阅读说明：本技术 用于制造3d细胞结构的生物打印机 (Bio-printer for manufacturing 3D cell structures ) 是由 艾丹·帕特里克·奥马霍尼 朱里奥·塞萨尔·卡德拉·里贝罗 塞缪尔·詹姆斯·迈尔斯 基兰·约瑟夫 于 2018-12-07 设计创作，主要内容包括：一种用于制造三维(3D)细胞结构的生物打印机,该生物打印机包括一个或多个用于容纳流体样品的储存容器；用于容纳样品容器并支撑将在其上打印3D细胞结构的基板的打印台；与一个或多个储存容器流体连通的样品加载系统,所述的样品加载系统配置成将样品从样品容器加载到一个或多个储存容器中；与样品加载系统流体连通的泵,所述的泵配置成将样品从样品容器中抽出并将样品泵入一个或多个储存容器中；以及与一个或多个储存容器流体连通的液滴分配系统,所述的液滴分配系统配置成将来自一个或多个储存容器的样品液滴打印到由打印台支撑的基板上。(A bioprinter for fabricating a three-dimensional (3D) cellular structure, the bioprinter comprising one or more storage containers for holding a fluid sample; a printing station for receiving the sample container and supporting a substrate on which the 3D cell structure is to be printed; a sample loading system in fluid communication with the one or more storage containers, the sample loading system configured to load a sample from the sample container into the one or more storage containers; a pump in fluid communication with the sample loading system, the pump configured to draw sample from the sample container and pump the sample into the one or more storage containers; and a droplet dispensing system in fluid communication with the one or more storage containers, the droplet dispensing system configured to print sample droplets from the one or more storage containers onto a substrate supported by the print station.)

1. A bioprinter for fabricating a three-dimensional (3D) cellular structure, the bioprinter comprising:

one or more storage containers for storing a fluid sample;

a printing station for placing a sample container and supporting a substrate on which the 3D cell structures are to be printed;

a sample loading system in fluid communication with the one or more storage containers, the sample loading system configured to load a sample from the sample container into the one or more storage containers;

a pump in fluid communication with the sample loading system, the pump configured to draw sample from the sample container and pump the sample into the one or more storage containers; and

a droplet dispensing system in fluid communication with the one or more storage containers, the droplet dispensing system configured to print sample droplets from the one or more storage containers onto a substrate supported by the print station.

2. The bioprinter of claim 1, further comprising a housing enclosing the one or more storage containers, the print station, the sample container, the sample loading system, the pump, and the droplet dispensing system.

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

4. The bioprinter of claim 3, wherein the air flow system comprises a fan to induce the laminar air flow 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, wherein the sample loading system comprises a needle for insertion into a sample container, the pump being configured to draw fluid through the needle when the needle is inserted into a sample container.

7. The bioprinter of claim 6, further comprising a first positioning unit connected to the needle, the first positioning unit configured to insert and withdraw the needle into and from a sample container.

8. The bioprinter of claim 6 or 7, further comprising a second positioning unit having a track, the second positioning unit connected 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.

9. The bioprinter of claim 8, further comprising a third positioning unit having a track, the third positioning unit coupled to the print station and configured to move the print station along the track of the third positioning unit.

10. The bioprinter of claim 9, wherein the track of the second positioning unit extends substantially perpendicular to the track of the third positioning unit.

11. The bioprinter of any one of claims 1 to 10, comprising a plurality of storage containers, and the sample loading system is configured to load a sample from the sample container into any one of the plurality of storage containers.

12. The bioprinter of claim 11, wherein the sample container is a tray having a plurality of sample wells configured to hold a sample, and the sample loading system is configured to load a sample from any of the sample wells into any of the storage containers.

13. The bioprinter of claim 12, further comprising a waste container configured to receive waste from the sample loading system.

14. The bioprinter of claim 13, wherein the waste container is disposed on the tray.

15. The bioprinter of any one of claims 1 to 14, wherein the pump is configured to draw the sample from one of the storage containers and pump the sample out of the sample loading system.

16. The bioprinter of any one of claims 1 to 15, further comprising a pressure regulator fluidly connected to each storage container to regulate pressure in each storage container.

17. The bioprinter of claim 16, further comprising a selector valve in fluid communication with the pump, the sample loading system, each storage container, and the pressure regulator, the selector valve configured to selectively connect the pump in fluid communication with the sample loading system and each storage container.

18. The bioprinter of claim 16 or 17, wherein the pressure regulator is removably connected in fluid communication to a compressed air supply.

19. A method of making a three-dimensional cellular structure comprising depositing one or more droplets of a sample using the bioprinter of any one of claims 1 to 18.

20. A method of making a three-dimensional cellular structure, the method comprising:

providing the bioprinter of any one of claims 1 to 18;

providing a substrate for a print station;

providing a sample container to a print station, the sample container comprising a sample;

loading the sample into one of the storage containers by the sample loading system;

the sample is printed from the storage container onto a substrate using a droplet dispensing system to form a three-dimensional cellular structure.

Technical Field

The present technology relates to bioprinters for fabricating three-dimensional (3D) cell structures, methods of preparing bioprinted 3D cell structures, and bioprinted 3D cell structures.

RELATED APPLICATIONS

This application is based on and claims priority from australian provisional patent application No.2017904946 filed 2017, 12, 8, the contents of which are incorporated herein by reference in their entirety.

Background

The main force of in vitro cell biology is cell culture, where primary or immortalized cells are simply plated on plastic or glass surfaces. Many cellular properties (e.g., cell proliferation, differentiation and response to external stimuli) are fundamentally different for cells found in two-dimensional (2D) and 3D environments in vivo. Especially for drug development and sophisticated medical procedures, cell culture conditions that better reflect the 3D animal environment and thus limit the number of failed animal experiments would be of great advantage.

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

For this reason, 3D multicellular models are generally considered superior models of in vivo systems compared to the more popular 2D cell cultures.

Furthermore, most cellular structures in multicellular biology are organized in three dimensions. A number of studies report the use of 3D bioprinting techniques to print cells (reviewed in (Murphy and Atala, 2014)).

There are many commercially available 3D bioprinters, for example: of EnvisionTEC BioScaffolder by GeSiM; bio X of Cellink; of RegenHUBioBot 2 from BioBot. Commercially available 3D bioprinters are most often based on micro-extrusion, thermal inkjet or piezo inkjet technologies. Commercially available 3D bioprinters typically use ink cartridges (e.g., ink cartridges)

Syringe barrel) for loading the substance into the printer. Each cartridge is typically associated with a printhead. Maintaining sterility during cartridge filling, handling, installation and removal is a challenge.

