding reservoir 120 for directing the sample 100 into the holding reservoir 120; a droplet dispensing system 25 in fluid communication with the holding reservoir 120; the droplet dispensing system 25 adapted to print sample droplets 101 from the holding reservoir 120 onto a substrate 125; a control system 40 for controlling the operation of the sample loading system 20, the selector valve 30, and the droplet dispensing system 40; a laminar airflow system 50; and a housing 60 that contains the sample loading system 20, the selector valve 30, the droplet dispensing system 40, and the laminar airflow system 50.

[0106] Sample Loading System 1-4, the sample loading system 20 is adapted to access the sample 100 contained in one or more sample containers 110 and is in fluid communication with one or more holding reservoirs 120. The holding reservoirs 120 are configured to hold the sample 100 from the sample containers 110. The sample containers 110 can be standard chromatography vials 111 having caps 112 including rubber septa 113 used in laboratories for sample storage and transport. The vials 111 are typically manufactured from glass, plastic, or any suitable material capable of maintaining a sterile environment within the vial 111. The vials 111 are made in multiple sizes to accommodate various samples 100, typically smaller vials that store about 5 ml and larger vials that store about 10 ml. The sample loading system 20 may include one sample container 110, or multiple sample containers 110, depending on the 3D cell construct to be printed by the bioprinter 10.

[0107] Each retention reservoir 120 is adapted to store a sample 100 received from one of the sample containers 110. Each retention reservoir 120 is fabricated from an elongated tube 122 wound within a reservoir housing 121. The elongated tube 122 may be coiled and contained within the housing 121. The elongated tube 122 is a spool of flexible tubing 122. In certain embodiments, the flexible tube 122 is made from polytetrafluoroethylene (PTFE) tubing or other suitable materials such as fluorinated ethylene propylene (FEP), ethyl trifluoroethylene (ETFE), polyether ether ketone (PEEK), silicone, thermoplastic elastomer (TPE), or stainless steel. In alternative embodiments, each retention reservoir 120 is a container having an inlet, an outlet, and a storage cavity for storing the sample 100. The bioprinter 10 can include one or more retention reservoirs 120 corresponding to one or more individual different samples 100.

[0108] The sample 100 in each sample container 110 may be a cell suspension, water, ethanol, bio-ink, activator, washing solution, flushing fluid, cell culture medium, or drugs dispersed in solution, which are described in detail below. The sample 100 stored in the sample container 110 may or may not be sterile.

[0109] The sample loading system 20 comprises at least one needle 130 insertable into each sample vial 111 and in fluid communication with one or more holding reservoirs 120. In the embodiment shown in the drawings, there is a single needle 130. The needle 130 is 50 mm long, beveled tip, 16 gauge, and manufactured from stainless steel. The needle 130 may be operatively associated with the sample loading system 20 to remove the sample 100 from each vial 111. The needle 130 is movable in the z-direction to insert the needle 130 into the vial 111 from above by a first positioning unit 140. The first positioning unit 140 is a miniature electric linear actuator 140a with a stroke of 60 mm driven by a lead screw 140b coupled to a stepper motor 140c as shown in Figures 3 and 4.

[0110] The bioprinter 10 includes a flow selector valve 30 that directs the samples 100 taken from the sample containers 110 to a holding reservoir 120. This ensures that each sample 100 taken from each vial 111 is held in a separate holding reservoir 120 to separate each sample 100 to maintain sterility.

[0111] The sample loading system 20 and droplet dispensing system 25 of the bioprinter 10 are connected by tubing 150. The tubing 150 is selected from a number of different materials, diameters and lengths based on its desired location and functional requirements. The tubing 150 connecting the sample loading system 20 to each retention reservoir 120 is 2.16 mm inner diameter, 3.175 mm outer diameter PTFE tubing. The elongated tubing 122 within each retention reservoir 120 is 1 / 8" PTFE tubing.

[0112] To prime each holding reservoir 120 with sample 100, the sample 100 moves from a vial 111 and through a selector valve 30 using a pump 160. The pump 160 may be a positive displacement pump, such as a peristaltic pump, a diaphragm pump, or a syringe pump. The pump 160 is connected to a selector valve 30 with an appropriate channel 31 for directing the sample 100 to the appropriate (and isolated) holding reservoir 120. The valve 30 is a low pressure flow-through selector valve 30 manufactured by VICI Valco Instruments Co. The flow selector valve 30 has a number of channels 31, such as 4, 6, 8, 10, 12 or 16 channels. The flow selector valve 30 has a common inlet connected to the pump 160 and the needle 130. Once a channel 31 is selected, the selected channel 31 is fluidly connected to the pump 160 and the needle 130. When a channel 31 is not selected, that channel 31 is fluidly connected to pressurized air from an air pressure regulator 171 in pressure regulator manifold 170. The pressure in each holding reservoir 120 or channel 31 is set independently by a respective regulator 171 in regulator manifold 170. Each regulator 171 in regulator manifold 170 is connected to selector valve 30 using 4 mm nylon tubing and 1 / 8" PTFE tubing. The numbering of the pressure regulators 171 in regulator manifold 170 is equal to the numbering of the holding reservoirs 120 or channels 31. This allows pressure to be set independently for each valve 252 of droplet dispensing system 25, which means that bioprinter 10 can support a wide range of liquid viscosities in each valve 252.

