3D bioprinter apparatus, kits, method, and systems for 3D printing tissue models with bioinks containing living cells

Abstract

One aspect described herein is a method of 3D printing a tissue model in a contained volume of photocrosslinkable bioink located on a print surface. The bioink may contain living cells. The method may comprise maintaining, with the print surface, a cell viability temperature of the contained volume; locating an objective lens vertically in a bottom working plane within the contained volume and successive working planes located above the bottom working plane by moving the lens vertically relative to the surface; and in the bottom working plane and each successive working plane, moving the voxel horizontally by directing the laser beam into the objective lens at different angles at different times; focusing, with the objective lens, at each different angle and time, the laser beam into a voxel; polymerizing, with the voxel, a portion of the contained volume at each horizontal location; and moving the contained volume horizontally.

Description

[0001] 3D BIOPRINTER APPARATUS, KITS, METHOD, AND SYSTEMS FOR 3D PRINTING TISSUE MODELS WITH BIOINKS CONTAINING LIVING CELLS

[0002] TECHINCAL FIELD

[0003] Aspects of this disclosure relate generally to 3D bioprinter apparatus, kits, methods, and systems. Particular aspects relate to 3D printing tissue models with bioinks containing living cells.

[0004] BACKGROUND

[0005] Tissue engineers routinely use living cells, biologically active molecules, and biomaterials to create functional tissues. Hydrogels are 3D networks composed of polymeric materials (natural and / or synthetic) capable of absorbing a considerable amount of water and swelling in aqueous medium, while maintaining physical integrity due to the presence of hydrophilic functional groups, such as OH, COOH, SO3H and CONH2. The use of hydrogels as scaffolding materials by tissue engineers has recently drawn much attention because their physicochemical properties can be tuned to match the properties of natural tissues. Some hydrogels may be applied as scaffolding material because they are similar to natural extracellular matrices in appearance and composition.

[0006] Extrusion-based bioprinting is a known method of applying hydrogels as scaffolding material. It involves using a 3D bioprinter apparatus to construct a scaffold layer-by-layer using a bioink dispensed from an extrusion nozzle in continuous filaments. Extrusion-based bioprinting faces challenges when printing complex structures, making it difficult or impossible to print some structures. Hollow structures, for example, cannot be produced using extrusion-based bioprinters since the addition of voids in the layers may result in collapsing of the construct during printing. In addition, the resolution of extrusion-based bioprinter systems is approximately 200 microns, which does not mimic human tissue structure.

[0007] SUMMARY

[0008] Methods of 3D printing a tissue model in a contained volume of photocrosslinkable bioink located on a print surface are described. The photocrosslinkable bioink may contain living cells. By way of example, aspects of the described methods may comprise: maintaining, with the print surface, a cell viability temperature of the contained volume; locating a field of view of an objective lens vertically in a bottom working plane within the contained volume and successive working planes located above the bottom working plane by moving the objective lens vertically relative to the print surface; and in the bottom working plane and each successive working plane, moving the voxel horizontally in the field of view by directing, with a galvo scan head, the laser beam into the objective lens at different angles at different times; focusing, with the objective lens, at each different angle and time, the laser beam into a voxel located at a horizontal location in the field of view; polymerizing, with the voxel, a portion of the contained volume with two photon polymerization at each horizontal location; and moving the contained volume horizontally relative to the field of view.

[0009] The method may comprise removably securing the contained volume to the print surface. The print surface may comprise an aperture; and the method may comprise removably securing the contained volume to the print surface above the aperture. The method may comprise: locating the contained volume of the photocrosslinkable bioink in a transparent container; removably securing the transparent container to the print surface above the aperture; and aligning the aperture with a bottom surface of the transparent print container so that a light beam may be transilluminable through the aperture, the transparent container, and the photocrosslinkable bioink. Maintaining the cell viability temperature may comprise one of transferring heat away from the print surface or transferring heat into the print surface. Maintaining the cell viability temperature may comprise directing a flow of electricity to a thermoelectric cooler that is thermally coupled to a bottom portion of the print surface. The thermoelectric cooler may be thermally coupled to a base plate and the method may comprise thermally isolating the print surface from the base plate and one of: transferring heat away from the thermoelectric cooler with the base plate; or transferring heat into the thermoelectric cooler with the base plate. The method may comprise circulating a heat transfer fluid through a heat exchanger that is thermally coupled to the base plate.

[0010] The method may comprise: causing, with a controller, a laser generator to output the laser beam; directing, with one or more mirrors, the laser beam into the galvo scan head; causing, with the controller, moveable mirrors of the galvo scan head to direct the laser beam into the objective lens at the different angles at the different times; and directing, with a hot mirror, the laser beam from the galvo scan head and into the objective lens at each different angle and time. The laser beam may comprise a high intensity pulsed laser beam. The laser generator may comprise a femtosecond laser or a Ti: Sapphire laser. The method may comprise applying, with the laser generator, a group delay dispersion compensation to the laser beam. Applying the group delay dispersion compensation may comprise applying, with the laser generator, opposite and equal dispersions to the laser beam. The method may comprise passing the laser beam through an integrated dispersion compensation unit of the laser generator.

[0011] The method may comprise measuring a power level of the laser beam with an internal power meter of the laser generator. The method may comprise outputting, with the internal power meter, power level data for the laser beam to the controller while 3D printing the tissue model. The method may comprise diverting, with a splitter, a portion of the laser beam into a power meter; measuring, with the power meter, a power level of the laser beam before directing it into the galvo scan head; and outputting, with the power meter, power level data for the laser beam while 3D printing the tissue model. The method may comprise directing the laser beam through an isolator and into a modulator; preventing, with the isolator, back reflections of the laser beam; and modifying, with the modulator, the laser beam before the galvo scan head.

[0012] The modulator may comprise an acousto-optic modulator; and modifying the laser beam may comprise diffracting or shifting, with the acousto-optic modulator, a frequency of the laser beam using sound waves. The modular may comprise an acousto-optic modulator; and modifying the laser beam may comprise of diffracting, with the acousto-optic modulator, the laser beam into multiple orders; and controlling, with the acousto-optic modulator, an average power output of the laser beam. The multiple orders may include 0thorder laser beams and 1storder laser beams; and the method may comprise: collecting the 1storder laser beams; controlling the 1storder laser beams; and directing the 0thorder laser beams into a beam trap.

[0013] Controlling the 1storder beams may comprise one of causing the 1storder beams to polymerize the portion of the contained volume; preventing polymerization of voided portions of the contained volume; and preventing over-polymerization of the portion of the contained volume. Causing the 1storder beams to polymerize may comprise switching the acousto-optic modulator into an ON state; and directing the 1storder beams into a beam expander. Preventing polymerization may comprise: switching the acousto-optic modulator into an OFF state; and directing 1storder beams into a beam trap. The method may comprise: receiving print data associated with a tissue model and a target average power of the laser beam in the bottom working plane and each successive working plane; and switching the acousto-optic modulator into the ON state or the OFF state responsive to one or both of the target average power and the print data.

[0014] Controlling the average power output may comprise: producing, with the acousto-optic modulator, a diffraction pattern that splits the laser beam into a 0thorder beam and a 1storder beam; modifying, with the acousto-optic modulator, an optical power of the 0thorder beam or the 1storder beam based on the target average power of the laser beam in the bottom working plane and each successive working plane; directing the 1storder beam into a first beam expander; and directing the 0thorder beam into the beam dump. The method may comprise expanding, with the first beam expander, the laser beam before the galvo scan head.

[0015] The method may comprise expanding, with a second beam expander, the laser beam before the objective lens. The second beam expander may comprise a first lens and a second lens; and expanding the laser may comprise directing the laser beam sequentially through the first lens and the second lens. The galvo scan head may comprise a plurality of moveable mirrors; and directing the laser beam and may comprise directing, with the plurality of moveable mirrors, the laser beam into the objective lens at the different angles and times. Directing the laser beam may comprise directing, with a plurality of moveable mirrors, the laser beam into scanning optics located between the galvo scan head and the objective lens. The scanning optics may comprise a scan lens and a tube lens. Focusing the laser beam into the voxel may comprise realizing a target average power of the laser beam at the voxel at each horizontal location.

[0016] The method may comprise moving a moveable power meter to a measurement location between the objective lens and the print surface; at the measurement location, measuring, with the moveable power meter, power level data for the laser beam after exiting the objective lens; and outputting, with the moveable power meter, the power level data, and moving the moveable power meter away from the measurement location.

[0017] Moving the moveable power meter may comprise causing an electric motor to rotate the moveable power meter into and out of the measurement location. Locating the field of view of the objective lens in the bottom working plane and each successive working plane may comprise moving the objective lens vertically while maintaining a vertical position of the print surface. The method may comprise moving the objective lens vertically in predetermined increments. Moving the contained volume horizontally relative to the field of view may comprise: moving the print bed in a first horizontal direction relative to the objective lens; and moving the print bed in a second horizontal direction relative to the objective lens, wherein the first direction and second directions are parallel to the bottom working plane and each successive working plane.

[0018] The method may comprise: 3D printing a first tile of the tissue model by completing the directing, focusing, and polymerizing steps in the field of view; moving the print bed in at least one of the first horizontal direction and the second horizontal direction until an edge of the field of view is aligned with an edge of the first tile of the tissue model; and after moving the print bed, 3D printing a second tile of the tissue model by completing the directing, focusing, and polymerizing steps in the field of view. The method may comprise stitching a portion of the edge of the first tile of the tissue model together with the second tile of the tissue model by completing the directing, focusing, and polymerizing steps at the edge of the field of view that is aligned with the edge of the first tile of the tissue model. The method may comprise 3D printing a first tile of the tissue model by causing the galvo scan head to move the voxel within a first portion of the contained volume in the field of view by directing the laser beam into the objective lens at the different angles and times. The method may comprise 3D printing additional portions of the tissue model by moving each additional portion of the contained volume into the field of view and causing the galvo scan head to move the voxel within each additional portion when the laser beam is directed into the objective lens at the different angles and times.

[0019] The method may comprise: directing, with a light source, a light beam through the contained volume; focusing the light beam onto an image sensor of the camera; and generating, with the image sensor, a digital image of the tissue model. The method may comprise outputting, with the camera, the digital image to a device in data communication with the camera while 3D printing the tissue model. The light source may comprise an LED, a collector lens, and condenser lens; and the method may comprise: generating, with the LED, the light beam; focusing, with collector lens, the light beam onto the condenser lens; and directing, with the condenser lens, the light beam through the contained volume and the photocrosslinkable bioink in a direction toward the camera.

[0020] The print surface may comprise an aperture; and the method may comprise: positioning the contained volume on the print surface over the aperture; and directing the light beam into the contained volume through the aperture. The method may comprise directing, with the condenser lens, a portion of the light beam through the aperture, through the contained volume, through the objective lens, and into the camera. The method may comprise passing the light beam through the objective lens in a first direction while directing the laser beam through the objective lens in a second direction that is generally opposite to the first direction. The method may comprise: reflecting, with an optical element, the laser beam from the galvo scan head and into the objective lens in the first direction; and transmitting, with the optical element, the focused beam through an objective lens and into the camera in the second direction. The method may comprise: reflecting, with a hot mirror, first wavelengths of the laser beam from the galvo scan head and into the objective lens in the first direction; and transmitting, with the hot mirror, second wavelengths of the focused beam from the objective lens and into the camera in the second direction.

[0021] Apparatus for 3D printing a tissue model in a contained volume of photocrosslinkable bioink also are described herein. The photocrosslinkable bioink may contain living cells. By way of example, aspects of the described apparatus may comprise: a print surface operable to maintain a cell viability temperature of the contained volume; an objective lens operable to focus a laser beam into a voxel located in a field of view of the objective lens; a vertical actuator operable to locate the field of view vertically in a bottom working plane within the contained volume and successive working planes located above the bottom working plane by moving the objective lens vertically relative to the print surface; a galvo scan head operable to move the voxel horizontally in the field of view by directing the laser beam toward different locations on the objective lens at different times; and a horizontal actuator operable to move the contained volume horizontally relative to the field of view, wherein, in the bottom working plane and each successive working plane, the galvo scan head causes: the objective lens, at each different location and time, to focus the laser beam into the voxel at a horizontal location in the field of view; and the voxel to polymerize a portion of the contained volume with two photon polymerization at each horizontal location; and the horizontal actuator moves the contained volume relative to the field of view.

[0022] The apparatus may comprise mounting arms operable to removably secure the contained volume to the print surface. The print surface may comprise an aperture; and the mounting arms may be operable to removably secure the contained volume to the print surface above the aperture. The contained volume of the photocrosslinkable bioink may be located in a transparent print container; the mounting arms may be operable to removably secure the transparent print container to the print surface above the aperture; and the aperture may be aligned with a bottom surface of the transparent printer container so that a light beam is transilluminable through the aperture, the transparent print container, and the photocrosslinkable bioink.

[0023] The apparatus may comprise a temperature control system operable to transfer heat away from the print surface or transfer heat to the print surface. The temperature control system may comprise a thermoelectric cooler that is thermally coupled to a bottom portion of the print surface. The apparatus may comprise a base plate, wherein: the print surface is thermally isolated from the base plate; and the thermoelectric cooler is thermally coupled to the base plate and operable to: transfer heat away from the thermoelectric cooler with the base plate; or transfer heat to the thermoelectric cooler with the base plate. The apparatus may comprise a heat exchanger that is thermally coupled to the base plate and operable to receive a heat transfer fluid circulated therethrough.

[0024] The apparatus may comprise a controller in data communication with the laser generator and the galvo scan head; the galvo scan head may comprise moveable mirrors operable with the controller to direct the laser beam into the objective lens at the different angles at the different times; and the apparatus may comprise: one or more mirrors operable to direct the laser beam from the laser generator and into the galvo scan head at the different angles at the different times; and a hot mirror operable to direct the laser beam from the galvo scan head and into the objective lens at each different angle and time.

[0025] The laser beam may comprise a high intensity pulsed laser beam. The laser generator may comprise a femtosecond laser or a Ti: Sapphire laser. The laser generator may be operable to apply a group delay dispersion to the laser beam. The laser generator may be operable to apply the group delay dispersion by applying opposite and equal dispersions to the laser beam. The laser generator may comprise an integrated dispersion compensation unit. The laser generator may comprise an internal power meter operable to measure a power level of the laser beam before it reaches the galvo scan head. The internal power meter may be operable to output data associated with the power level of the laser beam to the controller while 3D printing the tissue model. The apparatus may comprise: an external power meter; and a splitter operable to divert a portion of the laser beam into an external power meter; the external power meter being operable to: measure a power of the laser beam before the galvo scan head; and output data associated with the power level of the laser beam to the controller while 3D printing the tissue model.