The design of organ or tissue architecture 3D models for 3D bioprinting applications is based mainly on:

1) non-invasive medical imaging techniques for data collection, such as Computed Tomography (CT) and Magnetic Resonance Imaging (MRI); and

2) computer aided design and computer aided manufacturing (CAD-CAM) tools and mathematical modeling for information digitization, generation of 3D rendering models and generation of 2D cross-sectional images (Murphy and Atala, 2014; horn and Harrysson, 2012).

Tools and techniques are needed to facilitate scalable, repeatable and cost-effective application of 3D cell culture models in drug discovery, personalized medicine and general cell biology.

The present inventors have developed devices, systems, and methods for making in vitro 3D cell culture assays and arrays thereof.

SUMMARY

In a first aspect, the present invention provides a bioprinter for fabricating a three-dimensional (3D) cellular structure, the bioprinter comprising:

one or more storage containers storing a fluid sample;

a print station (print stage) that houses the sample container and supports a substrate on which the 3D cell structures are to be printed;

a sample loading system in fluid communication with the one or more storage containers, the sample loading system configured to load a sample from the sample container into the one or more storage containers;

a pump in fluid communication with the sample loading system, the pump configured to draw sample from the sample container and pump the sample into the one or more storage containers; and

a droplet dispensing system in fluid communication with the one or more storage containers, the droplet dispensing system configured to print sample droplets from the one or more storage containers onto a substrate supported by the print station.

In one embodiment, the bioprinter further comprises a housing enclosing one or more of a storage container, a print station, a sample holder, a sample loading system, a pump, and a droplet dispensing system.

In one embodiment, the bioprinter further comprises an air flow system disposed in the housing, the air flow system configured to induce a laminar air flow within the housing.

In one embodiment, the air flow system includes a fan to induce laminar air flow in the housing.

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

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

In one embodiment, the bioprinter further comprises a first positioning unit connected 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.

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

In one embodiment, the bioprinter further comprises a third positioning unit having a track, the third positioning unit being connected to the print table and configured to move the print table along the track of the third positioning unit.

In one embodiment, the track of the second positioning unit extends substantially perpendicular to the track of the third positioning unit.

In one embodiment, the bioprinter further comprises a plurality of storage containers, and the sample loading system is configured to load the sample from the sample container into any one of the plurality of storage containers.

In one embodiment, the sample container is a tray having a plurality of sample wells, the sample wells are configured to contain a sample, and the sample loading system is configured to load the sample from any one of the sample wells into any one of the storage containers. In one embodiment, the bioprinter further comprises a waste container configured to receive waste from the sample loading system.

In one embodiment, the waste container is disposed on the tray.

In one embodiment, the pump is configured to draw the sample from one of the storage containers and pump the sample out of the sample loading system.

In one embodiment, the bioprinter further comprises a pressure regulator fluidly connected to each of the storage containers to regulate the pressure in each of the storage containers.

In one embodiment, the bioprinter further comprises a selector valve in fluid communication with the pump, the sample loading system, each storage container, and the pressure regulator, the selector valve configured to selectively connect the pump in fluid communication with the sample loading system and each storage container.

In one embodiment, the pressure regulator is removably in fluid communication with a compressed air supply source.

In a second aspect, the invention provides a method of manufacturing a three-dimensional cellular structure comprising depositing one or more droplets of a sample using the bioprinter of the first aspect.

In a third aspect, the present invention provides a method of making a three-dimensional cellular structure, the method comprising:

providing the bioprinter of the first aspect;

providing a substrate to a print station;

providing a sample container to a print station, said sample container comprising a sample;

loading the sample into a storage container by the sample loading system; and

the sample is printed from the storage container onto a substrate using a droplet dispensing system to form a three-dimensional cellular structure.

The invention also discloses a bioprinter for manufacturing a 3D cell structure, the bioprinter comprising:

a sample loading system for loading a sample from a sample container into a storage container;

a selector valve in fluid communication with the reservoir for directing the sample into the reservoir;

a droplet dispensing system in fluid communication with the reservoir for printing droplets of the sample from the reservoir onto the substrate;

A control system to control operation of the sample loading system, the selection valve, and the droplet dispensing system;

laminar air flow systems (laminar air low systems); and

a housing enclosing the sample loading system, the selection valve, the droplet dispensing system, and the laminar air flow system.

In one embodiment, the sample loading system includes a plurality of sample containers and a plurality of storage containers for holding samples from each sample container.

In one embodiment, the plurality of sample containers are housed in a movable sample tray.

In one embodiment, the movable sample tray includes 10 positions for holding sample containers in an array.

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

In one embodiment, the removable sample tray further comprises a waste container for receiving waste generated by flushing the sample loading system.

In one embodiment, the movable sample tray further comprises a cleaning receptacle for cleaning the sample loading system, the selection valve and the droplet dispensing system.

In one embodiment, the sample loading system includes a needle for insertion into the sample container, a pump operatively connected to the needle for transferring the sample in the sample container to the storage container.

In one embodiment, the pump is a positive displacement pump.

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

In one embodiment, the pump is reversibly operable for resuspending the sample in the sample container.

In one embodiment, the sample loading system further comprises a first positioning unit operatively connected to the needle for positioning the needle to pierce or not pierce the sample container.

In one embodiment, the sample in the sample container may be a cell suspension, water, ethanol, bio-ink, an activator, a cleaning solution, a rinse, a cell culture medium, or a drug dispersed in a solution.

In one embodiment, the sample in the sample container is sterile.

In one embodiment, the sample loading system further comprises a second positioning unit operatively connected to the needle for positioning the needle in two dimensions.

In one embodiment, the storage container is an elongated tube.

In one embodiment, the elongate tube is coiled and enclosed within the chamber.

In one embodiment, the storage container is formed by a spool of flexible tubing.

In one embodiment, the flexible tube is made of Polytetrafluoroethylene (PTFE) tubing.

In one embodiment, the droplet dispensing system includes at least one printhead operably connected to a plurality of storage containers and adapted to dispense droplets of sample from each storage container onto the substrate.

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

In one embodiment, each valve is a micro-solenoid valve.

In one embodiment, the sample is stored in a storage container upstream of the micro-solenoid valve.

In one embodiment, each storage container has a corresponding print head.

In one embodiment, each storage vessel is connected to a pressure regulator.

In one embodiment, a compressed air supply is connected to the regulator manifold.

In one embodiment, each micro-solenoid valve is connected to each pressure regulator.

In one embodiment, the droplet dispensing system includes a plurality of pressure regulators in a regulator manifold, at least one check valve, wherein a compressed air supply source is operably connected to each pressure regulator in the regulator manifold.