[0113] A pressure regulator 171 in a regulator manifold 170 of the sample loading system 20 independently controls the supply pressure to each channel 31 of the selector valve 30 and to the holding reservoir 120. The pressure regulator manifold 170 is operatively connected to a compressed air supply inlet 180 and a static pressure reservoir (not shown). The bioprinter 10 includes an air filter 190 in the housing 60 for filtering air from the compressed air supply inlet 180. A pump 160 is operatively coupled to the needle 130 for transferring the sample 100 in the sample container 110 to the holding reservoir 120. The pump 160 may be operably reversible for resuspension of the sample 100 in the sample container 110.

[0114] The seal 114 is formed by the rubber septum 113 of each sample container 110 and is punctured using a needle 130 driven by the first positioning unit 140 upon operation of the first positioning unit 140. The first positioning unit 140 is operated by the control system 40 and a robotic linear actuator 140a. The control system 40 positions the first positioning unit 140 by moving a stepper motor 140c on the linear actuator 140a a desired number of steps. The first positioning unit 140 is operatively coupled to the needle 130 to position the needle 130 into and out of puncturing engagement with the sample container 110. The channel 31 is selected on the selector valve 30, the micro solenoid valve 252 is opened, and the pump 160 is turned on to move the sample 100 from the sample vial 111, through the needle 130, the tube 150, the pump 160, the selector valve 30, and into the holding reservoir 120. The pump 160 is then turned off and the micro solenoid valve 252 is closed. Channel 31 is deselected on the flow selector valve 30. The pressure is then set by the respective regulator 171 in the regulator manifold 170 and the micro solenoid valve 252 is repeatedly fired until all the air has left the tubing line 150 and the sample 100 has been expelled from the holding reservoir 120. The above process is repeated to prime each holding reservoir 120 to be used.

[0115] The sample loading system 20 further comprises a second positioning unit 141 operably coupled to the needle 130 and the print head 250 of the droplet dispensing unit 25 for positioning the needle 130 and the print head 250 in a two-dimensional space above the sample container 110 and the substrate 125. The second positioning unit 141 is configured to move the needle 130 and the print head 250 along a track 142. The second positioning unit 141 may be a three-axis motion control stage unit. The second positioning unit 141 is a belt-driven linear actuator 141a with a stroke of 300 mm. The belt 141b is a toothed belt and is driven by a stepper motor 141c.

[0116] To print sample droplets 101, one or more holding reservoirs 120 and micro solenoid valves 252 are primed as described above, and sample droplets 101 are fired from one or more nozzles 253 of a print head 250 of a droplet dispensing system 25 and deposited on a substrate 125 in a predetermined manner controlled by computer control software 40. This droplet dispensing system 25 is described in more detail below.

[0117] To clean the bioprinter 10, the sample loading system 20 can be used to move cleaning agent from the sample vial 111 using the needle 130, through the tubing line 150, the selector valve 30, and into the holding reservoir 120, as described above. The cleaning agent is expelled from the nozzle 253 of the droplet dispensing system 25 into the waste container 205. This process is repeated for other cleaning chemicals, such as 70% ethanol and water. Cleaning of the bioprinter 10 is complete when all the water has been flushed through the lines and only air is ejected from the nozzle 253 of the droplet dispensing system 25, as described in more detail below.

[0118] To resuspend the sample in the vial 111, the needle 130 may be moved toward the respective sample vial 111 using the first and second positioning units 140 and 141 until the needle 130 punctures the sample vial septum 113 and engages the sample 100. The cell-containing sample 100 in the sample vial 111 is moved from the sample vial 111 through the needle 130 and tubing 150 using the peristaltic pump 160. The cell-containing sample 100 is moved in the opposite direction (i.e., toward the sample vial 111) using the peristaltic pump 160 in reverse. This process of moving the cell-containing sample from and toward the vial 111 through the needle 130 and tubing can be repeated as necessary.

[0119] Sample Tray The sample containers 110 may be housed in a removable sample tray 200, which may be sterile. Any suitable number of vials 111 may be housed in the removable sample tray 200, although it is envisioned that up to 14 vials 111 may be housed in the removable sample tray 200. The removable sample tray 200 has one or more sample container housings 201 adapted to store different sizes of sample containers 110, such as vials 111 and a waste container 205. The removable sample tray 200 as shown in FIG. 5 has a lid 202 and a tray 203. The removable sample tray 200 may be manufactured from plastic or other suitable materials. The removable sample tray 200 is loadable into a recess 129 of a print stage 128 of the bioprinter 10 via a hinged door 210, as shown in FIGS. 13 and 16. In an alternative embodiment, the sample containers 110 are loadable into the removable sample tray 200. The removable sample tray 200 provides a substantially sterile environment for storing the vials 111, and each of the vials 111 is sterile.

[0120] It is also contemplated that, instead of a removable sample tray 200, the sample container housing 201 may be integrated into the print stage 128. In this case, each sample container 110 may be loaded into the print stage 128 via a hinged door 210 of the bioprinter 10.

[0121] The removable sample tray 200, which houses the sample containers 110, may further include a waste container 205 for receiving waste when flushing the sample loading system 20. The removable tray 200 may further include a wash container 204 for washing the sample loading system 20, the selector valve 30, and the droplet dispensing system 25.