[0026] The apparatus may comprise an isolator operable to prevent back reflections of the laser beam; and a modulator operable to modify the laser beam before the galvo scan head. The modulator may comprise an acousto-optic modulator operable to diffract or shift a frequency of the laser beam using sound waves. The modulator may comprise an acousto-optic modulator operable to diffract the laser beam into multiple orders and control an average power output of the laser beam. The multiple orders may include 0thorder laser beams and 1storder laser beams; and the acousto- optic modulator may be operable to: collect the 1storder laser beams; control the 1storder laser beams; and direct the 0thorder laser beams into a beam trap. The apparatus may comprise a controller operable to cause the 1storder beams to polymerize the portion of the contained volume; prevent polymerization of voided portions of the contained volume; and prevent overpolymerization of the portion of the contained volume. The controller may be operable to cause the 1storder beams to polymerize by switching the acousto-optic modulator into an ON state that directs the 1storder beams into a beam expander. The controller may be operable to prevent polymerization by switching the acousto-optic modulator into an OFF state that directs the laser beam into a beam trap.

[0027] The controller may be operable to: receive print data associated with a tissue model and a target average power of the laser beam in the bottom working plane and each successive working plane; and switch the acousto-optic modulator into the ON state or the OFF state responsive to one or both of the target average power and the print data. The acousto-optic modulator may be operable to: produce a diffraction pattern that splits the laser beam into a 0thorder beam and a 1storder beam; modify an optical power of the 0thorder beam or the 1storder beam based on the target average power of the laser beam in the bottom working plane and each successive working plane; direct the 1storder beam into a beam expander; and direct the 0thorder beam into the beam dump. The beam expander may be operable to expand the laser beam before the galvo scan head. The second beam expander operable to expand the laser beam before the objective lens. The second beam expander may comprise a first lens and a second lens; and the laser beam may be directed sequentially through the first lens and the second lens. The galvo scan head may comprise a plurality of moveable mirrors operable to direct the laser beam into the objective lens at the different angles. The apparatus may comprise scanning optics located between the galvo scan head and the objective lens. The plurality of moveable mirrors may be operable to direct the laser beam through the scanning optics and into the objective lens. The scanning optics may comprise a scan lens and a tube lens.

[0028] The apparatus may comprise a controller operable with the laser generator to realize a target average power of the laser beam at the voxel at each horizontal location. The apparatus may comprise a moveable power meter that, before or during the 3D printing of the tissue model, is: movable to a measurement location between the objective lens and the print surface; operable at the measurement location to measure a power level of the laser beam after exiting the objective lens and output data associated with the power level of the laser beam to the controller; and after outputting the data, moveable away from the measurement location. The apparatus may comprise an electric motor, wherein the controller is operable to cause the electric motor to rotate the power meter into and out of the location between the objective lens and the print surface. The horizontal actuator may be operable to maintain a vertical position of the print surface while the vertical actuator moves the objective lens vertically to locate the field of view of the objective lens in the bottom working plane and each successive working plane. The vertical actuator may be operable to move the objective lens vertically in predetermined increments.

[0029] The horizontal actuator may be operable to: move the print bed in a first horizontal direction relative to the objective lens; and move the print bed in a second horizontal direction relative to the objective lens, wherein the first direction and second directions are parallel to the bottom working plane and each successive working plane. The controller may be operable to: 3D print a first tile of the tissue model in the field of view by communicating with one or more of the laser generator, the print surface, the vertical actuator, and the galvo scan head; cause the horizontal actuator to move the print bed in at least one of the first horizontal direction and the second horizontal direction until an edge of the field of view is aligned with an edge of the first tile of the tissue model; and after moving the print bed, 3D printing a second tile of the tissue model by further communicating with one or more of the laser generator, the print surface, the vertical actuator, and the galvo scan head. The horizontal actuator may comprise a first linear actuator stacked on top of a second linear actuator; and the controller may be operable to cause the first linear actuator to move the print bed in the first horizontal direction and the second linear actuator to move the print bed in the second horizontal direction. The galvo scan head may be operable to 3D print a first tile of the tissue model by moving the voxel within a first portion of the contained volume in the field of view when the laser beam is directed into the objective lens at the different angles and times. The horizontal actuator may be operable to move additional portions of the contained volume into the field of view; and the galvo scan head may be operable to 3D print additional portions of the tissue model by moving the voxel within each additional portion of the contained volume in the field of view when the laser beam is directed into the objective lens at the different angles and times.

[0030] The apparatus may comprise: a camera comprising an image sensor; and a light source operable to direct a light beam through the contained volume and focus the light beam onto the image sensor of the camera, wherein the image sensor is operable to generate a digital image of the tissue model. The camera may be operable to output the digital image to a display while the apparatus is 3D printing the tissue model. The light source may comprise: an LED operable to generate the light beam; a condenser lens; and a collector lens operable to focus the light beam onto the condenser lens, the condenser lens being operable to direct the light beam through the contained volume and the photocrosslinkable bioink in a direction toward the camera.

[0031] The print surface may comprise an aperture; the contained volume may be positioned above the aperture; and the light beam may be directed through the aperture. The condenser lens may be operable to direct a portion of the light beam through the aperture, through the contained volume, through the objective lens, and into the camera. The light beam may pass through the objective lens in a first direction and the laser beam may pass through the objective lens in a second direction that is generally opposite to the first direction. The apparatus may comprise an optical element operable: to reflect the laser beam from the galvo scan head and into the objective lens in the first direction; and transmit the focused beam through objective lens and into the camera in the second direction. The apparatus may comprise a hot mirror operable to: reflect first wavelengths of the laser beam from the galvo scan head and into the objective lens in the first direction; and transmit second wavelengths of the focused beam from the objective lens and into the camera in the second direction. Related 3D bioprinter apparatus, kits, method, and systems also are described.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which are incorporated in and constitute part of this disclosure, illustrate exemplary aspects that, together with the written descriptions, serve to explain the principles of this disclosure. Numerous aspects are shown conceptually in the drawings and particularly described, pointed out, and taught in the written descriptions. Some structural and operational aspects may be better understood by referencing the written portions together with the accompanying drawings, of which:

[0034] FIG. 1 depicts a perspective view of an exemplary 3D bioprinter apparatus.

[0035] FIG. 2 depicts a conceptual view of a 3D bioprinter system comprising the FIG. 1 apparatus.

[0036] FIG. 3 depicts a top-down view of an exemplary laser system.

[0037] FIG. 4 depicts a perspective view an exemplary print surface, objective lens, power meter, and related components.

[0038] FIG. 5 depicts a partial side view of an exemplary galvo scan head, objective lens, imaging system, and related components.

[0039] FIG. 6 depicts a perspective view of an exemplary imaging source.

[0040] FIG. 7 depicts an exemplary image output of an exemplary tissue model.

[0041] FIG. 8 depicts an exemplary print container.

[0042] FIG. 9 depicts a perspective view of an exemplary print surface and movement system.

[0043] FIG. 10 depicts a perspective view of an exemplary print surface.

[0044] FIG. 11 depicts a partial exploded view of the FIG. 9 print surface.

[0045] FIG. 12 depicts a plate of the FIG. 9 print surface. FIG. 13 depicts plates for the FIG. 9 print surface.

[0046] FIG. 14 depicts a perspective view of a moveable power meter.

[0047] FIG. 15 images of two-phase polymerization and single-phase polymerization.

[0048] FIG. 16 depicts an exemplary 3D bioprinting method.

[0049] FIG. 17 depicts exemplary specifications for a laser source.

[0050] FIG. 18 depicts exemplary specifications for a movement system.

[0051] FIG. 19 depicts additional specifications for a FIG. 18 movement system.

[0052] FIG. 20 depicts exemplary delay settings.

[0053] Aspects of the examples illustrated in the drawings may be explained further by way of citations to the drawing and element numbers in the text of the description. The drawings and any citations thereto are provided for illustration purposes, and to further clarify the description of the present disclosure and are not intended to limit the present disclosure unless claimed.

[0054] DETAILED DESCRIPTION

[0055] Aspects of the present disclosure are not limited to the exemplary structural details and component arrangements described in this description and shown in the accompanying drawings. Many aspects of this disclosure may be applicable to other aspects and / or capable of being practiced or carried out in various variants of use, including the examples described herein.

[0056] Throughout the written descriptions, specific details are set forth to provide a more thorough understanding to persons of ordinary skill in the art. For convenience and ease of description, some well-known aspects may be described conceptually to avoid unnecessarily obscuring the focus of this disclosure. In this regard, the written descriptions and accompanying drawings should be interpreted as illustrative rather than restrictive, enabling rather than limiting. Exemplary aspects of this disclosure reference 3D bioprinter apparatus, kits, methods, and systems for 3D printing tissue models with bioinks containing living cells. For example, some aspects are described with reference to a 3D bioprinter apparatus comprising mirrors, a galvo scan head, an objective lens, related optics, a print surface, and related components that are operable, with a controller, to 3D print a tissue model within a contained volume of photocrosslinkable bioink positioned on the print surface, in which the photocrosslinkable bioink contains living cells and the print surface is operable to maintain a cell viability temperature of the contained volume before, during, and after the 3D printing process. As another example, some aspects are described with reference to 3D bioprinting methods that are performable with a controller to automate and modify different printing processes for using different bioinks to print different types of tissue models containing different types of living cells. As a further example, some aspects are described with reference to 3D bioprinting systems comprising examples of the above-referenced 3D bioprinter apparatus together with a laser generator, an imaging system, an automated power meter, the controller, sensors, and related components for using different photocrosslinkable bioinks to print different types of tissue models containing different types of living cells. Unless claimed, the descriptions provided in this disclosure are for convenience and not intended to limit this disclosure. Accordingly, any aspects described in this disclosure with reference to the 3D bioprinter apparatus, bioinks, kits, methods, and / or systems described herein may be similarly utilized with any comparable 3D printer apparatus, bioinks, kits, methods, and / or systems.

[0057] Several exemplary reference axes are described, including a lateral axis X-X, a longitudinal axis Y-Y, and a vertical axis Z-Z. Some elements and / or movements thereof are described relative to these axes, such as a first movement direction along one of axes X-X, Y-Y, and Z-Z that is opposite of a second movement direction along said one of axes X-X, Y-Y, and Z-Z. Axis Z-Z is generally shown as being vertical relative to the page so that terms like above, up, upward, upper or below, down, downward, lower may be oriented along axis Z-Z and described relative to a top and bottom of the page. Lateral axis X-X and longitudinal axis Y-Y may define a horizontal working plane that is generally parallel to a ground surface Various elements may be described herein as being movable along or about vertical axis Z-Z in directions toward and away from the horizontal working plane. As a further example, some objects may be described as “elongated,” meaning that they have a length greater than a width along a reference axis like one of axes X-X, Y-Y, and Z- Z. Additional axes, movements, and forces may be similarly described with reference any one of axes X-X, Y-Y, and Z-Z. These relative terms are provided for convenience and do not limit this disclosure unless claimed.

[0058] Inclusive terms such as “comprises,” “comprising,” “includes,” “including,” and variations thereof, are intended to cover a non-exclusive inclusion, such that aspects of any apparatus, kit, method, and system described herein, or element(s) thereof described as comprising a list of elements does not include only those elements but may include other elements not expressly listed and / or inherent thereto. Unless stated otherwise, the term “exemplary” means “example” rather than “ideal.” Various terms of approximation may be used, including “approximately” and “generally.” Approximately means “roughly” or within 10% of a stated number or outcome and generally means “usually” or more than a 50% probability of a stated number or outcome.

[0059] Connective terms such as “attached to,” “attachable to,” and “attaching” are intended to generically describe a structural connection between two or more elements. Some structural connections may be “rigidly attached” so that the connected elements are generally non-rotatable relative to one another, as when the elements are formed together (e.g., cast, bolted, and / or welded) and cannot be rotated independently without deflecting relative to one another or being damaged. Other structural connections may be “rotatably or movably attached” so that the connected elements are coupled together to permit movements relative to one another, as when the elements are pinned together (e.g., with any type of rotating, sliding, and / or telescoping connection) and can be rotated or moved freely and independently without damage. Unless stated otherwise, these exemplary connective terms and their modifiers may comprise any such variations.

[0060] Aspects of an exemplary processor are described. Functional terms such as “processing,” “computing,” “calculating,” “determining,” “displaying,” and the like, may refer to actions and processes performable by the processor, which may comprise any type of software and / or hardware. The software may comprise program objects (e.g., lines of codes) executable to perform various functions. Each program object may comprise a sequence of operations leading to a desired result, such as an algorithm and / or instructions for interfacing with an Al-powered data analytics platform. The operations may require or involve physical manipulations of physical quantities, such as electrical or magnetic signals capable of being stored, transferred, combined, compared, and otherwise manipulated. The signals may be described conceptually as bits, characters, elements, numbers, symbols, terms, values, or the like.

[0061] The hardware of the processor may comprise any known technologies for storing the program objects and any data associated therewith. For example, the program objects may be stored in any machine (e.g., computer) readable storage medium in communication with the processor, including any mechanism for storing or transmitting data and information in a form readable by a machine (e.g., a computer). Exemplary storage mediums may comprise read only memory (“ROM”); random access memory (“RAM”); erasable programmable ROMs (“EPROMs”); electrically erasable programmable ROMs (“EEPROMs”); magnetic or optical cards or disks; flash memory devices; and / or any electrical, optical, acoustical, or other form of propagated signals (e.g., carrier waves, infrared signals, digital signals, etc.).

[0062] In keeping with above, the processor may be operable with one or more remote processor(s) or and / or sensor(s) over a wireless network, such an iPhone or other iOS device, an Android phone or other Android device, a Bluetooth or Wi-Fi enabled sensor, or the like.

[0063] Some aspects of the present disclosure are described with reference to methods, steps of which may be performable with the processor. To help orient the reader, some methods may be described with reference to a conceptual drawing, such as a flowchart with boxes interconnected by arrows. Each box may represent a particular step or technology. The boxes may be combined, interconnected, and / or interchanged to provide options for additional modifications according to this disclosure. The arrows may define an exemplary sequence of operation for the steps, the order of which may be important. For example, a particular order of the steps may describe a sequence of operation that is performable by the computing element to realize specific processing benefits, such as improving a computational performance and / or an operational efficiency.

[0064] General aspects of this disclosure are now described with reference to exemplary 3D bioprinter apparatus, kits, methods, and systems shown in FIGs. 1-20 as being operable to create biomimetic tissue constructs or “tissue models” 2 (e.g., FIG. 7) by directing a laser beam 1 into a contained volume 3 of a photocrosslinkable bioink 4 containing living cells (e.g., FIG. 15). In a human body, for example, vasculature 5 (e.g., FIG. 7) are responsible for delivering nutrients and oxygen to living cells, ensuring their survival. The diffusion limit of oxygen in many tissues is approximately 200 microns, making it difficult or impossible for existing bioprinters to create a tissue model 2 that is optimized for oxygen diffusion because they cannot develop small-scale features. Because of the improvements described herein, aspects of the exemplary 3D bioprinter apparatus, kits, methods, and systems of this disclosure may be operable, electronically via a controller, to cause laser beam 1 to create or “print” complex tissue models 2 comprising complex, small-scale features like vasculature 5 (e.g., FIG. 7) in a precise, repeatable manner that allows the living cells in contained volume 3 of photocrosslinkable bioink 4 to experience enhanced cell survival and growth during and after printing.