In one embodiment, a pressure regulator is connected to the selector valve.

In one embodiment, a sample loading system is used to transfer a sample from a sample container to a storage container and a droplet dispensing system is operated to transfer a sample from a storage container to a printhead, with a pressure regulator of the droplet dispensing system and a selector valve of the sample loading system being operatively operated to transfer a sample from a storage container to a printhead.

In one embodiment, the droplet dispensing system further comprises a print station for supporting the substrate.

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

In one embodiment, the droplet dispensing system further comprises a third positioning unit operatively connected to the print table for positioning the print table in two-dimensional space.

In one embodiment, the control system records the identity of the sample in the sample container based on user input.

In one embodiment, the control system includes a non-transitory computer readable medium including programming instructions for operating the bioprinter.

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

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

In one embodiment, the fan is a centrifugal fan.

In one embodiment, a fan draws air into the front of the housing from under the print station, around the sample loading system, and through one or more filters, and out of the housing.

In one embodiment, the fan draws air through a front door of the bioprinter housing.

In one embodiment, the laminar flow system includes two filters, one for exhausting air and one for receiving air toward the print station.

In one embodiment, each filter is a High Efficiency Particulate Air (HEPA) filter.

In one embodiment, each filter receives about 50% of the airflow.

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

In one embodiment, the removable sample tray may be loaded into the bioprinter through a door.

In one embodiment, the movable sample tray has a lid.

In one embodiment, the housing has a recess for receiving the sample tray cover and a cover for the multi-well plate.

In one embodiment, the sample container may be loaded into a removable sample tray inside the bioprinter.

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

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

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. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this specification.

In order that the invention may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.

Brief description of the drawings

Fig. 1 is a rear perspective view of a bioprinter for fabricating 3D cell structures, showing a sample loading system.

FIG. 2 is a front perspective view of the sample loading system of FIG. 1;

FIG. 3 is a front perspective view of the sample loading system showing a plurality of sample containers, each containing a sample;

FIG. 4 is a rear perspective view of the sample loading system of FIG. 3;

FIG. 5 is a perspective view of a movable sample tray used in the sample loading system;

FIG. 6 is a front perspective view of a bioprinter equipped with a laminar flow system;

FIG. 7 is a rear perspective view of the bioprinter, showing only the laminar air flow system;

FIG. 8 is a rear perspective view of the bioprinter, showing only the laminar air flow system of FIG. 7;

FIGS. 9 and 10 illustrate a bioprinter having a transparent panel showing the air flow path of a laminar air flow system;

FIG. 11 is a top perspective view of the bioprinter showing a positioning unit for the droplet dispensing system and the sample loading system;

FIG. 12 is a side perspective view of a bioprinter having a plurality of storage containers and a compressed air supply system;

FIG. 13 is a front perspective view of the bioprinter of FIG. 11 with a layered air flow system installed;

FIG. 14 is a rear perspective view of the bioprinter of FIG. 13;

FIG. 15 is a rear perspective view of a bioprinter having a single storage container;

FIG. 16 is a front perspective view of the assembled bioprinter;

fig. 17 is a rear perspective view of the bioprinter of fig. 16;

FIG. 18 is a schematic flow diagram of a bioprinter assembly;

FIG. 19 is a side view of each component of the bioprinter, showing the air flow paths between the components of the laminar air flow system;

FIG. 20 is a flow chart of a bioprinter operatively associated with a control system computer;

FIG. 21 is an exemplary graphical user interface of control system software implemented on a computer, illustrating a 3D cell structure design;

FIG. 22 is an exemplary graphical user interface of control system software implemented on a computer showing printing of 3D cell structures on a multi-well plate; and

fig. 23 is a schematic illustration of a flow chart for designing a complete 3D cell structure with a bioprinter.

Detailed Description

As shown in the figure, the present invention discloses a bioprinter 10 for manufacturing 3D cell structures. The bioprinter 10 includes a sample loading system 20 for loading a sample 100 from a sample container 110 into a storage container 120; and a selector valve 30 in fluid communication with the reservoir 120 for directing the sample 100 into the reservoir 120; a droplet dispensing system 25 in fluid communication with the reservoir 120, the droplet dispensing system 25 adapted to print a droplet of sample 101 from the reservoir 120 onto a substrate 125; a control system 40 to control the operation of the sample loading system 20, the selection valve 30 and the droplet dispensing system 40; a laminar airflow system 50; a housing 60 for housing the sample loading system 20, the selector valve 30, the droplet dispensing system 40 and the laminar air flow system 50.

Sample loading system

Referring to fig. 1-4, the sample loading system 20 is adapted to acquire a sample 100 contained in one or more sample containers 110 and is in fluid communication with one or more storage containers 120. The storage container 120 is configured to store the sample 100 from the sample container 110. The sample container 110 may be a standard chromatography vial 111 having a lid 112, the lid 112 comprising a rubber septum 113 for storing and transporting samples in a laboratory. The vial 111 is typically made of glass, plastic, or any suitable material that enables the sterile environment to be maintained in the vial 111. Vials 111 may be made in a variety of sizes to hold various samples 100, typically with smaller vials storing about 5ml and larger vials storing about 10 ml. Depending on the 3D cell configuration to be printed by the bioprinter 10, the sample loading system 20 may include one sample container 110 or a plurality of sample containers 110.

Each storage container 120 is adapted to store a sample 100 received from one of the sample containers 110. Each storage container 120 is made of an elongated tube 122 wrapped inside a housing 121 of the storage container. Elongate tube 122 may be coiled and enclosed within housing 121. The elongate tube 122 is a spool of flexible tubing 122. In certain embodiments, the flexible tubing 122 is made of Polytetrafluoroethylene (PTFE) tubing or other suitable material, such as Fluorinated Ethylene Propylene (FEP), ethyltrifluoroethylene (ETFE), Polyetheretherketone (PEEK), silicone, thermoplastic elastomer (TPE), or stainless steel. In an alternative embodiment, each storage container 120 has an inlet, an outlet, and a storage chamber for storing the sample 100. The bioprinter 10 may include one or more storage containers 120 corresponding to one or more individual and distinct samples 100.

The sample 100 in each sample container 110 may be a cell suspension, water, ethanol, bio-ink, an activator, a cleaning solution, a washing solution, a cell culture medium, or a drug dispersed in a solution, which will be described in detail below. The sample 100 stored in the sample container 110 may or may not be sterile.