[0122] Droplet Dispensing System 11-15, droplet dispensing system 25 includes a print head 250 operatively coupled to a plurality of retention reservoirs 120 and adapted to dispense sample droplets 101 from each retention reservoir 120 onto substrate 125. At least one print head 250 may be an array of valves 251. The array of valves 251 may include a plurality of micro solenoid valves 252. The micro solenoid valves 252 may be VHS series solenoid valves manufactured by The Lee Company. Each micro solenoid valve 252 contains a nozzle 253 with an orifice diameter of 0.003", 0.005" or 0.007". Each micro solenoid valve 252 is opened by applying a spike and hold voltage across the solenoid coil. The spike voltage is 24V and the hold voltage is 5V. The duration of the spike voltage is 0.2-0.5ms. When the voltage is switched off the valve 252 returns to the closed position.

[0123] Each nozzle 253 may be a jeweled orifice dispensing nozzle 253 controlled by a microcontroller, i.e., control system 40. The sample 100 is stored in a holding reservoir 120 upstream of the micro solenoid valves 252 and the nozzles 253. The inner diameter of the jeweled orifice nozzles 253 can be from 127 to 254 μm, depending on the fluid viscosity and the desired droplet volume of the sample droplets 101.

[0124] The droplet dispensing system 25 includes a compressed air supply inlet 180 operably coupled to the pressure regulator manifold 170 via an air filter 190. The air moves the sample 100 around the sample loading system 20 and the droplet dispensing system 25 for dispensing through the nozzles 253 of the print head 250. Also, the desired sample droplet 101 volume can be adjusted using the back pressure set by the pressure regulators 171 of the regulator manifold 170, and the opening time of the respective valves 252. Typically, the back pressure is set at a pressure between 1 and 60 psi, the opening time of the valves 251 is 0.3 ms or longer, and the droplet volume is between 1 and 500 nl.

[0125] The droplet dispensing system 25 may further include a third positioning unit 300 operably coupled to the print stage 128, the third positioning unit 300 for positioning the print stage 128 in two-dimensional space. The third positioning unit 300 is configured to move the print stage 128 along a track 301. The track 142 extends perpendicular to the track 301. Referring to FIG. 13, the print stage 128 supports the substrate 125 and has a recess 129 configured to removably receive the sample tray 200. The three-axis motion control stage can accurately position the droplet dispensing system in all three (X, Y and Z) dimensions with a resolution of 10 μm.

[0126] In a sterile environment, each of the activator, bio-ink, and cell-containing bio-ink or cell-ink (i.e., sample 100) is slowly loaded into a suitable holding reservoir 120 using the sample loading system 20, avoiding the generation of small air bubbles.

[0127] The bioprinter is equipped with a power source in the form of a 24V DC power supply (not shown).

[0128] The compressed air supply inlet 180 is supplied from an air compressor (not shown). The air compressor can provide an air pressure of 3 to 10 bar. The compressed air can be supplied from a standard compressed air line, such as is common in laboratories.

[0129] The tubing 150 in the droplet dispensing system 25 is 40 mm 1 / 16" Teflon tubing. This tubing 150 connects the holding reservoir 120 to an array of valves 251 in the print head 250.

[0130] The sample loading system 20 can automatically load the sample 100 into the holding reservoir 120 for printing. This system has several advantages over current state-of-the-art bioprinting systems. First, the bio-ink can be stored in easy-to-handle sample containers 110, such as glass or plastic vials 111. These sample containers 110 are easily sterilized before filling with the bio-ink sample. An end user, such as a biologist, deposits cells in a suitable vial 111 using conventional methods, e.g., a pipette. Depositing the cells inside the bio-ink vial 111 can be done inside a biosafety cabinet to ensure that the sample 100 is not contaminated. After depositing the cells, the vial 111 can be placed in the appropriate location in the bioprinter 10.

[0131] The sample loading system 20 allows for the loading of bioink and cell-containing bioink from individual vials 111 sealed with rubber septa 113. This is achieved using a needle 130 positioned using a z-axis linear actuator 140. The needle 130 is fluidly connected to a positive displacement pump 160. Once the tip of the needle 130 pierces the septum 113 and is positioned inside the vial 111, the pump 160 engages and the fluid sample 100 is pumped out of the vial 111.

[0132] The print head 250 may include multiple electronic pressure regulators 171 that are individually adjustable to print a wide range of viscosities, droplet sizes, etc. based on user input, sample composition, and / or desired cellular composition. The electronic pressure regulators 171 are operably connected to the array of valves 251.

[0133] A bank of pressure regulators 171 (it is envisioned that bioprinter 10 may include ten since there are a maximum of ten holding reservoirs 120) is contained within a pressure regulator manifold 170. The function of manifold 170 is to distribute pressurized air to each regulator 171 from an external air compressor connected to a supply inlet 180. In Figures 11 and 12, only a single regulator 171 and one side of manifold 170 are shown. In Figures 14 and 15, a bank of ten regulators 171 is shown.

[0134] An exemplary embodiment of the sample loading system of the bioprinter 10 includes loading the sample 100 into the holding reservoir 120 as follows: 1. Move the selector valve 30 to the selected channel 31; 2. Open the micro solenoid valve 252; 3. Using the x-axis and y-axis actuators, position the needle 130 above the vial 111; 4. Insert the lower needle 130 into the vial 111 using the z-axis actuator to pierce the vial septum 113; 5. Engage the peristaltic pump 160; 6. Pumping fluid from vial 111 through selector valve 30 and into tube holding reservoir 120; 7. Stopping the pump 160 when the fluid reaches the nozzle of the micro solenoid valve 252; 8. Close the micro solenoid valve 252.