[0065] By way of example, an exemplary 3D bioprinter apparatus 100 is shown in FIGs. 1 and / or 2 as comprising optical components that are operable with laser beam 1 to utilize two-photon polymerization (or “2PP”) within a 3D bioprinting method for 3D printing tissue models 2 featuring small-scale 3D structures, such as micro- or nanostructures with complex 3D geometries including lumens, valves, void spaces, etc.), at high spatial resolution. One exemplary 3D bioprinting method 300 is shown in FIG. 16 as comprising steps for utilizing 2PP to print tissue models 2 with different small-scale 3D structures. As shown in FIG. 7, vasculature 5 is one example of a small-scale 3D structure that is printable with 3D bioprinter apparatus 100 and / or 3D bioprinting method 300. A layer of vasculature 5 is shown in FIG. 7 after being printed in a portion of contained volume 3 of photocrosslinkable bioink 4. Any small-scale structures like vasculature 5 may be similarly printed with 3D bioprinter apparatus 100 and / or 3D bioprinting method 300. As shown in FIG. 7, for example, by utilizing 3D bioprinter apparatus 100 and related systems to print tissue models with photocrosslinkable bioink 4 via 2PP according to one or more steps of 3D bioprinting method 300, the resulting tissue models 2 may have small-scale 3D structures or features thereof printed with micro- and / or nanoscale resolution, such as scaffolds displaying micro- and / or nanoscale features that are optimized for diffusing oxygen through tissue model 2 to sustain its living cells, like vasculature 5 and other circulatory structures.

[0066] As shown at “A” in FIG. 15, for example, laser beam 1 may be a high intensity pulsed laser beam and polymerization of photocrosslinkable bio ink 4 may happen at a focal point of laser beam 1, a point described herein as voxel 6 (hence the “VoxCell™” in Applicant’s name). 3D bioprinter apparatus 100 may be configured to form voxel 6 in contained volume 3 of photocrosslinkable bioink 4 and move voxel 6 to different locations in contained volume 3. Nonlinear absorption of two photons via 2PP may happen at each location of voxel 6 in contained volume 3, at similar or different power levels, causing polymerization of photocrosslinkable bioink 4 at each location by crosslinking a portion of photocrosslinkable bioink 4 with sub-micron resolution. By way of comparison, an example of single-photon absorption or “ 1PP” is shown at “B” in FIG. 15, in which laser beam 1 is depicted as lacking a focal point like voxel 6 at A in FIG. 15, meaning that polymerization of photocrosslinkable bioink 4 via 1PP cannot be precisely controlled and / or will not happen anywhere for some bioinks 4 because the power levels of laser beam 1 are too low.

[0067] One advantage of utilizing 3D bioprinter apparatus 100 and related systems to print tissue models 2 with photocrosslinkable bioink 4 via 2PP according to 3D bioprinting method 300 is that small- scale 3D structures may be unsupported during the printing because each polymerized region is located in and supported in three dimensions by a portion of contained volume 3, eliminating the need for any supports (e.g., posts) during the printing process, like those typically required by existing printing systems utilizing fused deposition modeling (FDM). Accordingly, because polymerization may occur at any different locations in contained volume 3 by causing 3D bioprinter apparatus 100 to move voxel 6 to each different location, allowing different portions of photocrosslinkable bioink 4 to be crosslinked at different times in rapid succession, various small- scale 3D structures may be printed with 3D bioprinter apparatus 100 and / or with 3D bioprinting method 300 to have complex geometric forms, including irregular shapes with circular structures optimized for oxygen diffusion like vasculature 5.

[0068] The advantages of utilizing 3D bioprinter apparatus 100 and / or 3D bioprinting method 300 to print different tissue models 2 with different photocrosslinkable bioinks 4 are numerous in comparison to existing 3D bioprinter apparatus, kits, methods, and / or systems, like those utilizing extrusion, FDM, stereolithography (SLA), or other technologies that are not optimized for use with bioinks 4 containing living cells. For example, in complement to their higher-resolution and support-free printing capabilities, 3D bioprinter apparatus 100 and / or 3D bioprinting method 300 also may be utilized to print smaller-scale 3D structures (e.g., nanoscale structures) within existing small-scale 3D structures (e.g., existing microscale structures); with directional independence, meaning in a bottom-up configuration or a top-down configuration in terrestrial settings and any other direction off planet; using a broad variety of bioinks 4; with cell survival and growth rates that are generally impossible to obtain with existing bioprinters.

[0069] As shown in FIGs. 1-14, 3D bioprinter apparatus 100 may utilize laser beam 1 to create complex tissue models 2 (e.g., FIG. 7) within contained volume 3 of photocrosslinkable bioink 4 by printing different small-scale 3D structures via 2PP, such as precise micro- or nanostructures with high spatial resolution, like scaffolds displaying micro- to nano-scale vasculature 5. Different types of photocrosslinkable bioink 4 and 3D printable materials may be used with laser beam 1, including any type of photocrosslinkable bioinks or resins, the bioinks described in International PCT App. No. PCT / CA2023 / 051679, filed December 15, 2023; and / or any bioink or resin including the polymers described in International PCT App. No. PCT / CA2023 / 050192, filed February 14, 2023, the entireties of which are hereby incorporated by reference.

[0070] Additional aspects of 3D bioprinter apparatus 100 are now described in detail. As shown FIGs. 1 and / or 2, 3D bioprinter apparatus 100 may comprise a laser system 110, a movement system 111, a print surface 112, an imaging system 113, a moveable power meter 114, a control system 115 (e.g. FIG. 2), and a support frame 116 (e.g., FIG. 1). As described herein, any combination of these elements may be sold together with 3D bioprinter apparatus 100, as part of 3D bioprinting system including related components, and / or as components of a 3D bioprinting kit, such as with an amount of photocrosslinkable bioink 4 (e.g., FIG. 15) suitable for printing a predictable amount of tissue models 2 (e.g., IL).

[0071] Laser System 110

[0072] Laser system 110 may comprise a plurality of optical components for generating laser beam 1, focusing laser beam 1 into voxel 6, and locating voxel 6 at different locations within contained volume 3 of photocrosslinkable bioink 4. As shown in FIGs. 1 and / or 2, laser system 110 may comprise a laser source 120, one or more power meters, an isolator 121, a modulator 122, a beam expander 124, a galvo scan head 125, and an objective lens 126, examples of which are now described. Laser source 120 may be operable with control system 115 to generate and output laser beam 1 into galvo scan head 125. As shown in FIG. 2, laser source 120 may output laser beam 1 as a high intensity pulsed laser beam that causes polymerization of photocrosslinkable bioink 4 to happen within contained volume 3 of photocrosslinkable bioink 4 at voxel 6, near instantly, where nonlinear absorption of two photons occurs. Locating voxel 6 at different locations in contained volume 3 therefore allows for near-instant crosslinking of photocrosslinkable bioink 4 at each different location. As described further below, because crosslinking happens very quickly, 3D bioprinter apparatus 100 may be operable to print small-scale structures with high spatial resolution, such as scaffolds displaying micro- to nano-scale features of vasculature 5, including circular structures like those of vasculature 5, by causing laser system 110 to move voxel 6 horizontally relative to contained volume 3 and further causing movement system 111 to move volume 3 horizontally and / or vertically relative to voxel 6.

[0073] Laser source 120 may comprise a femtosecond laser source, such as a tuneable Ti: Sapphire laser; and laser beam 1 may comprise a polymerizing beam produced therewith, such as a high intensity pulsed laser beam. As shown in FIG. 2, for 2PP applications, laser source 120 may be tuned to generate short pulses of laser beam 1 at a working plane above print surface 112, such as a bottom plane within contained volume 3 (e.g., a first working plane) and successive planes located above the bottom plane (e.g., each successive plane being a second working plane located above or below the first working plane in a print direction, such as top-down, bottom-up, or other direction). As shown in FIG. 2, laser source 120 may be operable with control system 115 to maintain a high average power output of laser beam 1 at each working plane. Exemplary specifications for laser source 120 are shown in FIG. 17 to facilitate operation of 3D bioprinter apparatus 100 according to this disclosure. By way of example, laser source 120 may comprise a tuneable Ti: Sapphire laser such as the Coherent Chameleon Vision-S Ti: Sapphire Laser described generally at https: / / www.coherent.com / content / dam / coherent / site / en / resources / datasheet / lasers / COHR_Cha meleonVision-S DS Ol 19_3.pdf, the entirety of which are hereby incorporated by reference.

[0074] Parameters of laser beam 1 such as pulse width and average power attainable at voxel 6 may be tuned to maximize print speeds of 3D bioprinter apparatus 100. Each optical component of laser system 110 may introduce dispersion, herein referred to as Group Delay Dispersion or “GDD”, that may be detrimental to the available peak intensity of laser beam 1 at voxel 6, potentially limiting print speeds. For example, a lower peak intensity of laser beam 1 at voxel 6 may reduce print speeds by reducing the speed at which voxel 6 may be moved with laser system 110 while inducing photopolymerization. The overall total GDD of laser system 110 may provide a reliable measure of how much its optical components may have broadened the pulse width of laser beam 1. Some optical components of laser system 110 have been selected to reduce its overall total GDD, thereby maximizing print speeds.

[0075] As shown in FIG. 2, laser source 120 may comprise an integrated dispersion compensation unit that maintains a short pulse width of laser beam 1 at a working plane and allows the user to apply custom forms of GDD compensation to laser beam 1. Laser source 120 of FIG. 2 may thus serve as a pre-compensation unit. For example, one form of GDD compensation may comprise causing laser source 120 to apply opposite and equal dispersions to laser beam 1 before it reaches modulator 122, beam expander 124, galvo scan head 125, and / or objective lens 126, allowing a fully restored pulse width of laser beam 1 to be realized at voxel 6. As a further example, as shown in FIG. 2, laser system 110 may comprise a plurality of mirrors M1-M7, each of which may comprise an ultrafast-enhanced silver mirrors for beam steering and reducing GDD.

[0076] It may be necessary to measure an average optical power of laser beam 1 continuously and / or at regular intervals during a printing process, such as during 3D bioprinting method 300. As shown in FIG. 2, the one or more power meters of laser system 110 may comprise a power meter that is integrated into laser source 120 and operable to output measurements of optical power to control system 115, providing a first opportunity to measure the average optical power of laser beam 1 prior to objective lens 126. As further shown in FIG. 2, laser beam 1 may be output from laser source 120 (e.g., from its integrated dispersion compensation unit) toward modulator 122; and the one or more power meters of laser system 110 also may comprise an external power meter that is positioned between laser source 120 and galvo scan head 125 and operable to output measurements of optical power to control system 115, providing another (e.g., second) or alternative opportunity to measure the average optical power of laser beam 1 prior to objective lens 126. As shown in FIG. 2, the external power meter of the one or more power meters of laser system 110 may comprise a splitter 127, an iris 128, and a power measurement device 129. Laser beam 1 may be directed first through splitter 127, which may divert and direct a portion of laser beam 1 into iris 128 and towards power measurement device 129. As shown in FIG. 2, power measurement device 129 may be operable to output measurement data associated with laser beam 1 during the printing process, such as by outputting continuous measurements to control system 115 during 3D bioprinting method 300.

[0077] The remainder of laser beam 1 may be directed from splitter 127, through isolator 121, and into modulator 122. As shown in FIG. 2, isolator 121 may comprise a Faraday isolator operable to prevent back reflections and provide laser beam 1 with mode-locked stability. Modulator 122 may comprise an acousto-optic modulator or “AOM” that uses an acousto-optic effect to diffract and / or shift a frequency of laser beam 1 using sound waves at particular power levels in the radio spectrum or radiofrequency. As shown in FIG. 2, modulator 122 may comprise a piezoelectric transducer which is attached to a material such as glass and utilizes an oscillating electric signal vibrating, creates sound waves that serve as moving periodic planes of expansion and compression that change an index of refraction of laser beam 1. For example, the sound waves may cause incoming laser beam 1 to scatter (e.g., via Brillouin scattering) off the resulting periodic index modulation, causing interference occurs similar to Bragg diffraction. In this example, the interaction may be thought of as a three-wave mixing process resulting in Sum-frequency generation or Differencefrequency generation between phonons and photons.

[0078] Modulator 122 may thus be operable to diffract laser beam 1 into multiple orders and control its average power output. As shown in FIGs. 2 and / or 3, laser beam 1 may be diffracted with modulator 122 to (i) collect 1st order beams and optimize them for forming tissue models by polymerizing targeted portions of photocrosslinkable bioink 4 in volume 3; (ii) prevent polymerization of portions of photocrosslinkable bioink 4 when laser beam 1 is passing over planned void regions in the tissue model, such as when printing vasculature 5; and / or (iii) prevent over-polymerization of the targeted portions of photocrosslinkable bioink 4 due to laser dwelling from direction reversing. Modulator 122 may cover a 700-1100 nm wavelength range; be made out of TeO2; and have an optical transmission of greater than 95%, an active aperture of 1.5x2 mm2, and a maximum rise / fall time of 192 ns. By way of example, modulator 122 may comprise an AOM from AA Opto-Electronic, such as the MT80-A1.5-IR described at http: / / www.aaoptoelectronic.com / wp-content / uploads / 2018 / 08 / MT80-Al.5-xx-edl-18.pdf, the entirety of which is hereby incorporated by reference.

[0079] As shown in FIGs. 2 and 3, modulator 122 may be switchable between an “ON” state that permits polymerization of photocrosslinkable bioink 4 by directing the 1st order beams collected into beam expander 124 via mirrors Ml, M2, and M3; and an “OFF” state that prevents polymerization of photocrosslinkable bioink 4 by transmitting laser beam 1 into a beam trap 130, without diffracting it into multiple orders, via mirrors Ml and M7. During normal use, control system 115 may automatically switch modulator 122 between the ON and OFF states responsive to different data sources. For example, the user may input an average power input and the state of modulator 122 may be automatically and / or manually controlled by control system 115 according to printing data associated with a particular tissue model for a particular patient.

[0080] As shown in FIG. 2, modulator 122 may serve as a laser shutter and power attenuation device. For example, when the AOM is switched into the ON state: (i) modulator 122 may produce a diffraction pattern that splits laser beam 1 into at least two orders, such as a Oth order beam and a 1st order beam; and (ii) a radiofrequency or RF power of the AOM may be adjusted to transfer more optical power to one of the 1 st order beam or the Oth order beam, depending on how much optical power is desired at the working plane. Conversely, when the AOM is switched into the OFF state, laser beam 1 may be transmitted into beam trap 130. As shown in FIG. 2, after passing through modulator 122 when in the ON state, the 1 st order beam may strike Ml, M2, and M3 (e.g., three 45-degree ultrafast-enhanced silver mirrors) before entering beam expander 124; whereas the Oth order laser beam 1 may strike mirror M7 (e.g., a silver d-shaped pick-off mirror) and be directed into beam trap 130.