The sample loading system 20 includes at least one needle 130, the needle 130 being insertable into each sample vial 111 and in fluid communication with one or more storage containers 120. In the embodiment shown in the drawings, there is a single needle 130. Needle 130 is 50 mm long, beveled tip, gauge 16, made of stainless steel. A 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 the first positioning unit 140. The first positioning unit 140 is a miniature electric linear actuator 140a with a stroke of 60mm driven by a lead screw 140b connected to a stepper motor 140c, as shown in fig. 3 and 4.

The bioprinter 10 includes a flow selector valve 30, which flow selector valve 30 directs a sample 100 taken from a sample container 110 into a storage container 120. This allows each sample 100 taken from each vial 111 to be held in a separate storage container 120 to isolate each sample 100 to maintain sterile conditions.

The sample loading system 20 and the droplet dispensing system 25 of the bioprinter 10 are connected by a conduit 150. The conduit 150 is selected from a variety 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 storage container 120 is a PTFE tube with an inner diameter of 2.16mm and an outer diameter of 3.175 mm. The elongated tube 122 in each storage container 120 is an 1/8 "PTFE tube.

To fill each storage container 120 with sample 100, the sample 100 is moved from the vial 111 to the selector valve 30 and through the selector valve 30 using the pump 160. The pump 160 may be a volumetric pump, such as a peristaltic pump, a diaphragm pump, or a syringe pump. The pump 160 is connected to a selector valve 30, which selector valve 30 comprises a suitable channel 31 for directing the sample 100 into a suitable (and isolated) holding vessel 120. Valve 30 is a low pressure flow-through selector valve 30 manufactured by VICI Valeo Instruments Co inc. The flow selector valve 30 includes a plurality 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. When 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 with compressed air from the air pressure regulator 171 in the pressure regulator manifold 170. The pressure in each storage vessel 120 or channel 31 is independently set by a corresponding regulator 171 in the regulator manifold 170. Each regulator 171 in regulator manifold 170 is connected to selector valve 30 using 4mm nylon tubing and 1/8 "PTFE tubing. The number of pressure regulators 171 in the regulator manifold 170 is equal to the number of storage vessels 120 or channels 31. This allows for independent setting of pressure for each valve 252 of the drop dispensing system 25, which means that the bioprinter 10 can support a variety of different fluid viscosities in each valve 252.

A pressure regulator 171 in the regulator manifold 170 of the sample loading system 20 independently controls the pressure in each channel 31 and the storage vessel 120 fed to the selector valve 30. 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 within the housing 60 for filtering air from the compressed air supply inlet 180. A pump 160 is operatively connected to the needle 130 to transfer the sample 100 in the sample container 110 to the storage container 120. The pump 160 may be reversibly operated to resuspend the sample 100 in the sample container 110.

The seal 114 is formed by the rubber septum 113 of each sample container 110 and is pierced with the 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 the robotic linear actuator 140 a. The control system 40 positions the first positioning unit 140 by moving the stepper motor 140c on the linear actuator 140a desired number of steps. The first positioning unit 140 is operatively connected to the needle 130 to position the needle 130 to pierce the sample container 110 or not to pierce 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 bottle 111 to the storage container 120 through the needle 130, the tubing 150, the pump 160, the selector valve 30. The pump 160 is then turned off and the micro-solenoid valve 252 is closed. The channel 31 is deselected on the flow selector valve 30. Pressure is then set by the corresponding regulator 171 of the regulator manifold 170 and the micro solenoid valves 252 are repeatedly triggered until all air is exhausted from the lines 150 and the sample 100 is ejected from the storage container 120. The above process is repeated to fill each storage container 120 to be used.

The sample loading system 20 further comprises a second positioning unit 141, the second positioning unit 141 being operatively connected to the needle 130 and the print head 250 of the droplet dispensing unit 25, the second positioning unit 141 being adapted to position the needle 130 and the print head 250 in a two-dimensional space above the sample container 110 and the substrate 125 needle. The second positioning unit 141 is configured to move the needle 130 and the print head 250 along the rail 142. The second positioning unit 141 may be a three-axis motion console 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 stepping motor 141 c.

To print the sample droplets 101, one or more storage containers 120 and micro-solenoid valves 252 are prepared (printed) as described above, and the sample droplets 101 are ejected from one or more nozzles 253 of the printhead 250 of the droplet dispensing system 25 to be deposited on the substrate 125 in a predetermined manner controlled by the computer control software 40. The droplet dispensing system 25 is described in more detail below.

To clean the bioprinter 10, using the sample loading system 20 as described above, the cleaning solvent in the sample container 111 can be removed using the needle 130 and passed through the conduit 150, the selector valve 30, and the storage container 120. The cleaning agent is ejected from the nozzle 253 of the droplet dispensing system 25 into the waste container 205. This process is repeated for other cleaning chemicals (e.g., 70% ethanol and water). Cleaning of the bioprinter 10 is complete when all of the water has been flushed through the line and only air is ejected from the nozzles 253 of the drop dispensing system 25, as will be described in detail below.

To resuspend the sample in the sample vial 111, the needle 130 may be moved towards the respective sample vial 111 using the first positioning unit 140 and the second positioning unit 141 until the needle 130 pierces the septum 113 of the sample vial and contacts the sample 100. The cell-containing sample 100 in the sample vial 111 is removed from the sample vial 111 using a peristaltic pump 160 through the needle 130 and the tubing 150. The peristaltic pump 160 is used in reverse to move the cell-containing sample 100 in the opposite direction (i.e., toward the sample vial 111). This process of removing the cell-containing sample from the sample bottle 111 and moving it to the sample bottle 111 through the needle 130 and the catheter may be repeated as necessary.

Sample plate

The sample containers 110 may be contained in a removable sample tray 200, and the removable sample tray 200 may be sterile. It is contemplated that up to 14 vials 111 may be contained in the movable sample tray 200, but any suitable number of vials 111 may be contained in the movable 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 waste containers 205. The removable sample tray 200 as shown in fig. 5 has a lid 202 and a tray 203. The movable sample tray 200 may be made of plastic or other suitable material. As shown in fig. 13 and 16, the movable sample tray 200 may be loaded into the recess 129 of the print station 128 of the bioprinter 10 through a hinged door 210. In an alternative embodiment, the sample containers 110 may be loaded into the movable sample tray 200. The removable sample tray 200 provides a sterile environment for storing the vials 111 and each vial 111 is sterile.

It is also contemplated that instead of a movable sample tray 200, the sample container housing 201 may be integral with the print station 128. In this case, each sample container 110 would be loadable into the print station 128 through the hinged door 210 of the bioprinter 10.