[0135] Another exemplary embodiment of the sample loading system 20 of the bioprinter 10, particularly for cleaning and sterilization, includes the following steps: 1. Place the micro solenoid valve 252 over the waste spittoon or vial; 2. Empty any fluid remaining after the print job into a waste container 205; 3. Move selector valve 30 to the selected channel for cleaning; 4. Using the x-axis and y-axis actuators, position the needle 130 above the vial 111 containing ethanol; 5. Insert the lower needle 130 into the vial 111 using the z-axis actuator to pierce the vial septum; 6. Open the micro solenoid valve 252; 7. Engage the peristaltic pump; 8. Pump ethanol from vial 111 through selector valve 30 and open micro solenoid valve 252; 9. Stop the pump when enough ethanol has passed through the open micro solenoid valve 252; 10. Close the micro solenoid valve 252; 11. Repeat the treatment with detergent; 12. Repeat the treatment with water.

[0136] The bioprinter 10 is adapted to print on many types of substrates 125, such as microwell plates and petri dishes. Referring to FIG. 20, the substrate 125 can be heated to 37° C. to aid in cell growth using a temperature control unit 280. Both temperature control units 280 regulate the temperature inside the bioprinter 10 based on the need for 3D cell-constructed conditions required for optimal growth conditions. The temperature control unit 280 is adjustable from 36° C. to 38° C. to regulate the temperature of the print head 250, the substrate 125 positioned on the print stage 128, and / or the interior of the bioprinter 10.

[0137] The substrate 125, which is placed and supported on the print stage, may be a multi-well plate 126. The substrate 125 may be a biocompatible consumable used to encapsulate and culture the printed cell structures. These substrates may include: · Microtiter plates with different well configurations (6, 12, 24, 48, 96 and 384 wells); · Microtiter plates with coverslip bottoms in different well configurations (6, 12, 24, 48, 96 and 384 wells); · Cover slips and microscope slides; Fluoro dishes of various sizes; and · Chamber slides with different chamber configurations (1, 2, 4, 8 and 16).

[0138] To clean the tubing lines 150, the array of valves 251 and the nozzles 253, a cleaning agent can be transferred from the sample vial 111 to and through the valves 252 and nozzles 253 using the droplet dispensing system 25, as described above. The cleaning agent is squirted from the nozzles 253 into the waste container 205. This process is repeated for other cleaning chemicals, such as 70% ethanol and water. Cleaning of the tubing lines 150 and print head 250 is finished when all the water has been washed through the lines 150, the array of valves 251 and the nozzles 253 and only air is being expelled from the nozzles 253.

[0139] Laminar Flow System 6-10, the bioprinter 10 further includes a laminar flow system 50 as shown in FIGS. 6-10. Sterility and operator safety are primary concerns in 3D bioprinting applications. This is typically achieved by placing the bioprinter in a biosafety cabinet or clean room. Typically, biosafety cabinets and clean rooms are considered valuable and expensive space in tissue culture labs. Thus, a solution is needed to minimize the use of biosafety cabinet and clean room space in 3D bioprinting applications.

[0140] An integrated laminar flow system 50 is integrated into the bioprinter 10 to provide a sterile environment for bioprinting of cells and 3D tissue culture models without the need for biosafety cabinets or clean room facilities. Additionally, the operator is protected using directional airflow to draw air from the external environment through the front access.

[0141] The laminar airflow system 50 includes a chamber or enclosure with a metal frame 500, e.g., stainless steel, with a metal grille at the base to allow the contaminated airflow to be drawn into an electrically powered blower or centrifugal fan 510. The contaminated air is pumped using the fan 510 through a duct inlet 520 and into a positive pressure chamber 535 consisting of two high efficiency particle arresting (HEPA) filters 525 and 530. The HEPA filters 525 and 530 are capable of removing at least 99% of particles from the contaminated airflow. One HEPA filter 525 acts as an exhaust to the outside environment, while the other HEPA filter 530 recirculates the airflow to the sterile chamber 535. Each filter is expected to account for approximately 50% of the airflow. FIG. 21 shows the flow of air through the duct inlet 520 via the blower fan 510, through the HEPA filters 525 and 530, into the bioprinter 10, and then exhausted or recycled. The airflow from the recirculating HEPA filter 525 to the sterile chamber 535 provides a unidirectional downward airflow into the sterile chamber 535 with a typical velocity of 0.45 m / s. This airflow provides a uniform clean airflow over the bioprinted sample droplets 101, significantly reducing the risk of particle contamination within the sample 100.

[0142] During bio-printing operations, the front access hinged door 210 must be closed to reduce the risk of particle contamination. Therefore, the blower fan 510 can be reduced in flow rate by lowering the blower RPM. The reduced airflow in the sterilization chamber 535 reduces the dehydration effects on the print head 250 and the sample 100. Additionally, it reduces the effect of airflow obstructing the sample droplets 101 during flight from the print head 250 to the print substrate 125.