[0081] As shown in FIG. 2, the 1st order beam may bypass mirror M7, strike mirror M3, and be directed into beam expander 124. As also shown in FIG. 2, beam expander 124 may function as Keplerian beam expander comprising adjustable lenses that may be optimized to expand laser beam 1 by a multiplier sufficient for meeting certain requirements of galvo scan head 125, such as by 1.68X. As shown in FIG. 2, beam expander 124 be operable with control system 115 to attenuate a power of laser beam 1 with control system 115 at different times, such as when aligning voxel 6 with contained volume 3 before the printing begins. As shown in FIGs. 1 and 2, laser beam 1 may exit beam expander 124 and be directed by mirror M4 toward galvo scan head 125.

[0082] Galvo scan head 125 may be operable to direct laser beam 1 toward a beam location that is centered on a back side of objective lens 126 at different angles at different times. As shown in FIG. 2, galvo scan head 125 may comprise laser-directing mirrors and be operable with galvo scanning optics 132. The laser-directing mirrors may comprise a plurality of small, motorized mirrors that are located inside a housing of galvo scan head 125 (e.g., shown as rectangular housing in FIG. 5) and operable with control system 115 to rapidly direct laser beam 1 toward the beam location on the back side of objective lens 126 at the different angles and different times with high degrees of accuracy and precision. By way of example, galvo scan head 125 may comprise a Thorlabs XG210-AG like those described at https: / / www.thorlabs.com / newgrouppage9. cfm?objectgroup_id=14124, the entirety of which is hereby incorporated by reference.

[0083] As shown in FIG. 2, the laser-directing mirrors of galvo scan head 125 may be operable with control system 115 to move voxel 6 horizontally in a field of view of objective lens 126 by directing laser beam 1 into the beam location of objective lens 126 at the different angles and different times, at speeds of metres per second (e.g., up to approximately 7 m / s) making it possible for objective lens 126 to rapidly move voxel 6 by converting angular motions of laser beam 1 into linear and / or horizonal motions of voxel 6 at correspondingly high speeds (e.g., up to approximately 700 mm / s). Control system 115 may be operable with galvo scan head 125 to further enhance printing speeds. As shown in FIG. 2, control system 115 may increase printing speeds by causing galvo scan head 125 to direct laser beam 1 toward objective lens 126 at different angular trajectories to realize increased acceleration when printing portions of tissue model 2, such as scaffolding for the curved and / or cylindrical portions of vasculature 5.

[0084] As shown in FIGs. 2 and 5, galvo scanning 132 optics may be operable to expand laser beam 1 by a first expansion factor to ensure that it overfills a back aperture of objective lens 126 by a second expansion factor. As shown in FIG. 5, the second beam expansion factor may be calculated based on a set of requirements for laser beam 1 prior to entering galvo scanning optics 132. As shown in FIG. 2, for 3D bioprinter apparatus 100, a first expansion factor of 4.167 may be required for laser beam 1 prior to entering galvo scanning optics 132 to ensure that laser beam 1 overfills a back aperture of the back surface of objective lens 126 by a second expansion factor of 1.11.

[0085] As shown in FIGs. 2 and 5, galvo scanning optics 132 may comprise a scan lens 133 and a tube lens 134 operable as a pair to expand laser beam 1 and provide telecentricity. Scan lens 133 and tube lens 134 may expand laser beam 1 based on a difference between their respective focal lengths. By way of example, scan lens 133 may have a focal length of approximately 50 mm and tube lens 134 may have a focal length of approximately 200 mm, leading to a beam expansion factor of 200 / 50 = 4. As a further example, scan lens 133 may comprise a 50 mm FL scan lens, like those sold by Thorlabs as (i) an SL50-2P2 and described at https: / / www.thorlabs.com / thorproduct.cfm?partnumber=SL50-2P2 or (ii) an XG210-AG and described at https: / / www.thorlabs.com / newgrouppage9. cfm?objectgroup_id=14124; and tube lens 134 may comprise a 200 mm FL laser scanning tube lens, like those sold by Thorlabs as a TTL200MP2 and described at https: / / www.thorlabs.com / thorproduct.cfm?partnumber=TTL200MP2, the entireties of which are hereby incorporated by reference.

[0086] Laser system 110 may utilize low GDD optical components and pre-compensating dispersion optics (e.g., of laser source 120) to increase the available peak intensity of laser beam 1 at voxel 6. Locating galvo scan head 125 after modulator 122 and beam expander 124 as shown in FIG. 2 may allow 3D bioprinter apparatus 100 to realize faster movement speeds of laser beam 1 during polymerization, leading to dramatic (e.g., 30x) increases in fabrication throughput. As shown in FIG. 2, galvo scan head 125 may move voxel 6 at speeds of 700 mm / s, making it possible to maximize fabrication throughput once parameter goals for laser beam 1 are met.

[0087] In part because of movement speeds obtainable with galvo scan head 125, tests indicate that 3D bioprinter apparatus 100 and / or 3D bioprinting method 300 may allow printing of an exemplary tissue model 2 within a contained volume 3 of 0.00877 mm3with stages within approximately 59 minutes at a printing speed of 2.5 mm / s. As a further example, during further testing, by adjusting settings of galvo scan head 125, the same 3D bioprinter apparatus 100 was able to print the same tissue model 2 within approximately 30.00 min. at 5 mm / s; 15.167 min. at 10 mm / s; 7.82 min. at 20 mm / s; 5.37 min. at 30 mm / s; 3.40 min. at 50 mm / s; 2.55 min. at 70 mm / s; and 2.00 min. at 90 mm / s. Even with high speeds obtainable with galvo scan head 125, a maximum printing speed of 3D bioprinter apparatus 100 may still be limited by the average laser power of laser beam 1 at voxel 6 and / or a minimum laser exposure threshold or “light dose" required to crosslink photocrosslinkable bioink 4, both of which are independent of galvo scan head 125.

[0088] Objective lens 126 may be operable to focus laser beam 1 into a voxel 6 located in a field of view of objective lens 126. As shown in FIGs. 1, 4, 5, and / or 15, voxel 6 may be moved horizontally in the field of view of objective lens 126 by directing, with galvo scan head 125, laser beam 1 toward a beam location that is centered on a back side of objective lens 126 at different angles at different times. At each different angle and time, objective lens 126 may focus laser beam 1 into a voxel 6 that is located in contained volume 3, at a horizontal location in the field of view of objective lens 126; thereby polymerizing, with voxel 6, a portion of contained volume 3 with 2PP at each horizontal location within the field of view of objective lens 126.

[0089] As described below, aspects of movement system 111 may be operable to move objective lens 126 relative to contained volume 3 in three dimensions (e.g., horizontal X, Y; and vertical Z dimensions), making it possible to print different layers of tissue model 2 in three dimensions by polymerizing different portions of contained volume 3 with voxel 6 via 2PP at different horizontal locations and / or in different vertical working planes of laser beam 1.

[0090] Objective lens 126 may form laser beam 1 into voxel 6 within contained volume 3 of photocrosslinkable bioink 4 at the different times, allowing tissue model 2 to be printed layer-by- layer. As shown in FIG. 2, objective lens 126 may be the final optical component of laser system 110 that laser beam 1 passes through before reaching contained volume 3. Galvo scan head 125 may direct laser beam 1 at different angles on objective lens 126 at different times. At each different angle and time, objective lens 126 may focus laser beam 1 into voxel 6 at a different horizontal location in its field of view, allowing laser beam 1 to print a layer of tissue model 2 by polymerizing a portion of photocrosslinkable bioink 4 via 2PP at each different horizontal location in its field of view. As shown in FIGs. 2 and 5, objective lens 126 may be a 10X objective lens that is infinity corrected, has a working distance of between 16.0 mm and 17.2 mm, and has a compatible tube lens focal length of 200 mm. By way of example, objective lens 126 may comprise a Nikon CFI Plan Fluor 10X / 0.3 or a Nikon Plan Fluorite Objective 4X / 0.13 like those described at https: / / downloads.microscope.healthcare.nikon.com / phase4 / literature / Brochures / Microscope- objectives_2CE-MPHK-5.pdf, the entirety of which is hereby incorporated by reference.

[0091] Movement System 111

[0092] Aspects of movement system 111 may be operable with control system 115 to physically move objective lens 126 in a first direction relative to contained volume 3 (e.g., vertically) and / or contained volume 3 in a second direction relative to voxel 6 (e.g., horizontally). As shown in FIGs. 1, 2, 4, and / or 6, movement system 111 may comprise a vertical or first actuator 140 and a horizontal or second actuator 141.

[0093] Aspects of first actuator 140 may be operable with control system 115 to move objective lens 126 in the first or vertical direction relative to contained volume 3. As shown in FIGs. 4 and 8, support frame 116 may comprise rigid structures that are made of aluminum and / or steel and interconnected to one another and a table or floor. First actuator 140 may be mounted to a portion of support frame 116 and electronically operable with control system 115 to move objective lens 126 in the first direction (e.g., vertically, along a Z-axis) relative to contained volume 3. For example, first actuator 140 may be electronically operable with control system 115 to move objective lens 126 in the first direction to locate a field of view of objective lens 126 in a bottom plane within contained volume 3 and successive planes located within contained volume 3 above the bottom plane.

[0094] Aspects of first actuator 140 may be designed and optimized for high-precision positioning of voxel 6 within contained volume 3. For example, first actuator 140 may comprise a low friction, preloaded, backlash-free ball screw drive that provides smooth high-speed and motion bidirectional repeatability with minimal heat induced position drift. As shown in FIGs. 4 and 8, objective lens 126 may be lighter than the combined weight of contained volume 3, print surface 112, and second actuator 141, making it easier for with first actuator 140 to move objective lens 126 in the first direction (e.g., vertically, with less torque) that it would be to move print surface 112 and second actuator 141 in the first direction. The reduced loading requirements associated with moving objective lens 126 in the first direction by itself may help to increase printing speeds for 3D bioprinter apparatus 100, allowing it to print vasculature 5 at higher speeds. Exemplary specifications for first actuator 140 are shown in FIG. 18 to facilitate operation of 3D bioprinter apparatus 100 according to this disclosure. By way of example, first actuator 140 may comprise a Newport VP-25XA motorized linear stage like those sold and described at https: / / www.newport.eom / p / VP-25XA, the entirety of which is hereby incorporated by reference.

[0095] Second actuator 141 may be operable with control system 115 to move contained volume 3 in the second or horizontal direction relative to objective lens 126. As shown in FIG. 4, contained volume 3 may be mounted on second actuator 141 and moveable horizontally therewith in a first horizontal or “X” direction and / or a second horizontal or “Y” direction responsive to control signals from control system 140. For example, second actuator 141 may be operable to move contained volume 3 in the X and / or Y directions relative to the field of view for objective lens 126, allowing tissue model 2 to be printed portion-by-portion and layer-by-layer, in which the portions and layers are stitched together by overlapping different fields of view for objective lens 126 when printing.

[0096] As shown in FIGs. 4 and 8, second actuator 141 may comprise a first motorized linear stage or first stage 142 stacked on top of a second motorized linear stage or second stage 143. Control system 115 may be operable to cause first stage 142 to move print surface 112 in the X direction relative to objective lens 126; and cause second stage 143 to move print surface 112 in the Y direction relative to objective lens 126. For example, in a bottom-up printing configuration, the X and Y directions may be first and second horizontal directions that are located in a bottom working plane within contained volume 3 and successive working planes located within contained volume 3 at locations above the bottom working plane. As a further example, in a top-down configuration, for comparison, the X and Y directions may be first and second horizontal directions that are located in a top working plane within contained volume 3 and successive horizontal planes located within contained volume 3 at locations below the top working plane.

[0097] Each stage 142, 143 may be designed with FEM-optimized base for the highest rigidity and thermal performance, using high strength and stable aluminum alloy, preferably with components like long life and high load recirculating ball bearings, high force linear motors, and high precision linear encoders. As shown in FIGs. 4 and 8, contained volume 3 may be mounted to first stage 142 via print surface 112 and moveable therewith in the first horizontal or X direction within each working plane, each of which may be mounted to second stage 143 and moveable therewith in the second horizontal or Y direction within each working plane, or vice versa. Stages 142, 143 may be independently operable with control system 115, allowing for precise movements of contained volume 3 in the X and / or Y directions relative to objective lens 126 and voxel 6 formed therewith. Exemplary specifications for stages 142 and 143 are shown in FIG. 19 to facilitate operation of 3D bioprinter apparatus 100 according to this disclosure. By way of example, each of first stage 142 and second stage 143 may comprise a Newport MLT50 motorized linear stage like those sold and described at https: / / www.newport.eom / p / MLT50, the entirety of which is hereby incorporated by reference.

[0098] Aspects of laser system 110, print surface 112, and movement system 111 may thus be operable to generate laser beam 1, focus it into voxel 6, and locate voxel 6 in contained volume 3 of photocrosslinkable bioink 4. In complement, once voxel 6 has been formed in contained volume 3, the combined X, Y, and / or Z movements of voxel 6 provided by laser system 110 and / or movement system 111 may render 3D bioprinter apparatus 100 operable to and / or 3D bioprinting method 300 performable to 3D print and stitch together portions of tissue model 2 in photocrosslinkable bioink 4 at voxel 6 via 2PP by allowing for smooth, high-speed movements of voxel 6 and contained volume 3 in three dimensions (e.g., X, Y, and / or Z) relative to one another.

[0099] Print surface 112

[0100] Aspects of print surface 112 are now described. When printing tissue constructs containing human cells, it may be desirable or necessary to maintain photocrosslinkable bioink 4 at or approximate to a cell viability temperature appropriate for maintaining the viability of living cells in contained volume 3 of photocrosslinkable bioink 4 so that tissue model 2 may be printed with the living cells. To print the exemplary tissue model 2 depicted in FIG. 7 (e.g., a tissue model 2 with vasculature 5), it may be necessary to keep photocrosslinkable bioink 4 at a low temperature during a printing process (e.g., like 3D bioprinting method 300 of FIG. 16) to maintain cell viability, such that a cell viability temperature of between approximately 4°C and approximately 10°C may be appropriate depending on the type of photocrosslinkable bioink 4 used and related system conditions. Print surface 112 may be operable to maintain the viability of the living cells by maintaining the cell viability temperature of contained volume 3 of photocrosslinkable bioink 4 before, during, and / or after the printing process.

[0101] As shown in FIGs. 8 and / or 10, contained volume 3 may be contained in a print container 7; and print surface 112 may comprise a print container mount 150, a heat transfer plate 151, and a temperature control system 152. As shown in FIG. 8, print container 7 may comprise a standard lab container for cell handling, such as a petri dish, a well plate, or a PDMS container. By way of example, print container 7 may comprise an imaging dish for high-end microscopy, such as an Ibidi® 35 mm p-dish like those sold and described at https: / / ibidi.com / dishes / 8-dish-35-mm- high-ibitreat.html, the entirety of which is hereby incorporated by reference.