The removable sample tray 200 containing the sample containers 110 may further include a waste container 205 for receiving waste when the sample loading system 20 is flushed. The removable tray 200 may also include a cleaning receptacle 204 for cleaning the sample loading system 20, the selector valve 30, and the droplet dispensing system 25.

Droplet dispensing system

Referring to fig. 11-15, droplet-dispensing system 25 includes a printhead 250, the printhead 250 being operatively connected to a plurality of reservoirs 120 and adapted to dispense droplets of sample 101 from each reservoir 120 onto substrate 125. At least one printhead 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 valve 252 may be a VHS series solenoid valve manufactured by The Lee Company. Each micro-solenoid valve 252 includes a nozzle 253 having an orifice diameter of 0.003 ", 0.005", or 0.007 ". Each micro solenoid valve 252 is opened by applying a spike and holding a voltage across the solenoid coil. The peak voltage was 24V and the holding voltage was 5V. The duration of the spike voltage is between 0.2 and 0.5 ms. When the voltage is cut off, the micro solenoid valve 252 returns to the closed position.

Each nozzle 253 may be a jewel orifice dispensing nozzle 253 controlled by a microcontroller, i.e., the control system 40. The sample 100 is stored in the reservoir 120 upstream of the micro-solenoid valve 252 and the nozzle 253. The inner diameter of the jewel orifice nozzle 253 can be between 127 and 254 μm depending on the fluid viscosity and the desired drop volume of the sample drop 101.

The droplet dispensing system 25 includes a compressed air supply inlet 180, the compressed air supply inlet 180 being operatively connected to the pressure regulator manifold 170 through an air filter 190. The air moves the sample 100 within the sample loading system 20 and the droplet dispensing system 25 for dispensing through the nozzles 253 of the printhead 250. The desired sample droplet 101 volume can also be adjusted using the back pressure set by the pressure regulator 171 of the regulator manifold 170 and the opening time of the respective valve 252. Typically, the back pressure is set to a pressure between 1 and 60psi, the valve 251 is opened for 0.3ms or more, and the drop volume is 1 to 500 nl.

The droplet dispensing system 25 may further comprise a third positioning unit 300 operatively connected to the print station 128, the third positioning unit 300 for positioning the print station 128 in two dimensions. The third positioning unit 300 is configured to move the print table 128 along the rail 301. The rail 142 extends perpendicular to the rail 301. Referring to fig. 13, print station 128 supports substrate 125 and has a recess 129 configured to removably receive sample tray 200. The 3-axis motion control stage is capable of accurately positioning the droplet dispensing system in all three (X, Y and Z) dimensions with a resolution of 10 μm.

Each activator, bio-ink, and cell-containing bio-ink or cell-containing ink (i.e., sample 100) is slowly loaded into a suitable storage container 120 using the sample loading system 20 in a sterile environment to avoid the generation of small amounts of air bubbles.

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

The compressed air supply inlet 180 is supplied from an air compressor (not shown). The air compressor may provide an air pressure of 3 to 10 bar. The compressed air may be supplied from a common compressed air line commonly found in research laboratories.

The tubing 150 within the droplet dispensing system 25 is a 40mm 1/16 "Teflon (Teflon) tubing. The conduit 150 connects the reservoir 120 to the array of valves 251 of the printhead 250.

The sample loading system 20 may automatically load the sample 100 into the storage container 120 for printing. This system has several advantages over prior art bioprinting systems. First, the bio-ink may be stored in a sample container 110 that is easy to handle, such as a glass or plastic bottle 111. These sample containers 110 are easily sterilized prior to filling with the bio-ink sample. The end user, e.g., a biologist, uses a pipette or like conventional method to deposit its cells in the appropriate vial 111. The storage of cells in the bio-ink bottle 111 may be performed in a bio-safety cabinet to ensure that the sample 100 is not contaminated. After the cells are deposited, the vial 111 may be placed in position within the bioprinter 10.

Sample loading system 20 allows for the loading of bio-ink and bio-ink containing cells from a single vial 111 sealed with a rubber septum 113. This is accomplished by using a needle 130 positioned by a z-axis linear actuator 140. The needle 130 is fluidly connected to a volumetric pump 160. When the tip of the needle 130 pierces the septum 113 and is located inside the vial 111, the pump 160 is activated and the fluid sample 100 is withdrawn from the vial 111.

The printhead 250 may include a plurality of electronic pressure regulators 171 that may be individually adjusted to print a wide range of viscosities, droplet sizes, etc. based on user input, sample structure, and/or desired cell structure. The electronic pressure regulator 171 is operatively connected to an array of valves 251.

The pressure regulator manifold 170 houses a pressure regulator set 171 (it is contemplated that the bioprinter 10 may include 10 as there are up to 10 storage vessels 120). The function of the manifold 170 is to distribute compressed air from an external air compressor connected to the supply inlet 180 to each of the regulators 171. In fig. 11 and 12, only one side of a single regulator 171 and manifold 170 is shown. In fig. 14 and 15, a set of ten adjusters 171 is shown.

An exemplary embodiment of a sample loading system for bioprinter 10 includes the following steps for loading a sample 100 into a storage container 120:

1. Moving the selector valve 30 to the selected channel 31;

2. opening the micro solenoid valve 252;

3. positioning the needle 130 over the sample vial 111 using the x-axis and y-axis actuators;

4. inserting the needle 130 down into the vial 111 and piercing the septum 113 of the vial using the Z-axis actuator;

5. starting the peristaltic pump 160;

6. pumping fluid from vial 111 through selector valve 30 into storage container 120;

7. when the fluid reaches the nozzle of the micro solenoid valve 252, the pump 160 is stopped; and

8. the micro solenoid valve 252 is closed.

Another exemplary embodiment of the sample loading system 20 of the bioprinter 10, particularly for cleaning and sterilizing, includes the steps of:

1. placing the micro solenoid valve 252 on the waste spittoon or bottle;

2. pouring all the liquid remaining after the print job into the waste liquid container 205;

3. moving the selector valve 30 to the selected channel for cleaning;

4. the needle 130 was placed over the ethanol-filled sample vial 111 using x-axis and y-axis drives;

5. using a z-axis actuator to insert the needle 130 down into the vial 111 and pierce the vial septum;

6. opening the micro solenoid valve 252;

7. starting a peristaltic pump;

8. pumping ethanol from vial 111 through selection valve 30 and then opening micro solenoid valve 252;

9. the pump is stopped when sufficient ethanol passes through the micro-solenoid valve 252;

10. Closing the solenoid valve 252;

11. repeating the process with a detergent; and

12. the process was repeated with water.