[0143] Control Software Bioprinter 10 is controlled via custom software developed for printing biological assays. Control system 40 comprises control software including a non-transitory computer readable medium having programmed instructions for operating bioprinter 10. The non-transitory computer readable medium is located separately from bioprinter 10 and operably connectable thereto.

[0144] The software includes a graphical user interface (GUI) as shown in Figures 21 and 22. Through the GUI, the end user can select different assay printing routines and modify assay parameters such as droplet spacing and droplet volume. The user can also manually control the spatial position of the droplet dispensing system and create custom patterns of droplets. Further features of the software include routines for washing, priming, and purging the droplet dispensing system.

[0145] Bioprinting requires a 3D model of the object to be printed. In tissue engineering applications, this is typically created using engineering tools such as CAD / CAM software. These tools are expensive and have a high degree of complexity, forcing scientists to spend time and resources learning engineering tools. In 3D tissue culture applications, the complexity of the structures to be printed is lower. There is a need for a simple and intuitive method to create 3D structures for bioprinting in 3D tissue culture applications.

[0146] The software provided with the bioprinter 10 provides a method for designing each layer of the 3D structure to be printed. In one embodiment, a grid is provided for the user to draw the pattern for each layer of the structure. The material to be printed can be defined as a mixture of multiple materials dispensed from different nozzles 253 in the print head 250. For example, a hydrogel can be defined as droplets of bioink mixed with droplets of an activator.

[0147] Typical substrates used in biology laboratories for tissue culture are multi-well plates, such as 6, 12, 24, 48, 96 and 384 well plates. In one embodiment, an interface 670 is provided for printing predefined 3D structures inside each well on a multi-well plate. The user first selects a well or an array of wells and then selects a printing routine to be printed in those wells.

[0148] The custom software provides a user interface for the user to input where within the array they would like to bioprint a layer of the three-dimensional cell construct. A print preview button 671 is provided with the software prior to printing to allow the user to visualize where the cells will be printed and what the structure will be. A feature of the software is that it can control the droplet size of the bioprinter to vary how the cell construct is printed. The intent behind the layering of the cell construct is to mimic how biologists use z-stack layering in a microscope.

[0149] Typically, the bioprinter 10 prints 20-25 layers in building a 3D cellular construct, although the number of layers is controlled using the control system and associated control software.

[0150] A positioning unit 141 coupled to the print head 250 is controlled by computer controlled software to spatially position the valves 251 and nozzles 253 during each ejection of a sample droplet 101 of bio-ink, activator, cells, cell-ink, or combinations thereof. The computer controlled spatial positioning of the solenoid valves and nozzles, and the computer controlled droplet ejection from the valves 251 and nozzles 253 facilitates the generation of 3D tissue constructs.

[0151] The process of generating 3D tissue constructs was repeated at multiple locations on substrate 125 to generate an array of 3D tissue constructs.

[0152] The control system, either by user input or automatic recording, records the identity of each sample 100 in the sample container 110. The intent is to know which sample container 110 contains which sample 100 so that during printing, when the holding reservoir 120 stores the respective sample, the required sample is printed in the desired location.

[0153] Bioprinter Housing The bioprinter 10 comprises a housing 60 that encloses the sample loading system 20, the droplet dispensing system 25, and the laminar airflow system 50. The housing 60 is assembled from multiple panels made from steel, aluminum, and stainless steel and assembled with screw fasteners 600. The housing 60 further includes a hinged door 210 at the front to allow a user to access the sterilization chamber 535 of the bioprinter 10. A removable sample tray 200 can be loaded into the bioprinter 10 through the door 210. The front panel and hinged door 210 can be made from glass or clear plastic. Figures 16 and 17 show the bioprinter assembly 10.

[0154] method In operation, the bioprinter 10 has the following steps to transfer a sample from a sample container to a holding reservoir ready to be utilized by the print head to bioprint a 3D cellular construct: To prime the printer holding reservoir 120 and solenoid valve with fluid, fluid is moved from the sample container to and through the solenoid valve using the sample loading system. The appropriate channel is selected on the flow selector valve. The needle 130 is used to puncture the seal of the sample vial and open the solenoid valve. The peristaltic pump is activated to move the desired amount of fluid from the sample container through the needle, tubing, pump, tubing, flow selector valve and into the holding reservoir. The peristaltic pump is turned off and the solenoid valve is closed. The appropriate channel is deselected on the flow selector valve. The pressure is set by regulator 171 and solenoid valve 252 is repeatedly fired until all air is out of the lines and a droplet(s) of bio-ink, activator, cells, cell-ink, or combinations thereof is expelled from nozzle 253. The above process is repeated for each printer fluid reservoir and solenoid valve used.

[0155] Steps for loading bioink into the holding reservoir: 1. Move the selector valve to the selected channel; 2. Open the solenoid valve; 3. Use the x- and y-axis actuators to position the needle above the vial; 4. Using the z-axis actuator, lower the needle into the vial and pierce the vial septum; 5. Engage the peristaltic pump; 6. Pump fluid from the vial through the selector valve into the tubing holding reservoir; 7. Stop the pump when the fluid reaches the nozzle of the solenoid valve; 8. Close the solenoid valve.

[0156] To print the bio-ink, activator, cells, cell-ink, or combinations thereof from the solenoid valves, the printer fluid reservoirs and solenoid valves are primed as described above, causing droplets of the bio-ink, activator, cells, cell-ink, or combinations to be fired from the nozzles and deposited on the substrate in a predetermined manner controlled by computer controlled software.