[0102] Print container mount 150 may comprise one or more mounting arms 153 operable to maintain a position of print container 7 on heat transfer plate 151. As shown in FIGs. 9 and 10, one or more mounting arms 153 may comprise a first mounting arm located on one side of print container 7 and a second mounting arm located on the other side of container 7.

[0103] As shown in FIGs. 9 and 10, heat transfer plate 151 may comprise a plurality of plates and mounting systems that are interconnected to one another, temperature control system 152, and second actuator 141. Each plate of heat transfer plate 151 may be machined from aluminum and sized to improve the structural rigidity of print surface 112 as a unit, maintain a flatness of print container 7 when being moved in along the X and / or Y axis by second actuator 141, and / or maintain a squareness of print container 7 relative to objective lens 126 along the Z axis. As shown in FIG. 11, the different plates of heat transfer plate 151 may comprise a mounting plate 154, a base plate 155, a print container plate 156, and a heat exchanger cover plate 157.

[0104] As shown in FIG. 9, mounting plate 154 may be bolted to first stage 142 of second actuator 141 and movable relative objective lens 126 by operation of first stage 142 and / or second stage 143 with control system 115. Mounting plate 154 may be made of an insulating material with high mechanical strength, such as Delrin plastic. As shown in FIG. 9, mounting plate 154 may provide a rigid, thermally insulating spacer between heat transfer plate 151 and second actuator 141. Each mounting arm 153 may be mounted to base plate 155 by a screw with a biasing element (e.g., shown as a metal spring) operable to cause each mounting arm 153 to apply a biasing force to print container 7.

[0105] As shown in FIG. 12, base plate 155 may comprise an aperture 160, a first plurality of holes 161, a second plurality of holes 162, and rails 163. Aperture 160 may extend through base plate 155 and be slightly oversized to allow for trans-illumination of laser beam 1 through base plate 155 and minimize cut off light. A rearward portion of base plate 155 may be mounted to second actuator 141 and a forward portion of base plate 155 may be cantilevered outwardly from actuator 141. First plurality of holes 161 may be located on opposing edges of the rearward portion of base plate 155 and sized for bolting base plate 155 to mounting plate 154. Aperture 160 and second plurality of holes 162 may be located on the forward portion of base plate 155 and arranged in a plurality of different patterns to facilitate bolting of different elements to base plate 155. As shown in FIGs. 11 and 12, rails 163 may extend upwardly from base plate 155 and comprise additional holes for bolting heat exchanger cover plate 157 thereto. Because of thermal characteristics attributable to its material composition (e.g., aluminum), thickness, and / or holes 162, base plate 155 may be operable to maintain a temperature of photocrosslinkable bio ink 4 to within about 1 °C of a setpoint throughout the duration of the printing process, as measured during performance testing.

[0106] As shown in FIG. 13, print container plate 156 may be formed from a solid piece of material (e.g., aluminum) into the shape of a rectangular prism comprising an aperture 164, a plurality of mounting holes 165, and a bottom-facing opening (e.g., shown as opening upwardly). As described further with reference to imaging system 113, aperture 164 may define a light path extending through print container plate 156 and be slightly oversized to allow for transillumination of light beam 8 through print container plate 156 and minimize cut off light. As shown in FIGs. 11 and / or 13, plurality of mounting holes 165 may be located at the comers and along the edges of print container plate 156, allowing it to be bolted to the forward portion of base plate 155.

[0107] As shown in FIG. 11, temperature control system 152 may comprise a thermoelectric cooler (or “TEC”, also known as a thermoelectric heat pump or a Peltier module) 166, an insulating plate 167, and a heat exchanger 168. TEC 166 may comprise a first TEC responsible for cooling contained volume 3 (e.g., at right in FIG. 11) and a second TEC responsible for heating contained volume 3 (e.g., at left in FIG. 11). The first and second TECs may be spaced apart from one another by a distance (e.g., 25.4 mm) from the center of aperture 164. The distance may vary, although smaller distances may be preferred because reducing the distance between contained volume 3 and the first and second TECs 166 may reduce overall condensation buildup and temperature gradient across volume 3.

[0108] As shown in FIG. 11, insulating plate 167 may provide a rigid, thermally insulating spacer between the first and second TECs 166, limiting flows of thermal energy transmitted from the first TEC 166 to the second TEC 166 and vice versa. Insulating plate 167 may be made of an insulating material with high mechanical strength, such as Delrin plastic. As shown in FIG. 11, insulating plate may comprise a TEC opening 169, an aperture 170, and a plurality of mounting holes 171. Insulating plate 167 may comprise one TEC opening 169 for each TEC 166. For example, each TEC opening 169 may have a square shape that is sized to receive a corresponding square shape of one TEC 166 so that the first and second TECs 166 are spaced apart from one another. Aperture 170 may be coaxially aligned with apertures 160, 164 described above. As shown in FIG. 11, aperture 170 may define a light path extending through insulating plate 167 and be slightly oversized to allow for trans-illumination of light beam 8 through insulating plate 167 and minimize cut off light. Like plurality of mounting holes 165, plurality of mounting holes 171 may be located at the corners of insulating plate 167 and along the edges of plate 167 so that it may be bolted to the forward portion of base plate 155 with print container plate 156.

[0109] As described herein, aspects of base plate 155, print container plate 156, and insulating plate 167 may serve as a custom machined housing for TEC 166 that provide thermal conductivity through the desired surfaces (e.g., in directions parallel with laser beam 1) and thermal isolation between other surfaces (e.g., directions transverse with laser beam 1). By way of example, print container plate 156 may be made of aluminum to promote heat transfer into contained volume contained volume 3; and / or insulating plate 167 may be made of Delrin plastic to isolate the hot side of each TEC 166 from its respective cold side. Additionally, ensuring all the components of print surface 112 may be machined (e.g., including base plate 155, print container plate 156, and insulating plate 167) to improve the flatness of contained volume 3 in print container 7 by more accurately aligning the working plane of laser beam 1 with a top of print container plate 156 and / or a bottom of container 7.

[0110] To allow for customization, print container plate 156 may serve as a cover or for TEC 166 that is removable and / or replaceable with other lids for TECs, potentially allowing several different types of contained volumes 3 and / or containers 7 to be mounted to print container plate 156. One example of print container plate 156 is shown at top in FIG. 13 as described above. Another example of print container plate 156 is shown at bottom in FIG. 13 as a plate 156’ having an aperture 164’ surrounded by a recess sized to receive a petri dish or microscopic slide. In this example, the recess may interact and / or be interlocked with edges of container 7 to hold it in place.

[0111] Print surface 112 may utilize heat exchanger 168 to operate TEC(s) 166 with active coolant circulation, allowing temperature control system 152 to sustain the cell viability temperature of contained volume 3 for extended periods of time (e.g., hours). As shown in FIG. 11, heat exchanger 168 may comprise a block 172, an input port 173, and an exit port 174. Block 172 may comprise a low-profile heat transfer block formed from a thermally conductive material to define internal conduits, a first opening for input port 173, and a second opening for exit port 174. By way of example, block 172 may be milled from aluminium because of its high thermal conductivity and compatibility with base plate 155 and heat exchanger cover plate 157, both of which also may be milled from aluminum, eliminating the possibility of galvanic corrosion. As shown in FIG. 11, a hose may extend into the first opening to define input port 173, through the internal conduits, and out of the second opening to define exit port 174. The hose may be coupled to a liquid cooling system operable to transfer heat from block 172, into a fluid flowing through the hose (e.g., water), and out to the environment (e.g., to ambient air) air via a cooling fan.

[0112] As shown in FIGs. 10 and 11, heat exchanger cover plate 157 may be formed from a solid material like aluminum into the shape of a rectangular prism comprising a bottom-facing opening that opens upward to receive block 172, one or more rearward-facing openings that open rearwardly to receive input port 173 and exit port 174, and a plurality of holes 175. As shown in FIG. 11, heat exchanger cover plate 157 may be placed over rails 163 and block 172 by locating block 172 between rails 163 and locating them inside the bottom-facing facing opening. As shown in FIG. 11, plurality of holes 175 may be located at the corners and in a central portion of heat exchanger cover plate 157. Heat exchanger cover plate 157 may be bolted to rails 163 via the comer-located holes 175 to define an interior cavity located on the rearward portion of base plate 155. The central holes 175 may affect thermal characteristics of heat exchanger cover plate 157 and allow for mounting additional devices thereto. Like base plate 155, the thermal characteristics of cover plate 157 and / or rails 163 (e.g., attributable to their material composition (e.g., aluminum) and / or thickness) may help to maintain the temperature of contained volume 3 during the printing process.

[0113] Imaging system 113

[0114] Aspects of imaging system 113 are now described. As shown in FIGs. 2 and / or 5, imaging system 113 may comprise optical components and an image sensor that are operable with control system 115 to generate highly magnified, real-time images and / or videos of tissue model 2 during the printing process and cause the images and / or videos to be displayed on a monitor or other display device in data communication with control system 115. As described herein, imaging system 113 may comprise a light source 176, mirror M6, objective lens 126, mirror M5, and a camera 177.

[0115] Light source 176 may be operable with control system 115 to generate a light beam 8 and direct it into camera 177 through contained volume 3, laser beam 1, objective lens 126, and mirror M5. In complement, camera 177 may comprise an image sensor that receives light beam 8, generates the images and / or videos of tissue model 2 therewith, and output data including the images and / or videos to control system 115. By way of example, light source 176 may comprise a transmission Koehler illumination assembly that evenly illuminates contained volume 3 from underneath and is operable to adjust a brightness and / or contrast of light beam 8, making it possible to image and / or display features of tissue model 2 during the printing process (e.g., during 3D bioprinting method 300).

[0116] As shown in FIGs. 4, 5, and / or 6, light source 176 may comprise an LED 178, a lens 179, an aperture plate with an aperture 180, mirror M6, and a base structure 181. LED 178 may be operable with control system 115 to generate light beam 8 and direct it toward the aperture plate and through aperture 180. Lens 179 may be collector lens operable to focus light beam 8 and direct it through aperture 180 toward mirror M6. As shown in FIG. 6, mirror M6 may comprise a 45-degree mirror (e.g., another ultrafast-enhanced silver mirror) positioned to direct light beam 8 into base structure 181. As shown in FIGs. 4 and / or 6, base structure 181 may comprise a housing 182, a lumen 183, and a portion of support frame 116. Aspects of light source 176 may be located underneath housing

[0117] 182, which may be suspended above mirror M6 by the portion of support frame 116. As shown in FIGs. 2 and / or 4, lumen 183 may extend through housing 182 along axis Z-Z. A condenser lens may be located in lumen 183 (e.g., at the top portion of lumen 183 shown in FIG. 4) and operable to render light beam 8 into a parallel light beam 8 that illuminates the underside of contained volume 3. As shown in FIG. 2, base structure 181, housing 182, and lumen 183 may be positioned underneath objective lens 126 so that the condensing lens and parallel light beam 8 produced therewith may be coaxially aligned with and directed through objective lens 126 along axis Z-Z.

[0118] As shown in FIGs. 2 and / or 6, the optical path for imaging system 113 may start at LED 178, after which light beam 8 may pass through lens 179, through aperture 180, strike mirror M6 and be reflected upward through lumen 183 and the condenser lens contained therein, at which light beam 8 has come to focus. As shown in FIGs. 2, 4, and / or 5, the condenser lens in lumen 183 may transmit light beam 8 through contained volume 3 and toward objective lens 126 . As shown in FIG. 2, light beam 8 may pass through objective lens 126 and strike mirror M5 after exiting lens 26; and mirror M5 may comprise a soda-lime hot mirror that allows a first portion of light beam 8 (e.g., visible wavelengths) to pass therethrough to camera 177, reflects a second portion of light beam 8 (e.g., NIR wavelengths) away from camera 177, and directs laser beam 1 toward objective lens 126.

[0119] As shown in FIGs. 2 and / or 6, camera 177 may comprise a tube lens 184 and objective lens 126. Camera 177 may be selected based on the type of light beam 8 output from LED 178, such as a monochromatic camera operable with a narrow band of wavelengths. By way of example, camera 177 may comprise a Basler ace acA2440-75 um monochrome camera like those sold and described at https: / / www.baslerweb.com / en-us / shop / aca2440-75um / , the entirety of which is hereby incorporated by reference. The image sensor of camera 177 may comprise complementary metal oxide semiconductor (CMOS) sensor that is operable to create the images and / or videos. Tube lens 184 may be operable to generate the images and / or videos at a plane of an eyepiece diaphragm of camera 177 (e.g., within the so-called intermediate image plane) by focusing each image and / or frame into the CMOS sensor of camera 177. Control circuitry of camera 177 may be operable to output the images and / or videos to control system 115 before, during, and after the printing process.

[0120] As shown in FIG. 2, laser beam 1 may be output in a first direction along axis Z-Z (e.g., a downward vertical direction) and light beam 8 may be output in a second direction along axis Z- Z (e.g., an upward vertical direction), causing laser beam 1 to pass through light beam 8 and vice versa. Objective lens 126 may therefore serve a dual purpose within 3D bioprinter apparatus 100. As shown in FIG. 2, 3D bioprinter apparatus 100 may utilize objective lens 126 to both (i) form laser beam 1 into voxel 6 within contained volume 3 for printing purposes; and (ii) illuminate contained volume 3 with light beam 8 for imaging purposes so that the resulting images and / or videos are magnified and centered about voxel 6.

[0121] An exemplary digital image 185 output with camera 177 is shown in FIG. 7 at a micrometre scale as depicting a tissue model 2 with vasculature 5. As shown in FIG. 8, digital image 185 may be depicted via a display device 186, such as an LCD monitor in data communication with control system 115. The images and / or videos (e.g., like digital image 185 of FIG. 7 and / or a succession thereof) may be output continuously with camera 177, control system 115, display device 186, and / or another display device in real-time during the printing process.

[0122] Moveable Power Meter 114

[0123] Aspects of moveable power meter 114 are now described. Different means for measuring the average optical power of laser beam 1 are described herein. As shown in FIGs. 2 and 3 and described above, laser source 120 may comprise an integral power meter operable to output a continuous measurement of optical power to control system 115, providing a first opportunity to measure the average optical power of laser beam 1 prior to objective lens 126. As shown in FIGs. 2 and 3 and also described above, power measurement device 129 may provide another or alternative, second opportunity to measure the average optical power of laser beam 1 prior to objective lens 126, providing measurement data comparable to that output from the integral power meter of laser source 120. Laser source 120 and power measurement device 129 are shown as being located in advance of objective lens 126. Each optical element between laser source 120 and objective lens 126 may affect the optical power of laser beam 1 at voxel 6, suggesting that additional measurements of optical power may be necessary. As shown in FIG. 14 moveable power meter 114 may provide another or alternative opportunity to measure the average optical power of laser beam 1 after objective lens 126, preferably at regular intervals during a printing process for tissue model 2 (e.g., 3D bioprinting method 300), providing additional measurement data comparable to that output from laser source 120 and power measurement device 129.