The bioprinter 10 is adapted to print onto a variety of substrates 125, such as microplates and petri dishes. Referring to fig. 20, the substrate 125 may be heated to 37 ℃ using a temperature control unit 280 to assist in cell proliferation. The two temperature control units 280 regulate the temperature inside the bioprinter 10 based on the 3D cell building conditions required for optimal growth conditions. The temperature control unit 280 may be adjusted to between 36 ℃ and 38 ℃ to adjust the temperature of the printhead 250, the substrate 125 disposed on the print table 128, and/or the interior of the bioprinter 10.

The substrate 125 disposed on and supported by the print table may be a porous plate 126. The substrate 125 may be a biocompatible consumable for encapsulating and culturing printed cellular structures. These substrates may include:

microtiter plates of different well configuration (6, 12, 24, 48, 96 and 384 wells);

microtiter plates with different well structures at the bottom of the cover slip (6, 12, 24, 48, 96 and 384 wells);

coverslips and microscope slides;

fluorodish of various sizes; and

chamber culture slides (Chamber slides) of different Chamber configurations (1, 2, 4, 8 and 16).

To clean the tubing 150, array of valves 251, and nozzle 253, a droplet dispensing system 25 may be used to move the cleaning fluid from the sample bottle 111 to and through the valve 252 and nozzle 253, as described above. The cleaning agent is sprayed from the nozzle 253 into the waste container 205. This process is repeated for other cleaning chemicals (e.g., 70% ethanol and water). Cleaning of the conduit 150 and printhead 250 is accomplished when only air is expelled from the nozzles 253 after all water has been flushed through the conduit 150, the valves 251 of the array, and the nozzles 253.

Laminar flow system

Referring to fig. 6-10, the bioprinter 10 further includes a laminar flow system 50 as shown in fig. 6-10. Sterility and operator safety are major issues in 3D bioprinting applications. This is typically accomplished 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 laboratories. Therefore, there is a need for a solution that minimizes the use of bio-safe cabinets and clean room spaces in 3D bioprinting applications.

The integrated laminar flow system 50 integrated into the bioprinter 10 provides a sterile environment for bioprinting of cells and 3D tissue culture models without the need for a biosafety cabinet or clean room facility. In addition, air is drawn from the outside environment through the front tunnel using a directed air flow, thereby providing protection for the operator.

The laminar air flow system 50 comprises a chamber or housing having a metal frame 500 (e.g. stainless steel) and is provided with a metal grill at the bottom to allow the contaminated air flow to be drawn into an electric blower or centrifugal fan 510. The contaminated air is pumped by fan 510 through duct inlet 520 into plenum 535, plenum 535 consisting of two high efficiency particulate rejection (HEPA) filters 525 and 530. HEPA filters 525 and 530 may remove at least 99% of the particles from the contaminated air stream. One HEPA filter 525 serves as exhaust for the external environment and the other HEPA filter 530 circulates air to the sterile room 535. It is envisaged that each filter will absorb approximately 50% of the airflow. Fig. 21 shows the flow of air that enters the bioprinter 10 through the conduit inlet 520, through the blower 510, through the HEPA filters 525 and 530, and is exhausted or recirculated. The airflow from recirculation HEPA filter 525 to aseptic chamber 535 provides a one-way downward airflow to aseptic chamber 535 at a typical velocity of 0.45 m/s. This gas flow provides a uniform clean gas flow over the bioprinted sample droplet 101, thereby greatly reducing the risk of particle contamination in the sample 100.

During a bioprinting operation, the front access hinged door 210 must be closed to reduce the risk of particle contamination. Accordingly, the flow rate of the blower 510 can be reduced by reducing the rotational speed of the blower. The reduced gas flow in aseptic chamber 535 reduces the impact of dehydration on print head 250 and sample 100. In addition, it reduces the effect of air flow interfering with sample drop 101 during its flight from printhead 250 to print substrate 125.

Control software

The bioprinter 10 is controlled by developing custom software for printing biometrics. The control system 40 includes control software including a non-transitory computer readable medium having programming instructions for operating the bioprinter 10. The non-transitory computer readable medium is located separately from the bioprinter 10 and is operatively connected to the bioprinter 10.

The software includes a Graphical User Interface (GUI) as shown in fig. 21 and 22. The end user can select different assay printing programs via the GUI and alter assay parameters such as drop spacing and drop volume. The user can also manually control the spatial position of the droplet dispensing system and create custom patterns of droplets. Other functions of the software include cleaning, priming and rinsing procedures for the droplet dispensing system.

Bioprinting requires printing a 3D model of the object. For tissue engineering applications, they are typically created using engineering tools (e.g., CAD/CAM software). These tools are expensive and highly complex, forcing scientists to spend time and resources learning engineering tools. For 3D tissue culture applications, the complexity of the structures to be printed is low. There is a need for a simple and intuitive method to create 3D structures for bioprinting in 3D tissue culture applications.

The software accompanying the bioprinter 10 provides a method of designing each layer of the 3D structure to be printed. In one embodiment, a grid is provided for the user to draw a pattern for each layer of the structure. The material to be printed may be defined as a mixture of multiple materials dispensed from different nozzles 253 in the printhead 250. For example, a hydrogel may be defined as a droplet of bio-ink mixed with a droplet of activator.

Typical substrates for tissue culture in biological laboratories are multi-well plates, such as 6, 12, 24, 48, 96 and 384 well plates. In one embodiment, an interface 670 is provided to print previously defined 3D structures within each well on a multi-well plate. The user first selects a hole or array of holes and then selects the printing routine to print in those holes.

The customization software provides a user interface for the user to input a location on which to bioprint a layer of the 3D cell construct. A print preview button 671 is provided with the software prior to printing to allow the user to visualize the location of the print unit and what the configuration will be. One function of the software is that it can control the size of the drops of the bioprinter to alter the way the cell structure is printed. The intent behind the layering of cellular structures is to mimic how biologists use z-stack layering in microscopes.

Typically, the bioprinter 10 will print 20-25 layers when building a 3D cell structure, but the number of layers is controlled using a control system and associated control software.

A positioning unit 141, controlled by computer control software, connected to the print head 250 spatially positions the valves 251 and nozzles 253 each time a droplet 101 of bio-ink, activator, cell, cellular ink, or a combination thereof is ejected. The computer-controlled spatial positioning of the solenoid valves and nozzles and the computer-controlled droplet ejection of valves 251 and nozzles 253 facilitate the generation of 3D tissue structures.

To generate the array of 3D tissue structures, the process of generating the 3D tissue structures is repeated at a plurality of locations on the substrate 125.

The control system records the identity of each sample 100 in the sample container 110 either through user input or automatic recording. The purpose is to know which sample container 110 contains which sample 100 so that during printing, when the storage containers 120 store their respective samples, the necessary samples are printed to the desired location.