[0157] A positioning unit is coupled to the print head 250 and controlled by computer-controlled software to spatially position the solenoid valves and nozzles during each ejection of droplets of bio-ink, activator, cells, cell-ink, or combinations thereof. The computer-controlled spatial positioning of the solenoid valves and nozzles, and the computer-controlled ejection of droplets from the solenoid valves and nozzles, facilitates the generation of 3D tissue constructs.

[0158] To generate an array of 3D tissue constructs, the process of generating the 3D tissue constructs is repeated at multiple locations on the substrate.

[0159] Bioink Bioink is defined herein as an aqueous solution of one or more types of polymers in which cells can be suspended or housed. Upon activation or crosslinking, it produces a hydrogel structure with physical and chemical properties dictated by the chemical and physical composition of the bioink. Polymers are defined as an array of both synthetic and natural polymers, proteins and peptides. The polymers may be in their native state or may be chemically modified with amine or thiol reactive functional groups.

[0160] The synthetic polymer may include: Polysaccharides, for example polymers containing fructose, sucrose or glucose functional groups; · Non-ionic polymers, such as poly(ethylene glycol) (PEG), poly(hydroxyethyl methacrylate) (PHEMA), poly(ε-caprolactone) (PCL), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(NIPAAM) and poly(propylene fumarate) (PPF) and derivatives; · Polyelectrolytes - positively or negatively charged polymers, amphoteric and zwitterionic polymers; Polypeptide - a single chain of many amino acids (minimum two amino acids) held together by amide bonds; and · Nucleobase-Containing Synthetic Polymers Polymers that have nucleobase (adenine, thymine, guanine or cytosine) repeating units.

[0161] Natural polymers may include: Polysaccharides, such as alginate, chitosan, gellan gum, hyaluronic acid, agarose and glycosaminoglycans; Proteins, such as gelatin, fibrin and collagen; DNA and oligonucleotides, such as single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), DNA enzymes and aptamers; and · Basement membrane extract.

[0162] Amine reactive functional groups may include aldehyde, epoxy, N-hydroxysuccinimide (NHS) and 2-vinyl-4,4-dimethylazlactone (VDM).

[0163] Thiol-reactive functional groups may include alkenes, alkynes, azides, halogens, and cyanates.

[0164] The bioink used and found to be suitable was alginate (2 w / v%) dissolved in calcium-free DMEM supplemented with 10 v / v % FCS, L-glutamine and sodium pyruvate.

[0165] The bioink with dispersed SK-N-BE(2) neuroblastoma cells is referred to as cell-laden bioink.

[0166] Activator The activator is an aqueous solution containing either small molecules or polymers that interact with the bioink to form a hydrogel structure. The composition of the activator can be varied to control the physical properties of the resulting hydrogel. The type of activator used is highly dependent on the polymer used as well as the intended crosslinking process.

[0167] The activator may be selected from the following: Inorganic salts, such as calcium carbonate, calcium chloride, sodium chloride, magnesium sulfate; sodium hydroxide and barium chloride; Photoinitiators, such as 2,2-dimethoxy-2-phenylacetophenone (DMPA) and Irgacure; Polyelectrolytes - polymers with an opposite charge to the macromolecules in the bioink. This can be cationic, anionic, amphoteric and zwitterionic; Polypeptide - a single chain of many amino acids (minimum 2 amino acids) joined by amide bonds; DNA linker - a polymer with a nucleotide or DNA sequence that is complementary to that present on the bioink polymer; and Natural or synthetic polymers that contain amine or thiol groups either naturally or through chemical modification.

[0168] The activator used in the alginate bioink was 4 w / v% calcium chloride dissolved in MilliQ water.

[0169] Crosslinking or gelation This is the process in which individual polymer chains are linked together by an activator to form a hydrogel. Crosslinking processes can be classified as either chemical or physical crosslinking. Physical or non-covalent crosslinking may include: Ionic crosslinking - crosslinking by interaction of activators with opposite charges present in the polymer. Activators may include charged oligomers, ionic salts and ionic molecules; Hydrogen bonding - crosslinking by electrostatic attraction of polar molecules. In this case, the polymer and the activator have polar functional groups; Thermal crosslinking - crosslinking by rearrangement of polymer chains in response to a temperature change (heating or cooling); and · Hydrophobic interactions or van der Waals forces.

[0170] Chemical or covalent crosslinking involves a chemical reaction between the polymer and an activator. Types of reactions may include: Photocrosslinking, which accelerates the crosslinking reaction by UV or light irradiation; Michael-type addition reaction between thiols and vinyl-bearing polymers in aqueous media; · Schiff base reaction of amino and aldehyde groups; Diels-Alder reaction; Click chemistry; Aminolysis reactions to activate ester groups; and Enzymatic cross-linking.

[0171] Other examples of bioink and active agent combinations are shown in the table below: [Table 1]

[0172] Cell Ink Cell ink is an aqueous solution of one or more types of molecules or macromolecules in which cells are evenly suspended and remain suspended throughout the 3D bioprinting process. The concentration of the cell ink is optimized to prevent cells from settling, yet still maintain high cell viability.

[0173] The cell ink may be selected from the following: Small molecules such as glycerol; and · Polymers such as Ficoll™, dextran, alginate, gellan gum, methylcellulose and poly(vinylpyrrolidone) (PVP).