[0124] Automating movements of moveable power meter 114 may be desirable to reduce the amount of time require for setting up and / or modifying 3D bioprinter apparatus 100 during each printing process and / or therebetween. For example, automating the position of power meter 114 with respect to an optical axis of laser beam 1 after it passes through objective lens 126 (e.g., axis Z-Z) may result in less user interaction with 3D bioprinter apparatus 100 during each printing process, and ensure that data collection of the average optical power is consistent with each experiment. Since power readings for laser beam 1 are typically required frequently, such as at the start of the printing process for each layer of tissue model 2, automating movements of moveable power meter 114 helps to reduce the amount of manual intervention required to print tissue model 2.

[0125] As shown in FIG. 14 power meter 114 may comprise a stepper motor mount 188, a stepper motor 189, a shaft adapter 190, a homing arm 191, a limit switch 192, a post 194, an arm coupler 195, an arm 196, and a power sensor 197.

[0126] As shown in FIG. 14, stepper motor mount 188 may comprise an inverted U-shaped frame that is made of a rigid material (e.g., aluminum) and bolted to the table or floor to provide a portion of support frame 116 that is rigid enough provide a stable foundation for moveable power meter 114 that resists any reaction forces associated with causing stepper motor 189 to move rotating arm 196 and power sensor 197. Stepper motor 189 may comprise electric motor with a collar that is rigidly attached to an elevated surface of stepper motor mount 188 and an output shaft extending vertically through an opening in the elevated surface for attachment to shaft adapter 190.

[0127] As shown in FIG. 14, shaft adapter 190 may be attached the output shaft of stepper motor 189. For example, the output shaft of stepper motor 189 may have an outer diameter (e.g., 0.25”) and shaft adapter 190 may be attached thereto. Homing arm 191 may be attached to shaft adapter 190 and / or the output shaft of stepper motor 188 and rotatable therewith. As shown in FIG. 14, limit switch 192 may be mounted to stepper motor mount 188 and located in a rotational path of homing arm 191 so that contact therebetween may be used by control system 115 to home stepper motor 189, allowing power sensor 197 to be accurately positioned to intercept laser beam 1 after objective lens 126 and then removed from the path of laser beam 1 if its average power level is sufficient.

[0128] As shown in FIG. 14, vertical post 194 may have a second diameter (e.g., 0.50”) that is larger than the first diameter of the output shaft of stepper motor 189. Shaft adapter 190 may be rigidly attached to the output shaft and vertical post 194, allowing the output shaft of stepper motor 189 to rotate vertical post 194 via shaft adapter 190. Coupler 195 may comprise a right-angle clamp operable to rigidly attach vertical post 194 to horizontal post 196 to. As shown in FIG. 14, vertical post 194 may extend in a first direction that is parallel with the optical axis of laser beam 1 (e.g., along the Z axis) and post 196 may extend along a second direction that is transverse with the first direction (e.g., along the X axis and / or the Y axis).

[0129] Power sensor 197 may be attached to post 196. As shown in FIG. 14 power sensor 197 may comprise a mounting hole extending partially therethrough and the open end of horizontal post 196 may be inserted in the mounting hole and bolted to sensor 197. In this configuration, power sensor 197 may be perpendicular to laser beam 1, allowing for appropriate collection of measurement data associated with its average optical power. As shown in FIG. 14, stepper motor 189 may be operable with control system 115 to rotate post 194 in order to swing arm 196 and power sensor 197 attached thereto into and out of the optical path of laser beam 1.

[0130] Power sensor 197 may comprise a thermal power sensor that is operable output measurement data associated with the average optical power of laser beam 1. By way of example, power sensor 197 may comprise a Thorlabs S425C Thermal Power Sensor Head like those sold and described generally at https: / / www.thorlabs.com / thorproduct.cfm?partnumber=S425C, the entirety of which is hereby incorporated by reference. As shown in FIG. 14, power sensor 197 may be held at the working plane of laser beam 1 by horizontal post 196, allowing power sensor 197 to swing into the optical axis manually or via operation of stepper motor 189 when a power reading is desired. Manual readings require the user to move power sensor 197 and then return to a workstation (e.g., one running the Thorlabs Power Meter Console software) before reviewing the results, whereas using stepper motor 189 allows everything to be performed from the workstation, reducing the amount of manual intervention required. Control system 115

[0131] Operational aspects of 3D bioprinter apparatus 100 are now described with reference to control system 115. As show in FIG. 2, control system 115 may comprise any combination of hardware, firmware, and / or software that is operable to cause 3D bioprinter apparatus 100 to print different types of tissue models 2 with different types of contained volumes 3 and bioinks 4. Aspects of control system 115 may be located throughout 3D bioprinter apparatus 100 and in data communication therewith over a wired (e.g., as shown in FIG. 2) or wireless connection (e.g., via Wi-Fi). As described herein, the communicative capabilities of control system 115 may allow for precise control and tuning of 3D bioprinter apparatus 100, making it possible to print complex tissue models 2 at high printing speeds in different volumes 3 and / or bioinks 4, enhancing the translation between in vitro and in vivo results for tissue engineering and drug development.

[0132] The hardware, firmware, and / or software of control system 115 may cause voxel 6 to print tissue model 2 within contained volume 3 of photocrosslinkable bioink 4 by precisely controlling aspects of laser system 110, print surface 112, movement system 111, imaging system 113, and automated power meter 114, allowing 3D bioprinter apparatus 100 to perform any printing functions described herein. As shown in FIG. 2, control system 115 may comprise an operating unit 200, a modulator driver 201, a galvo controller 202, a movement controller 203, a stepper motor controller 204, a power supply 205, and various power and / or data connections between each of these elements and one or more elements of 3D bioprinter apparatus 100, such as laser system 110, print surface 112, movement system 111, imaging system 113, and / or moveable power meter 114.

[0133] Operating unit 200 may serve as a general-purpose computing device for 3D bioprinter apparatus 100, allowing it to control the printing functions described herein and perform computing processes related thereto. As shown in FIG. 2, operating unit 200 may comprise hardware (e.g., processing device, like a computer) and / or software operable therewith to: (i) cause print surface 112 to maintain a cell viability temperature of contained volume 3; (ii) cause movement system 111 to locate a field of view of objective lens 126 in a bottom plane within contained volume 3 and successive planes located above the bottom plane; and (iii) in the bottom plane and each successive plane, cause galvo scan head 125 to direct laser beam 1 toward objective lens 126 at different angles at different times in order to focus, with objective lens 126, at each different angle and time, laser beam 1 into a voxel 6 that is located at a horizontal location in the field of view and operable to polymerize a portion of volume 3 with two photon polymerization at each horizontal location; and (iv) after polymerizing volume 3 at each horizontal location in the field of view, cause movement system 111 to move volume 3 horizontally relative to the field of view.

[0134] As shown in FIG. 2, operating unit 200 and modulator driver 201 may be in data communication with modulator 122 and operable therewith to diffract laser beam 1 into multiple orders and control an average power output of laser beam 1. For example, the multiple orders may include 0thorder laser beams and 1storder laser beams; and modulator 122 may be operable with modulator driver 201 to collect the 1storder laser beams 1 ; control the 1storder laser beams 1 ; and direct the 0thorder laser beams 1 into beam trap 130. In these examples, operating unit 200 and modulator driver 201 may be operable with laser source 120, modulator 122, and related aspects of laser system 110 to cause the 1storder beams 1 to form polymerized portions of contained volume 3 at voxel 6; prevent polymerization of voided portions of contained volume 3, such as interior portions of vasculature 5 (e.g., FIG. 7); and prevent over-polymerization of the polymerized portions of volume 3.

[0135] Operating unit 200 may be operable with modulator driver 201 to cause the 1storder beams 1 to polymerize portions of contained volume 3 of photocrosslinkable bioink 4 by switching modulator 122 into an ON state that directs the 1storder beams into beam expander 124 and prevent polymerization by switching modulator 122 into an OFF state that directs laser beam 1 into beam trap 130. As shown in FIG. 2, modulator driver 201 may comprise a cabling and breakout board with processing components for switching modulator 122 into the ON and OFF states. The cabling and breakout board of modulator driver 201 may be installed in operating unit 200 and operable with its control software, centralizing aspects of control system 115.

[0136] As shown in FIG. 2, operating unit 200 and galvo controller 202 may be in data communication with galvo scan head 125 and operable therewith to direct laser beam 1 toward different locations on objective lens 126 at different times, allowing control system 115 and galvo scan head 125 to move voxel 6 horizontally in the field of view of objective lens 126. For example, galvo scan head 125 may comprise electronically moveable mirrors (e.g., via electric motors) that are operable with galvo controller 202 direct laser beam 1 toward the different locations at the different times. As shown in FIG. 2, operating unit 200, modulator driver 201, and galvo controller 202 may be operable with galvo scan head 125 and modulator 122 to provide time synchronized switching of modulator 122 and analog power control of laser source 120. For example, galvo controller 202 may comprise a digital-to-analog converter or DA converter (e.g., one meeting an XY2-100 standard) for switching modulator 122 and a laser connector for analog power control of laser source 120. For example, galvo controller 202 may be installed in operating unit 200 and placed in data communication with galvo scan head 125 and modulator 122 via circuitry of operating unit 200. By way of example, galvo controller 202 may comprise a Scanlab RTC6 PCIe galvo controller like those described at https: / / www.scanlab.de / sites / default / files / 2020- 08 / 12_RTC6%20control%20boards.pdf, the entirety ofwhich is hereby incorporated by reference.

[0137] A reliable data connection between galvo controller 202 and modulator 122 may be required to maintain its capabilities for time synchronized switching and analog laser power control. As shown in FIG. 2, modulator 122 may comprise a 15-pin DSUB breakout board, a laser controller of galvo controller 202 may comprise a corresponding 15-pin DSUB laser connector socket, and control system 115 may comprise a cable extend therebetween. When installed in operating unit 200 with modulator driver 201 and operable therewith, aspects of galvo controller 202 may be optimized to minimize latencies when switching modulator 122, such as a servo lag time of galvo scan head 125 and a runtime latency of the DA converter.

[0138] Because of its connection galvo controller 202, during normal use, the ON or OFF state of modulator 122 may be automatically controlled by modulator driver 201 and galvo controller 202 so that only an average power input is required. By way of example, modulator driver 201 may comprise a fixed frequency drive and galvo controller 202 may be interfaced with drive 201 to so that a 5 V TTL digital signal may sent to the fixed frequency driver to switch modulator 122 “ON” when polymerization is desired. By way of example, the fixed frequency driver of modulator 122 may comprise a Laboratory 110-230 VAC fixed frequency driver from AA Opto-Electronic (MODA80-D41k51k-344575), like those sold and described at http: / / www.aaoptoelectronic.com / our-products / rf-drivers-for-modulators-fixed-frequency- shifters / , the entirety of which are hereby incorporated by reference. In addition to the “ON” and “OFF” control of modulator 122 via modulator driver 201, operating unit 200 and galvo controller 202 also may be operable with modulator 122 to attenuate the average optical power of the 1st order beam 1 at the working plane. For example, a front of modulator driver 201 may comprise a RF power potentiometer that can be rotated to adjust the average power of the 1 st order beam 1 by reducing the diffraction efficiency. A voltage from 0-5 V may be output from galvo controller 202 to tune the average optical power to a specific value. As shown in FIG. 2, an analog output from galvo controller 202 may be connected to an analog input of modulator driver 202 and operable to set a corresponding average laser power based on a position of the potentiometer. For example, a target average power and voltage-power correlation table may be specified in a set of marking parameters managed by operating unit 200 (e.g., within the below- referenced 3D design data), which then reads the voltage-power correlation table and sets the appropriate voltage output on the analog pin to ensure that the correct optical power is obtained at the working plane. In this example, when the 5 V TTL signal output from galvo controller 202 goes high, the analog signal input to modulator driver 201 may control the diffraction efficiency of modulator 122 and subsequently the average power of laser beam 1 at the working plane.

[0139] Movement controller 203 may be in data communication with movement system 111 and operable therewith to move objective lens 126 and contained volume 3 relative to one another. As shown in FIG. 9, movement controller 203 may in data communication with first and second actuators 140, 141, and operable therewith to cause first actuator 140 to move objective lens 126 relative to print surface 112 in the first direction (e.g., vertically); and second actuator 141 to move contained volume 3 in the second direction (e.g., horizontally) relative to objective lens 126.

[0140] As shown in FIG. 5, a maximum size of a useable field of view for objective lens 126 may be approximately 0.99 x 0.99 mm2. The maximum size of the useable field of view objective lens 126 may be limited by aspects of galvo scan head 125, such as a by a diffraction limited field of view of scan lens 133. Movement controller 203 may be operable with operating unit 200 compensate for the maximum size of the usable field of view of objective lens 126 by using voxel 6 to print different sections or “stages” of a layer of tissue model 2 in each working plane and stitch the different stages together to fabricate large structures. The technique may be called “tile stitching” because the stages may be ordered so that each portion or “tile” of a printed tissue model 2 is both smaller than the aforementioned maximum size of the field of view of objective lens 126 and stitched together via voxel 6 with one or more adjacent tiles to print tissue model 2 as one solid structure.

[0141] As shown in FIG. 2, movement controller 203 and operating unit 200 may thus be operable with second actuator 141 to printer larger tissue models 2 (e.g., larger than approximately 0.99 x 0.99 mm2) by stitching different portions of tissue model 2 together within each working plane by moving contained volume 3 relative to lens 126 and its field of view. As shown in FIGs. 2 and / or 9, movement controller 203 may cause second actuator 141 to move print surface 112 in a first horizontal direction relative to objective lens 126 and / or a second horizontal direction relative to lens 126, wherein the first direction and second directions are parallel to each working plane.

[0142] Tile stitching may be used to print each layer of tissue model 2. For example, movement controller 203 may be operable with galvo scan head 125 and second actuator 141 to print a first tile of a layer of tissue model 2 by moving voxel 6 within a first portion of contained volume 3 in the field of view of objective lens 126 when laser beam 1 is directed into lens 126 at different angles and times. After which, in this example, movement controller 203 may be operable with galvo scan head 125 and second actuator 141 move additional portions of contained volume 3 into the field of view and print additional tiles of tissue model 2 in the layer by moving again voxel 6 within each additional portion of contained volume 3 in the field of view when laser beam 1 is again directed into objective lens 126 at different angles and times. In this example, movement controller 203 may cause: 3D bioprinter apparatus 100 to print the first tile in the layer by communicating with laser generator 120, print surface 112, first actuator 140, and / or galvo scan head 125 to locate voxel 6 in contained volume 3; second actuator 141 to move print surface 112 in one or more horizontal directions until an edge of the field of view is aligned with an edge of the first tile; and after moving print surface 112, cause 3D bioprinter apparatus 100 to print the second tile in the layer and stitch an edge of the second tile to an edge of the first tile in the layer by further communicating with laser generator 120, print surface 112, first actuator 140, and / or galvo scan head 125 to further locate voxel 6 in contained volume 3.