Biological printer shell

The bioprinter 10 includes a housing 60 that contains the sample loading system 20, the droplet dispensing system 25, and the laminar air flow system 50. The housing 60 is assembled from a number of panels made of steel, aluminum and stainless steel and assembled using screw fasteners 600. The housing 60 also includes a hinged door 210 at the front to allow a user to access the sterile room 535 of the bioprinter 10. The movable sample tray 200 may be loaded into the bioprinter 10 through a door 210. The front panel and the hinged door 210 may be made of glass or transparent plastic. Fig. 16 and 17 illustrate the bioprinter assembly 10.

Method of producing a composite material

In operation, the bioprinter 10 has the steps for transferring a sample from a sample container into a storage container, ready for bioprinting a 3D cell construct by a print head. To fill the printer's storage container 120 and solenoid valve with fluid, a sample loading system is used to move fluid from the sample container to and through the solenoid valve. The appropriate channel is selected on the flow selector valve. The needle 130 is used to pierce the seal of the sample vial and open the solenoid valve. The peristaltic pump is turned on and the desired amount of fluid is removed from the sample container through the needle, tubing, pump, tubing, flow selector valve, and into the storage container. The peristaltic pump is closed and the solenoid valve is closed. Deselecting the appropriate channel on the flow selector valve. The pressure is set by the regulator 171, the solenoid valve 252 is repeatedly ignited until all air is discharged out of the tubing, and then bio-ink droplets, activator droplets, cell-ink droplets, or a combination thereof are ejected from the nozzle 253. The above process is repeated for each printer fluid container and solenoid valve used.

Loading bio-ink into a storage container:

1. moving the selector valve to the selected channel;

2. opening the electromagnetic valve;

3. positioning a needle over the sample vial using an x-axis and y-axis actuator;

4. lowering the needle into the vial using the Z-axis actuator, piercing the vial septum;

5. starting a peristaltic pump;

6. pumping liquid from the vial through the selection valve into the storage container;

7. stopping the pump when the fluid reaches the solenoid valve nozzle; and

8. the solenoid valve is closed.

To print bio-ink, activator, cells, cellular ink, or combinations thereof from the solenoid valve, the printer reservoir and solenoid valve are primed using the steps described above, droplets of bio-ink, activator, cells, cellular ink, or combinations thereof are ejected from the nozzle in a predetermined manner, controlled by computer-controlled software, and deposited on a substrate.

The positioning unit is connected to the print head 250 and is controlled by computer control software to spatially position the solenoid valves and nozzles each time a droplet of bio-ink, activator, cell, cellular ink, or combinations thereof is ejected. The spatial positioning of the solenoid valve and nozzle, as well as the ejection of droplets from the solenoid valve and nozzle, is computer controlled to facilitate the generation of 3D tissue structures.

To produce an array of 3D tissue structures, the process of generating the 3D tissue structures is repeated at a plurality of locations on the substrate.

Bio-ink

In this specification, bio-ink is defined as an aqueous solution of one or more types of macromolecules in which cells can be suspended or contained. Upon activation or crosslinking, the bio-ink produces a hydrogel structure having its physical and chemical properties defined by the chemical and physical composition of the bio-ink. Macromolecules are defined as arrays of synthetic and natural polymers, proteins and peptides. The macromolecule may be in its native state or chemically modified with amine or thiol reactive functionalities.

The synthetic macromolecule may include:

polysaccharides, e.g. polymers containing fructose, sucrose or glucose functional groups

Nonionic polymers, such as polyethylene glycol (PEG), Polyhydroxyethylmethacrylate (PHEMA), poly-caprolactone (PCL), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), poly (NIPAAM) and polypropylene fumarate (PPF) and derivatives thereof;

polyelectrolytes-polymers carrying a positive or negative charge, amphoteric and zwitterionic polymers;

polypeptide-a single linear chain of a plurality of amino acids (at least 2 amino acids) bound together by amide bonds; and

Synthetic polymers containing nucleobases-polymers having nucleobase (adenine, thymine, guanine or cytosine) repeating units.

Natural macromolecules 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) DNase and aptamers; basement membrane extract.

The amine-reactive functional groups may include: aldehydes, epoxy groups, N-hydroxysuccinimide (NHS) and 2-vinyl-4, 4-dimethylazlactone (VDM).

The thiol-reactive functional groups may include: alkenes, alkynes, azides, halogens and cyanate esters.

The bio-inks used and found suitable were: alginate (2 w/v% concentration) was dissolved in calcium-free DMEM, supplemented with 10 v/v% FCS, L-glutamine and sodium pyruvate.

The bio-ink having dispersed SK-N-BE (2) neuroblastoma cells is referred to as a cell-containing bio-ink.

Activating agent

The activator is an aqueous solution comprising small or large molecules that interact with the bio-ink 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 depends to a large extent on the macromolecule used and the desired crosslinking process.

The activator may be selected from:

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;

polyelectrolyte-polymers, with opposite charge to the macromolecules in the bio-ink. It can be cationic, anionic, amphoteric oxide and zwitterionic;

polypeptide-a single linear chain consisting of a number of amino acids (at least 2 amino acids) bound together through amide bonds;

DNA linker-a macromolecule with a nucleotide or DNA sequence complementary to the nucleotide or DNA sequence present on the macromolecule of the bio-ink;

natural or synthetic macromolecules bearing amine or thiol groups, natural or chemically modified.

Activators for alginate bio-inks are: 4 w/v% calcium chloride dissolved in MilliQ water.

Crosslinking or gelling

This process is the process by which individual macromolecular chains are linked together by an activating agent to form a hydrogel. The crosslinking process can be classified as chemical or physical crosslinking. Physical crosslinking or non-covalent crosslinking may include:

ionic crosslinking-crosslinking by interaction of the macromolecule with the opposite charge in an activator. Activators can include charged oligomers, ionic salts, and ionic molecules;

Hydrogen bonding-crosslinking by electrostatic attraction of polar molecules. In this case, the macromolecule and the activator carry polar functional groups.

Temperature crosslinking-crosslinking by rearrangement of the macromolecular chains, which rearrangement is the response of the macromolecular chains to temperature changes (heating or cooling); and

hydrophobic interactions or van der waals forces.

Chemical or covalent crosslinking involves a chemical reaction between a macromolecule and an activator. The types of reactions may include:

photocrosslinking, the crosslinking reaction being accelerated by irradiation with ultraviolet light or light; michael-type (Michael) addition reaction between a thiol and a vinyl-bearing macromolecule in an aqueous medium;

schiff base reaction between amino and aldehyde group;

diels-alder reaction;

click chemistry;

ammonolysis of the active ester groups; and

enzymatic cross-linking.