[0174] Ficoll™ is a neutral, highly branched, high mass, hydrophilic polysaccharide that is readily soluble in aqueous solutions. Ficoll™ radii range from 2-7 nm and are prepared by reaction of polysaccharides with epichlorohydrin. Ficoll™ is a registered trademark owned by GE Healthcare.

[0175] The cell ink used was Ficoll™ 400 (10 w / v%) dissolved in PBS.

[0176] The cell ink with dispersed SK-N-BE(2) neuroblastoma cells is referred to as cell ink-containing cells.

[0177] Gellan gum is a water-soluble anionic polysaccharide produced by the bacterium Sphingomonas elodea (formerly Pseudomonas elodea).

[0178] cell culture solution Cell culture media are liquids that come into contact with cultured cells and are suitable for a variety of cell-related tasks. The preparation process involves careful analysis of salt and pH balance, incorporation of only biocompatible molecules, and sterilization.

[0179] Some of the cell culture solutions include: · Cell culture media such as Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Icecove's Modified Dulbecco's Medium (IMDM), Medium 199, Ham's F10, Ham's F12, McCoy's 5A and Rothwell Park Memorial Institute (RPMI) medium; · Growth supplements such as fetal calf serum (FCS), epidermal growth factor (EGF), basic fibroblast growth factor (bFBF), fibroblast growth factor (FBF), endothelial growth factor (ECGF), insulin-like growth factor 1 (IGF‐1) and platelet derived growth factor (PDGF); Biological buffers such as PBS, HEPES and CHES; Chelating and stabilizing solutions; and · Sterile MilliQ water.

[0180] Cell culture conditions The cells and 3D tissue culture models can be incubated, cultured and maintained using standard cell culture techniques. The 3D tissue culture models including the cells encapsulated in the hydrogel mold can be incubated under conditions that allow or maintain cell growth or spheroid formation. Cultivation is typically performed at about 37°C for at least 24 hours at a CO2 concentration of 5% for most animal and human cell lines. It will be understood that incubation can be performed under any suitable conditions, temperature and duration that allows the growth, maintenance or spheroid formation of the type of cells in the hydrogel mold.

[0181] Useful Solution Useful solutions are defined as solutions that do not come into contact with the cells, but are used to clean and sterilize all printer surfaces exposed to the cells. These solutions may include: · Ethanol in the correct concentration; · Sterile Milli-Q water; Cell culture media; cleaning agents; and Hydrogen peroxide solution (2% w / v maximum concentration).

[0182] Preparation of bioink First, the bioink is prepared by mixing the appropriate type and amount of polymers in an appropriate cell culture solution. After achieving homogeneity, the blank bioink is sterilized via both UV irradiation and filtration (0.22 μm filter). The bioink is then kept at 4 °C until further use.

[0183] 1. Preparation of Cells Harvest the cells by washing with PBS. Aspirate the PBS. Add trypsin and incubate at 37°C to dissociate the cells from the flask surface. Add tissue culture medium to collect the dissociated cells into a tube. Centrifuge the cells, aspirate the supernatant, and resuspend the pellet in fresh medium. Measure cell count by mixing equal volumes of cell suspension and trypan blue staining solution. Perform calculations to determine cell concentration. The desired number of cells can then be added to bioink, cell ink, or cell culture solution.

[0184] Preparation of activator The appropriate type and amount of molecules were dissolved in an appropriate cell culture solution, and the resulting solution was sterilized by UV irradiation and filtration before use.

[0185] Preparation of cell ink The appropriate type and amount of molecules were dissolved in a suitable cell culture solution. After achieving homogeneity, the resulting solution was sterilized by UV irradiation and filtration before use. The cell ink was then kept at room temperature until further use.

[0186] Cell collection Cultured cells of interest at a specific confluency are harvested according to an already established protocol. To create the cell-containing bioink or cell ink, the harvested cells are resuspended at a precise cell concentration to obtain a concentration of 250 million cells / ml in 200 μl of bioink or cell ink. The resulting cell pellet is then redispersed in a precise volume of bioink or cell ink. The cell-containing bioink or cell ink is then ready for use in the 3D bioprinter.

[0187] Printing the hydrogel mold The hydrogel templates could be printed using a drop-on-drop method, whereby droplets of the bioink and droplets of the activator were deposited on top of each other to generate the hydrogel. This process can be repeated and used to form 3D hydrogel structures by building up layers of hydrogel.

[0188] cell type 3D tissue culture models such as spheroids can be prepared from any suitable cell type including adherent cells such as mammalian liver cells, gastrointestinal cells, pancreatic cells, kidney cells, lung cells, tracheal cells, vascular cells, skeletal muscle cells, cardiac cells, skin cells, smooth muscle cells, connective tissue cells, corneal cells, urogenital cells, breast cells, germ cells, endothelial cells, epithelial cells, fibroblasts, neural cells, Schwann cells, adipocytes, bone cells, bone marrow cells, chondrocytes, pericytes, mesothelial cells, cells derived from endocrine tissues, stromal cells, stem cells, stem cell, progenitor cells, lymphatic cells, blood cells, endodermal derived cells, ectodermal derived cells, mesodermal derived cells, or combinations thereof.