[0143] Aspects of control system 115 may be in data communication with print surface 112 and operable therewith to maintain the viability of living cells in contained volume 3 of photocrosslinkable bioink 4. As shown in FIG. 9, operating unit 200 may in data communication with temperature control system 152 to maintain a cell viability temperature of contained volume 3 for extended periods of time (e.g., hours) by causing one side of TEC 166 to heat up or cool down and / or causing heat exchanger 168 to circulate the cooling fluid as required to manage excess heat from the other side of TEC 166.

[0144] Aspects of control system 115 may be in data communication with imaging system 113 and operable therewith to generate images and / or video of tissue model 2 during a printing process such as 3D bioprinting method 300. As shown in FIGs. 5 and 6, operating unit 200 may in data communication with light source 176, camera 177, and a display device (e.g., a screen) and operable therewith to cause light source 176 to generate light beam 8 and direct it into camera 177 through contained volume 3, after which operating unit 200 may subsequently cause camera 177 to generate the images and / or video and output them to the display device while simultaneous causing 3D bioprinter apparatus 100 to print tissue model 2.

[0145] Aspects of control system 115 may be in data communication with movable power meter 114 and operable therewith to measure an average power of laser beam 1 after objective lens 126. As shown in FIG. 14, operating and stepper motor controller 204 may be in data communication with stepper motor 189, limit switch 192, and power sensor 197 and operable therewith to cause stepper motor 189 to move power sensor 197 into a measurement location between objective lens 126 and print surface 112; receive power level data output from power sensor 197 at the measurement location; and after receiving the power level data and determining if further measurements are required, causing stepper motor 189 to move power sensor 197 away from the measurement location.

[0146] Power supply 205 may be selected based on power requirements of laser system 110, such as galvo scan head 125. For example, if galvo controller 202 comprises a Thorlabs XG210-AG, then power supply 205 may comprise a dual output + / - 15 V linear power supply with a 150 W max, a < 100 mV ripple, and a < 0.5% DC to 30 MHz noise. By way of example, power supply 205 may comprise a SolaHD SLD-15-3030-15T as described at https: / / www.digikey.com / en / products / detail / solahd / SLD-15-3030-15T / 10071952, the entirety of which is hereby incorporated by reference. As a general-purpose computing device for 3D bioprinter apparatus 100, operating unit 200 may comprise hardware and / or software operable to input 3D design data associated with tissue model

[0147] 2 from any data source via wired or wireless connection. The 3D design data may comprise geometrical data for tissue model 2, temperature specifications for contained volume 3 and / or photocrosslinkable bioink 4, average power requirements for laser 1 at voxel 6, layering and tile stitching instructions for movement system 111, and related parameters for causing 3D bioprinter apparatus 100 to print different types of tissue models 2 with different types of contained volumes

[0148] 3 and / or bioinks 4. For example, the 3D print data for each tissue model 2 may be generated externally (e.g., by vasculature design software) and uploaded to operating unit 200 to start the printing process.

[0149] In response to the 3D design data, the hardware and / or software of operating unit 200 may generate and output control signals causing aspects of 3D bioprinter apparatus 100 such as laser generator 120, modulator 122, galvo scan head 125, movement system 111, and / or print surface 112, causing them to perform the various printing functions described herein, such as maintaining the viability living cells in contained volume 3, generating laser beam 1, directing and focusing beam 1 into a voxel 6 located in volume 3, moving voxel 6 to different locations within volume 3, and / or moving volume 3 relative to objective lens 126. Because of operating unit 200, aspects of 3D bioprinter apparatus 100 may customized to print different tissue models 2 with different contained volumes 3 and / or bioinks 4 at high-speeds responsive to 3D design data from any source with little or no human interaction. By way of example, the software of unit 200 may comprise or be compatible with Direct Machining Pro or “DMC” as described and sold at https: / / directmachining.com / dmc- pro, the entirety of which is hereby incorporated by reference.

[0150] Aspects of control system 115 may be operable with laser system 110 responsive to the 3D design data. As shown in FIG. 2, operating unit 200 may be in data communication with modulator driver 201 and one or more power meters and operable therewith to: receive measurements of a target average power of laser beam 1; and switch modulator 122 into between the ON, OFF states responsive to the 3D design data and / or the measurements of target average power. For example, the power meter(s) may comprise the integrated power meter of laser generator 120, power measurement device 129, and / or moveable power meter 114; and operating unit 200 may be operable therewith to continuously and / or intermittently measure the average power of laser beam 1 before and after objective lens 126, apply customized forms of GDD compensation, and performing related quality control functions. As a further example, operating unit 200 and modulator driver 201 also may be operable to: produce a diffraction pattern that splits laser beam 1 into a 0thorder beam 1 and a 1storder beam 1; modify an optical power of the 0thorder beam or the 1storder beam based on a target average power of laser beam 1 in the bottom working plane and each successive working plane; direct the 1storder beam 1 into beam expander 124; and direct the 0thorder beam 1 into beam dump 130.

[0151] Aspects of control system 115 also may be operable with movement system 111 responsive to the 3D design data. For example, to adjust for different types of tissue models 2, contained volumes 3, and / or bioinks 4, the 3D print data may specify the use of similar or different layer thicknesses to realize different geometries (e.g., like vasculature 5 of FIG. 7); and movement controller 203 may be operable with first actuator 140 to move objective lens 126 in predetermined increments selected by operating unit 200 to control the layer thicknesses during the printing of each different geometry. As a further example, the 3D print data may specify particular tile sizes and shapes for each layer of tissue model 2; and movement controller 203 may be operable with second actuator 141 to move contained volume 3 for account for limitations of objective lens 126 if / when size and shape of a particular layer is larger than the field of view of lens 126.

[0152] Because of its versatility, the hardware and / or software of operating unit 200 also may be operable with galvo controller 202 to perform various quality control and / or correctional functions responsive to the 3D print data. For example, together with operating unit 200, galvo controller 202 may generate a marking correction file tissue model 2, the accuracy of which may aid printing in high-resolution and verifying print quality. As another example, together with operating unit 200, galvo controller 202 may execute a “sky writing” function when mark trajectories are required to be executed at a constant marking velocity, ensuring that light dosed applied by 2PP at voxel 6 is consist throughout model 2. As another example, the software of operating unit 200 may incorporate delays into the process to ensure that mark trajectories are not over-scanned, underscanned, burned-in, or oscillating. An exemplary set of delay settings for operating unit 200 are shown in FIG. 20 to facilitate operation of 3D bioprinter apparatus 100 according to this disclosure. Aspects of control system 115 also may be operable with imaging system 113 responsive to the 3D design data. For example, operating unit 200 may be in data communication with camera 177 (e.g., FIG. 2) and display device 186 (e.g., FIG. 7) and operable therewith to: display a user interface on display device 186 that an operator of 3D bioprinter apparatus 100 can use to capture the images and / or videos; generate a visual representation of tissue model 2 based on the 3D design data; project the visual representation on display device 186, such as by overlaying the representation into images and / or videos; and / or continuously compare the images and / or videos with tissue model 2 during the printing process, making camera 177 useful for quality control.

[0153] Aspects of 3D bioprinter apparatus 100 are now described with reference to an exemplary printing method that is shown in FIG. 16 and described herein as 3D bioprinting method 300 comprising steps for printing different tissue models 2 with different contained volumes 3 of photocrosslinkable bioinks 4. Although described with reference 3D bioprinter apparatus 100 and bioink 4, it is contemplated that aspects of 3D bioprinting method 300 may be similarly described with other types of bioprinters and / or temperature-reactive print materials.

[0154] In keeping with above, 3D bioprinting method 300 may comprise steps for 3D printing tissue model 2 in contained volume 3 of photocrosslinkable bioink 4 when located on print surface 112. As shown in FIG. 16, 3D bioprinting method 300 may comprise: (i) maintaining, with print surface 112, a cell viability temperature of contained volume 3 (a maintaining step 301); (ii) locating a field of view of objective lens 126 vertically in a bottom working plane within the contained volume 3 and successive working planes located above the bottom working plane by moving objective lens 126 vertically relative to print surface 112 (a locating step 302); and in the bottom working plane and each successive working plane, (iii) moving voxel 6 horizontally in the field of view by directing, with galvo scan head 125, laser beam 1 into objective lens 126 at different angles at different times (a first moving step 303); (iv) focusing, with objective lens 126, at each different angle and time, laser beam 1 into voxel 6 at a horizontal location in the field of view (a focusing step 304); (v) polymerizing, with voxel 6, a portion of contained volume 3 with 2PP at each horizontal location (a polymerizing step 305); and (vi) moving contained volume 3 horizontally relative to the field of view (a second moving step 306). When made, used, and / or performed according to this disclosure, aspects of 3D bioprinter apparatus 100 and related systems as well as contained volume 3 and photocrosslinkable bioink 4 described for use therewith, such as in a related kit; and / or the various methods of 3D printing tissues described in relation to apparatus 100 and related kits or systems, such as the steps of 3D bioprinting method 300 and any intermediate steps described with reference to the 3D bioprinter apparatus 100, may aid the development and utility of 3D printing complex tissue models 2 containing living cells operative to enhance the translation between in vitro and in vivo results in the fields of tissue engineering and drug development.

[0155] While principles of the present disclosure are described herein with reference to illustrative aspects for particular applications, the disclosure is not limited thereto. Those having ordinary skill in the art and access to this disclosure will recognize additional modifications, applications, aspects, and substitution of equivalents all fall in the scope of the described aspects. Accordingly, the present disclosure is not to be considered as limited by the foregoing description.

Claims

CLAIMS1. A method of 3D printing a tissue model in a contained volume of photocrosslinkable bioink located on a print surface, the photocrosslinkable bioink containing living cells, the method comprising: maintaining, with the print surface, a cell viability temperature of the contained volume; locating a field of view of an objective lens vertically in a bottom working plane within the contained volume and successive working planes located above the bottom working plane by moving the objective lens vertically relative to the print surface; and in the bottom working plane and each successive working plane, moving the voxel horizontally in the field of view by directing, with a galvo scan head, the laser beam into the objective lens at different angles at different times; focusing, with the objective lens, at each different angle and time, the laser beam into a voxel located at a horizontal location in the field of view; polymerizing, with the voxel, a portion of the contained volume with two photon polymerization at each horizontal location; and moving the contained volume horizontally relative to the field of view.

2. The method of claim 1, comprising removably securing the contained volume to the print surface.

3. The method of claim 2, wherein the print surface comprises an aperture and the method comprises removably securing the contained volume to the print surface above the aperture.

4. The method of claim 3, comprising: locating the contained volume of the photocrosslinkable bioink in a transparent container;removably securing the transparent container to the print surface above the aperture; and aligning the aperture with a bottom surface of the transparent print container so that a light beam is transilluminable through the aperture, the transparent container, and the photocrosslinkable bioink.

5. The method of claim 1, wherein maintaining the cell viability temperature comprises one of transferring heat away from the print surface; or transferring heat into the print surface.

6. The method of claim 1, wherein maintaining the cell viability temperature comprises directing a flow of electricity to a thermoelectric cooler that is thermally coupled to a bottom portion of the print surface.

7. The method of claim 6, wherein the thermoelectric cooler is thermally coupled to a base plate and the method comprises: thermally isolating the print surface from the base plate; and one of transferring heat away from the thermoelectric cooler with the base plate; or transferring heat into the thermoelectric cooler with the base plate.

8. The method of claim 7, comprising circulating a heat transfer fluid through a heat exchanger that is thermally coupled to the base plate.

9. The method of claim 1, comprising: causing, with a controller, a laser generator to output the laser beam;directing, with one or more mirrors, the laser beam into the galvo scan head; causing, with the controller, moveable mirrors of the galvo scan head to direct the laser beam into the objective lens at the different angles at the different times; and directing, with a hot mirror, the laser beam from the galvo scan head and into the objective lens at each different angle and time.

10. The method of claim 9, wherein the laser beam comprises a high intensity pulsed laser beam.

11. The method of claim 9, wherein the laser generator comprises: a femtosecond laser; or a Ti: Sapphire laser.

12. The method of claim 9, comprising applying, with the laser generator, a group delay dispersion compensation to the laser beam.

13. The method of claim 12, wherein applying the group delay dispersion compensation comprises applying, with the laser generator, opposite and equal dispersions to the laser beam.

14. The method of claim 9, comprising passing the laser beam through an integrated dispersion compensation unit of the laser generator.

15. The method of claim 9, comprising measuring a power level of the laser beam with an internal power meter of the laser generator.

16. The method of claim 15, comprising outputting, with the internal power meter, power level data for the laser beam to the controller while 3D printing the tissue model.

17. The method of claim 1, comprising: diverting, with a splitter, a portion of the laser beam into a power meter; measuring, with the power meter, a power level of the laser beam before directing it into the galvo scan head; and outputting, with the power meter, power level data for the laser beam while 3D printing the tissue model.

18. The method of claim 1, comprising: directing the laser beam through an isolator and into a modulator; preventing, with the isolator, back reflections of the laser beam; and modifying, with the modulator, the laser beam before the galvo scan head.

19. The method of claim 18, wherein the modulator comprises an acousto-optic modulator and modifying the laser beam comprises diffracting or shifting, with the acousto-optic modulator, a frequency of the laser beam using sound waves.

20. The method of claim 18, wherein the modulator comprises an acousto-optic modulator and modifying the laser beam comprises: diffracting, with the acousto-optic modulator, the laser beam into multiple orders; and controlling, with the acousto-optic modulator, an average power output of the laser beam.

21. The method of claim 20, wherein: the multiple orders include 0thorder laser beams and 1storder laser beams; and the method comprises: collecting the 1storder laser beams; controlling the 1storder laser beams; and directing the 0thorder laser beams into a beam trap.

22. The method of claim 21, wherein controlling the 1storder beams comprise one of: causing the 1storder beams to polymerize the portion of the contained volume; preventing polymerization of voided portions of the contained volume; and preventing over-polymerization of the portion of the contained volume.

23. The method of claim 22, wherein causing the 1storder beams to polymerize comprises: switching the acousto-optic modulator into an ON state; and directing the 1storder beams into a beam expander.

24. The method of claim 23, wherein preventing polymerization comprises: switching the acousto-optic modulator into an OFF state; and directing 1storder beams into a beam trap.

25. The method of claim 24, comprising: receiving print data associated with a tissue model and a target average power of the laser beam in the bottom working plane and each successive working plane; andswitching the acousto-optic modulator into the ON state or the OFF state responsive to one or both of the target average power and the print data.

26. The method of claim 20, wherein controlling the average power output comprises: producing, with the acousto-optic modulator, a diffraction pattern that splits the laser beam into a 0thorder beam and a 1storder beam; modifying, with the acousto-optic modulator, an optical power of the 0thorder beam or the 1storder beam based on the target average power of the laser beam in the bottom working plane and each successive working plane; directing the 1storder beam into a first beam expander; and directing the 0thorder beam into the beam dump.