The following table lists examples of other bio-ink and activator combinations:

cell ink

The cellular ink is an aqueous solution of one or more molecules or macromolecules in which the cells will be uniformly suspended throughout the 3D bioprinting process. The concentration of the cell ink is optimized to prevent cell sedimentation, but still maintain high cell viability.

The cellular ink may be selected from the following options:

Small molecules, such as glycerol; and

macromolecules, e.g. Ficoll^TMGlucose, alginate, gellan gum, methylcellulose and polyvinylpyrrolidone (PVP).

Ficoll^TMIs a neutral, highly branched, high-quality hydrophilic polysaccharide, and is easily soluble in aqueous solution. Ficoll^TMHas a radius in the range of 2-7nm and is prepared by reacting a polysaccharide with epichlorohydrin. Ficoll^TMIs a registered trademark owned by GE Healthcare.

The cell ink used was Ficoll dissolved in PBS^TM400 at a concentration of 10 w/v%.

The cell ink having dispersed SK-N-BE (2) neuroblastoma cells is referred to as cell-containing cell ink.

Gellan gum is a water-soluble anionic polysaccharide produced by the bacterium Sphingomonas sp.

Cell culture solution

The cell culture solution is a liquid that comes into contact with cultured cells and is suitable for various tasks relating to the cells. The preparation process includes precise analysis of salt and pH equilibration, and only adding biocompatible molecules and sterilizing.

Some cell culture fluids include:

cell culture Media, such as DMEM Medium (Dulbecco's Modified Eagle Medium), Minimal Essential Medium (MEM), IMDM Medium (Iscove's Modified Dulbecco's Medium), 199 Medium (Medium 199), Ham's F10, Ham's F12, McCoy's 5A Medium and RPMI Medium (Roswell Park Medium Institute);

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

sterilized MilliQ water.

Cell culture conditions

Cells and 3D tissue culture models can be incubated, cultured and maintained using standard cell culture techniques. The 3D tissue culture model includes cells encapsulated in a hydrogel mold, which can be incubated under conditions that allow or maintain cell growth or spheroid formation. For most animal and human cell lines, 5% CO is typically at about 37 ℃ C₂Incubate horizontally for at least 24 hours. It will be appreciated that incubation can be carried out under any suitable conditions, temperature and duration that allow for growth, maintenance or spheroid formation of one or more cells in the hydrogel mold.

Utility solutions

Utility solution is defined as a solution that does not come into contact with the cells but is used to clean and disinfect all printer surfaces exposed to the cells. These solutions may include:

Ethanol at the correct concentration;

sterile MilliQ water;

cell culture medium;

a detergent; and

hydrogen peroxide solution (maximum concentration 2 w/v%).

Preparation of bio-ink

Initially, bio-inks are prepared by mixing the correct type and amount of macromolecule in the appropriate cell culture fluid. After uniformity was achieved, the blank bio-ink was sterilized by uv irradiation and filtration (0.22 μm filter). The bio-ink was then kept at 4 ℃ until further use.

Cell preparation

Cells were harvested by washing with PBS. The PBS was aspirated. Trypsin was added and incubated at 37 ℃ to detach the cells from the flask surface. Tissue culture medium was added to collect the isolated cells into tubes. The cells were centrifuged, the supernatant aspirated and the pellet resuspended in fresh medium. Cell counts were performed by mixing equal volumes of cell suspension and trypan blue stain. Calculated to determine cell concentration. The desired number of cells may then be added to the bio-ink, cell ink, or to the cell culture fluid.

Preparation of the activators

The correct type and amount of molecules are dissolved in the appropriate cell culture fluid. Before use, the resulting solution was sterilized by UV irradiation and filtration.

Preparation of cell ink

The correct type and amount of molecules are dissolved in the appropriate cell culture fluid. After homogeneity is achieved, the resulting solution is sterilized by UV irradiation and filtration before use. The cell ink was then kept at room temperature until further use.

Cell collection

By following an established protocol, cultured cells of interest are harvested with a certain degree of fusion. To formulate a bio-ink or cell-ink containing cells, the harvested cells were resuspended at the correct cell concentration to yield a concentration of 2.5 billion cells/ml in 200 μ l of bio-ink or cell-ink. The resulting cell pellet is then re-dispersed in the correct volume of bio-ink or cellular ink. The bio-ink containing cells or the cellular ink can then be used in a 3D bioprinter.

Printing hydrogel mould

The hydrogel molds may be printed using a drop-through drop printing process, in which bio-ink droplets and activator droplets are deposited on top of each other to create the hydrogel. This process can be repeated and used to form a 3D hydrogel structure by building up a hydrogel layer.

Cell type

The 3D tissue culture model (e.g., a sphere) can be made from any suitable cell type, including adherent cells, such as mammalian hepatocytes, gastrointestinal tract cells, pancreatic cells, kidney cells, lung cells, tracheal cells, vascular cells, skeletal muscle cells, heart 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 tissue, stromal cells, stem cells, progenitor cells, lymphocytes, blood cells, endoderm derived cells, ectoderm derived cells, mesoderm derived cells, or a combination thereof.

Other cell types may include other eukaryotic cells (e.g., chinese hamster ovary), bacteria (e.g., helicobacter pylori), fungi (e.g., penicillium chrysogenum) and yeasts (e.g., Saccharomyces cerevisiae).

The cell line SK-N-BE (2) (neuroblastoma cell) has been successfully used in the course of 3D tissue culture models under various conditions. It should be understood that it is contemplated that other cell lines may also be used to prepare 3D tissue models as desired according to the developed process. Other cell lines used include DAOY (human medulloblastoma cancer cells), H460 (human non-small cell lung cancer) and p53R127H (human pancreatic cancer cells). 088 and 089 list other possible suitable cell lines.

The development of 3D bioprinting technology is to produce high density 3D tissue culture models encapsulated in hydrogel molds by drop-on-demand technology. In particular, 3D printing techniques are used to print biocompatible hydrogel molds using bio-inks and activators, which are constructed in a layer-by-layer manner to fabricate various 3D structures. During the manufacturing of the hydrogel mold, droplets of high cell density may be contained in the hydrogel mold.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Reference to the literature

Murphy,S.and Atala,A.(2014).3D bioprinting of tissues andorgans.Nature Biotechnol,32(8),pp 773-785.

Horn,T.and Harrysson,O.(2012).Overview of current additivemanufacturing technologies and selected applications.Sci.Prog,95,pp 255–282.