[0189] Additional cell types may include other eukaryotic cells (eg, Chinese hamster ovary), bacteria (eg, Helicobacter pylori), fungi (eg, Penicillium chrysogenum) and yeast (eg, Saccharomyces cerevisiae).

[0190] The cell line SK-N-BE(2) (neuroblastoma cells) has been used in the process of creating 3D tissue culture models under various conditions. It will be understood that other cell lines are expected to function as required in the 3D tissue models created by the developed process. Other cell lines used include DAOY (human medulloblastoma cancer cells), H460 (human non-small lung cancer) and p53R127H (human pancreatic cancer cells). Other cell lines that may be suitable are listed in 088 and 089.

[0191] A 3D bioprinting technique was developed to fabricate high-density 3D tissue culture models encapsulated in hydrogel molds by a drop-on-demand technique. Specifically, 3D printing technology was used to print biocompatible hydrogel molds with bioinks and activators that were built up layer-by-layer to fabricate various 3D structures. During the fabrication of the hydrogel molds, droplets with high cell density can be included in the hydrogel molds.

[0192] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the invention as illustrated in the specific embodiments without departing from the spirit or scope of the invention as broadly described, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0193] References Murphy, S. and Atala, A. (2014). 3D bioprinting of tissues and organs. Nature Biotechnol, 32(8), pp 773-785.

[0194] Horn, T. and Harrysson, O. (2012). Overview of current additive manufacturing technologies and selected applications. Sci. Prog, 95, pp 255-282.

Claims

1. 1. A bioprinter for producing three-dimensional (3D) cellular constructs, the bioprinter comprising: one or more holding reservoirs for holding the fluid sample; a print stage for holding the sample container and supporting the substrate on which the 3D cell construct is printed; a sample loading system in fluid communication with the one or more holding reservoirs, the sample loading system configured to automatically load a sample from a sample container into the one or more holding reservoirs; a pump in fluid communication with the sample loading system configured to draw the sample from a sample container and pump the sample into the one or more holding reservoirs; wherein the sample loading system includes a needle for insertion into a sample vessel, the pump configured to draw fluid through the needle when the needle is inserted into the sample vessel, and further including a first positioning unit coupled to the needle, the first positioning unit configured to insert the needle into the sample vessel and to withdraw the needle from the sample vessel; and a droplet dispensing system in fluid communication with the one or more holding reservoirs, the droplet dispensing system configured to print sample droplets from the one or more holding reservoirs onto a substrate supported by the print stage; wherein said droplet dispensing system is a separate entity from said sample loading system; Including, a bioprinter.

2. The bioprinter of claim 1 , further comprising a housing enclosing the one or more holding reservoirs, the print stage, the sample loading system, the pump, and the droplet dispensing system.

3. The bioprinter of claim 2 , further comprising an airflow system disposed within the housing, the airflow system configured to induce laminar airflow within the housing.

4. The bioprinter of claim 3 , wherein the airflow system comprises a fan for inducing the laminar airflow within the housing.

5. The bioprinter of claim 3 or 4, wherein the air flow system comprises at least one air filter.

6. The bioprinter of any one of claims 1 to 5, further comprising a second positioning unit having a track, the second positioning unit coupled to the needle and the droplet dispensing system and configured to move the needle and the droplet dispensing system along the track of the second positioning unit.

7. 7. The bioprinter of claim 6, further comprising a third positioning unit having a track, the third positioning unit coupled to the print stage and configured to move the print stage along the track of the third positioning unit.

8. The bioprinter of claim 7 , wherein the track of the second positioning unit extends substantially perpendicular to the track of the third positioning unit.

9. A bioprinter according to any one of claims 1 to 8, comprising a plurality of holding reservoirs, and the sample loading system configured to load a sample from the sample container into any one of the plurality of holding reservoirs.

10. 10. The bioprinter of claim 9, wherein the sample container is a tray having a plurality of sample wells, the sample wells configured to receive a sample, and the sample loading system is configured to load a sample from any one of the sample wells into any one of the retention reservoirs.

11. The bioprinter of claim 10 , further comprising a waste container configured to receive waste material from the sample loading system.

12. The bioprinter of claim 11 , wherein the waste container is provided on the tray.

13. A bioprinter according to any preceding claim, wherein the pump is configured to draw the sample from one of the holding reservoirs and pump the sample out of the sample loading system.

14. A bioprinter according to any preceding claim, further comprising a pressure regulator coupled in fluid communication to each retention reservoir for regulating the pressure within each retention reservoir.

15. 15. The bioprinter of claim 14, further comprising a selector valve in fluid communication with the pump, the sample loading system, each holding reservoir, and the pressure regulator, the selector valve configured to selectively connect the pump in fluid communication with the sample loading system and each holding reservoir.

16. 16. The bioprinter of claim 14 or 15, wherein the pressure regulator is removably coupled in fluid communication with a source of compressed air.

17. A method of producing a three-dimensional cell construct, comprising depositing droplets of one or more samples using a bioprinter according to any one of claims 1 to 16.

18. 1. A method for producing a three-dimensional cell construct, the method comprising: Providing a bioprinter according to any one of claims 1 to 16; providing a substrate to the print stage; providing a sample container to a print stage, the sample container containing a sample; loading a sample into one of the holding reservoirs with the sample loading system; and printing the sample from the holding reservoir onto the substrate using the droplet dispensing system to form the three-dimensional cell construct. A method comprising:

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