27. The method of claim 26, comprising expanding, with the first beam expander, the laser beam before the galvo scan head.

28. The method of claim 27, comprising expanding, with a second beam expander, the laser beam before the objective lens.

29. The method of claim 28, wherein: the second beam expander comprises a first lens and a second lens; and expanding the laser comprises directing the laser beam sequentially through the first lens and the second lens.

30. The method of claim 1, wherein the galvo scan head comprises a plurality of moveable mirrors and directing the laser beam comprises:directing, with the plurality of moveable mirrors, the laser beam into the objective lens at the different angles and times.

31. The method of claim 30, wherein directing the laser beam comprises directing, with the plurality of moveable mirrors, the laser beam into scanning optics located between the galvo scan head and the objective lens.

32. The method of claim 31, wherein the scanning optics comprise a scan lens and a tube lens.

33. The method of claim 1, wherein focusing the laser beam into the voxel comprises realizing a target average power of the laser beam at the voxel at each horizontal location.

34. The method of claim 1, comprising: moving a moveable power meter to a measurement location between the objective lens and the print surface; at the measurement location, measuring, with the moveable power meter, power level data for the laser beam after exiting the objective lens; outputting, with the moveable power meter, the power level data; and moving the moveable power meter away from the measurement location.

35. The method of claim 34, wherein moving the moveable power meter comprises causing an electric motor to rotate the moveable power meter into and out of the measurement location.

36. The method of claim 1, wherein locating the field of view of the objective lens in the bottom working plane and each successive working plane comprises moving the objective lens vertically while maintaining a vertical position of the print surface.

37. The method of claim 36, comprising moving the objective lens vertically in predetermined increments.

38. The method of claim 1, wherein moving the contained volume horizontally relative to the field of view comprises: moving the print bed in a first horizontal direction relative to the objective lens; and moving the print bed in a second horizontal direction relative to the objective lens, wherein the first direction and second directions are parallel to the bottom working plane and each successive working plane.

39. The method of claim 38, comprising:3D printing a first tile of the tissue model by completing the directing, focusing, and polymerizing steps in the field of view; moving the print bed in at least one of the first horizontal direction and the second horizontal direction until an edge of the field of view is aligned with an edge of the first tile of the tissue model; and after moving the print bed, 3D printing a second tile of the tissue model by completing the directing, focusing, and polymerizing steps in the field of view.

40. The method of claim 39, comprising stitching a portion of the edge of the first tile of the tissue model together with the second tile of the tissue model by completing the directing, focusing, and polymerizing steps at the edge of the field of view that is aligned with the edge of the first tile of the tissue model.

41. The method of claim 1, comprising:3D printing a first tile of the tissue model by causing the galvo scan head to move the voxel within a first portion of the contained volume in the field of view by directing the laser beam into the objective lens at the different angles and times.

42. The method of claim 41, comprising:3D printing additional portions of the tissue model by: moving each additional portion of the contained volume into the field of view; and causing the galvo scan head to move the voxel within each additional portion when the laser beam is directed into the objective lens at the different angles and times.

43. The method of claim 1, comprising: directing, with a light source, a light beam through the contained volume; focusing the light beam onto an image sensor of the camera; and generating, with the image sensor, a digital image of the tissue model44. The method of claim 43, comprising outputting, with the camera, the digital image to a device in data communication with the camera while 3D printing the tissue model.

45. The method of claim 43, wherein: the light source comprises an LED, a collector lens, and condenser lens; and the method comprises: generating, with the LED, the light beam; focusing, with collector lens, the light beam onto the condenser lens; anddirecting, with the condenser lens, the light beam through the contained volume and the photocrosslinkable bioink in a direction toward the camera.

46. The method of claim 43, wherein the print surface comprises an aperture and the method comprises: positioning the contained volume on the print surface over the aperture; and directing the light beam into the contained volume through the aperture.

47. The method of claim 46, comprising directing, with the condenser lens, a portion of the light beam through the aperture, through the contained volume, through the objective lens, and into the camera.

48. The method of claim 47, comprising passing the light beam through the objective lens in a first direction while directing the laser beam through the objective lens in a second direction that is generally opposite to the first direction.

49. The method of claim 48, comprising: reflecting, with an optical element, the laser beam from the galvo scan head and into the objective lens in the first direction; and transmitting, with the optical element, the focused beam through objective lens and into the camera in the second direction.

50. The method of claim 48, comprising: reflecting, with a hot mirror, first wavelengths of the laser beam from the galvo scan head and into the objective lens in the first direction; andtransmitting, with the hot mirror, second wavelengths of the focused beam from the objective lens and into the camera in the second direction.

51. An apparatus for 3D printing a tissue model in a contained volume of photocrosslinkable bioink containing living cells, the apparatus comprising: a print surface operable to maintain a cell viability temperature of the contained volume; an objective lens operable to focus a laser beam into a voxel located in a field of view of the objective lens; a vertical actuator operable to locate the field of view vertically in a bottom working plane within the contained volume and successive working planes located above the bottom working plane by moving the objective lens vertically relative to the print surface; a galvo scan head operable to move the voxel horizontally in the field of view by directing the laser beam toward different locations on the objective lens at different times; and a horizontal actuator operable to move the contained volume horizontally relative to the field of view, wherein, in the bottom working plane and each successive working plane, the galvo scan head causes: the objective lens, at each different location and time, to focus the laser beam into the voxel at a horizontal location in the field of view; and the voxel to polymerize a portion of the contained volume with two photon polymerization at each horizontal location; and the horizontal actuator moves the contained volume relative to the field of view.

52. The apparatus of claim 51, comprising mounting arms operable to removably secure the contained volume to the print surface.

53. The apparatus of claim 51, wherein the print surface comprises an aperture and the mounting arms are operable to removably secure the contained volume to the print surface above the aperture.

54. The apparatus of claim 53, wherein: the contained volume of the photocrosslinkable bioink is located in a transparent print container; the mounting arms are operable to removably secure the transparent print container to the print surface above the aperture; and the aperture is aligned with a bottom surface of the transparent printer container so that a light beam is transilluminable through the aperture, the transparent print container, and the photocrosslinkable bioink.

55. The apparatus of claim 51, comprising a temperature control system operable to: transfer heat away from the print surface; or transfer heat to the print surface.

56. The apparatus of claim 55, wherein the temperature control system comprises a thermoelectric cooler that is thermally coupled to a bottom portion of the print surface.

57. The apparatus of claim 56, comprising a base plate, wherein: the print surface is thermally isolated from the base plate; and thermoelectric cooler is thermally coupled to the base plate and operable to: transfer heat away from the thermoelectric cooler with the base plate; or transfer heat to the thermoelectric cooler with the base plate.

58. The apparatus of claim 57, comprising a heat exchanger that is thermally coupled to the base plate and operable to receive a heat transfer fluid circulated therethrough.

59. The apparatus of claim 51, comprising: a controller in data communication with the laser generator and the galvo scan head; the galvo scan head comprising moveable mirrors operable with the controller to direct the laser beam into the objective lens at the different angles at the different times; and the apparatus comprising: one or more mirrors operable to direct the laser beam from the laser generator and into the galvo scan head at the different angles at the different times; and a hot mirror operable to direct the laser beam from the galvo scan head and into the objective lens at each different angle and time.

60. The apparatus of claim 59, wherein the laser beam comprises a high intensity pulsed laser beam.

61. The apparatus of claim 60, wherein the laser generator comprises: a femtosecond laser; or a Ti: Sapphire laser.

62. The apparatus of claim 51, wherein the laser generator is operable to apply a group delay dispersion to the laser beam.

63. The apparatus of claim 53, wherein the laser generator is operable to apply the group delay dispersion by applying opposite and equal dispersions to the laser beam.

64. The apparatus of claim 51, wherein the laser generator comprises an integrated dispersion compensation unit.

65. The apparatus of claim 51, wherein the laser generator comprises an internal power meter operable to measure a power level of the laser beam before it reaches the galvo scan head.

66. The apparatus of claim 65, wherein the internal power meter is operable to output data associated with the power level of the laser beam to the controller while 3D printing the tissue model.

67. The apparatus of claim 51, comprising: an external power meter; and a splitter operable to divert a portion of the laser beam into an external power meter; the external power meter being operable to: measure a power of the laser beam before the galvo scan head; and output data associated with the power level of the laser beam to the controller while 3D printing the tissue model.

68. The apparatus of claim 51, comprising: an isolator operable to prevent back reflections of the laser beam; and a modulator operable to modify the laser beam before the galvo scan head.

69. The apparatus of claim 68, wherein the modulator comprises an acousto-optic modulator operable to diffract or shift a frequency of the laser beam using sound waves.

70. The apparatus of claim 68, wherein the modulator comprises an acousto-optic modulator operable to: diffract the laser beam into multiple orders; and control an average power output of the laser beam.

71. The apparatus of claim 70, wherein: the multiple orders include 0thorder laser beams and 1storder laser beams; and the acousto-optic modulator is operable to: collect the 1storder laser beams; control the 1storder laser beams; and direct the 0thorder laser beams into a beam trap.

72. The apparatus of claim 71, comprising a controller operable to: cause the 1storder beams to polymerize the portion of the contained volume; prevent polymerization of voided portions of the contained volume; and prevent over-polymerization of the portion of the contained volume.

73. The apparatus of claim 72, wherein the controller is operable to cause the 1storder beams to polymerize by switching the acousto-optic modulator into an ON state that directs the 1storder beams into a beam expander.

74. The apparatus of claim 73, wherein the controller is operable to prevent polymerization by switching the acousto-optic modulator into an OFF state that directs the laser beam into a beam trap.

75. The apparatus of claim 73, wherein the controller is operable to: receive print data associated with a tissue model and a target average power of the laser beam in the bottom working plane and each successive working plane; and switch the acousto-optic modulator into the ON state or the OFF state responsive to one or both of the target average power and the print data.

76. The apparatus of claim 70, wherein the acousto-optic modulator is operable to: produce a diffraction pattern that splits the laser beam into a 0thorder beam and a 1storder beam; modify an optical power of the 0thorder beam or the 1storder beam based on the target average power of the laser beam in the bottom working plane and each successive working plane; direct the 1storder beam into a beam expander; and direct the 0thorder beam into the beam dump.

77. The apparatus of claim 76, wherein the beam expander is operable to expand the laser beam before the galvo scan head.

78. The apparatus of claim 77, comprising a second beam expander operable to expand the laser beam before the objective lens.

79. The apparatus of claim 78, wherein: the second beam expander comprises a first lens and a second lens; and the laser beam is directed sequentially through the first lens and the second lens.

80. The apparatus of claim 51, wherein the galvo scan head comprises a plurality of moveable mirrors operable to direct the laser beam into the objective lens at the different angles.

81. The apparatus of claim 80, comprising scanning optics located between the galvo scan head and the objective lens, wherein the plurality of moveable mirrors are operable to direct the laser beam through the scanning optics and into the objective lens.

82. The apparatus of claim 81, wherein the scanning optics comprise a scan lens and a tube lens.

83. The apparatus of claim 51, wherein comprising a controller is operable with the laser generator to realize a target average power of the laser beam at the voxel at each horizontal location.

84. The apparatus of claim 83, comprising a moveable power meter that, before or during the 3D printing of the tissue model, is: movable to a measurement location between the objective lens and the print surface; operable at the measurement location to measure a power level of the laser beam after exiting the objective lens and output data associated with the power level of the laser beam to the controller; and after outputting the data, moveable away from the measurement location.

85. The apparatus of claim 84, comprising an electric motor, wherein the controller is operable to cause the electric motor to rotate the power meter into and out of the location between the objective lens and the print surface.

86. The apparatus of claim 51, wherein the horizontal actuator is operable to maintain a vertical position of the print surface while the vertical actuator moves the objective lens vertically to locate the field of view of the objective lens in the bottom working plane and each successive working plane.

87. The apparatus of claim 86, wherein the vertical actuator is operable to move the objective lens vertically in predetermined increments.

88. The apparatus of claim 1, wherein the horizontal actuator is operable to: move the print bed in a first horizontal direction relative to the objective lens; and move the print bed in a second horizontal direction relative to the objective lens, wherein the first direction and second directions are parallel to the bottom working plane and each successive working plane.

89. The apparatus of claim 88, wherein the controller is operable to:3D print a first tile of the tissue model in the field of view by communicating with one or more of the laser generator, the print surface, the vertical actuator, and the galvo scan head; cause the horizontal actuator to move the print bed in at least one of the first horizontal direction and the second horizontal direction until an edge of the field of view is aligned with an edge of the first tile of the tissue model; and after moving the print bed, 3D printing a second tile of the tissue model by further communicating with one or more of the laser generator, the print surface, the vertical actuator, and the galvo scan head.

90. The apparatus of claim 89, wherein:the horizontal actuator comprises a first linear actuator stacked on top of a second linear actuator; and the controller is operable to cause the first linear actuator to move the print bed in the first horizontal direction and the second linear actuator to move the print bed in the second horizontal direction.

91. The apparatus of claim 51, wherein the galvo scan head is operable to 3D print a first tile of the tissue model by moving the voxel within a first portion of the contained volume in the field of view when the laser beam is directed into the objective lens at the different angles and times.

92. The apparatus of claim 91, wherein: the horizontal actuator is operable to move additional portions of the contained volume into the field of view; and the galvo scan head is operable to 3D print additional portions of the tissue model by moving the voxel within each additional portion of the contained volume in the field of view when the laser beam is directed into the objective lens at the different angles and times.

93. The apparatus of claim 51, comprising: a camera comprising an image sensor; and a light source operable to direct a light beam through the contained volume and focus the light beam onto the image sensor of the camera, wherein the image sensor is operable to generate a digital image of the tissue model.

94. The apparatus of claim 93, wherein the camera is operable to output the digital image to a display while the apparatus is 3D printing the tissue model.

95. The apparatus of claim 93, wherein the light source comprises: an LED operable to generate the light beam; a condenser lens; and a collector lens operable to focus the light beam onto the condenser lens, the condenser lens being operable to direct the light beam through the contained volume and the photocrosslinkable bioink in a direction toward the camera.

96. The apparatus of claim 95, wherein: the print surface comprises an aperture; the contained volume is positioned above the aperture; and the light beam is directed through the aperture.

97. The apparatus of claim 96, wherein the condenser lens is operable to direct a portion of the light beam through the aperture, through the contained volume, through the objective lens, and into the camera.

98. The apparatus of claim 97, wherein the light beam passes through the objective lens in a first direction and the laser beam passes through the objective lens in a second direction that is generally opposite to the first direction.

99. The apparatus of claim 98, comprising an optical element operable to: reflect the laser beam from the galvo scan head and into the objective lens in the first direction; and transmit the focused beam through objective lens and into the camera in the second direction.

100. The apparatus of claim 98, comprising a hot mirror operable to: reflect first wavelengths of the laser beam from the galvo scan head and into the objective lens in the first direction; and transmit second wavelengths of the focused beam from the objective lens and into the camera in the second direction.