WO2022177496A1

WO2022177496A1 - Bioprinting workflow systems and methods

Info

Publication number: WO2022177496A1
Application number: PCT/SE2022/050172
Authority: WO
Inventors: Erik Gatenholm; Hector Martinez
Original assignee: Bico Group Ab; Cellink Bioprinting Ab
Priority date: 2021-02-17
Filing date: 2022-02-17
Publication date: 2022-08-25

Abstract

A modular approach to automating the bioprinting workflow, including any one or more of bioprinting, quality control monitoring, liquid dispensing such as bioink or cell media dispensing, incubating bioprinted constructs, and performing one or more assays on a bioprinted construct. According to this modular approach, limited user intervention or instruction is needed to prepare bioprinted constructs and perform testing (e.g. cosmetics, skin care products, drugs, etc.), transplantation, and/or high throughput drug discovery, screening, and/or toxicity testing. Due to the modular nature of this approach, multiple bioprinting and testing workflows can be performed by re-arranging, substituting, replacing, removing or adding one or more or multiple or duplicative processes/functionalities to a customizable workflow-based system of components.

Description

BIOPRINTING WORKFLOW SYSTEMS AND METHODS

Field of the Invention

The present invention relates to the field of bioprinters, and more particularly, to technology that provides a modular approach to automating the bioprinting workflow, including any one or more of bioprinting, quality control monitoring, cell media dispensing, incubation of printed constructs, assaying the constructs, etc.

BACKGROUND

Apparatuses, systems and methods for automation of dispensing, printing and incubation of cellular material are known from the prior art.

However, there is a need for improved apparatuses, systems and methods allowing for even higher capacity, thereby meeting current and future demands for provision of bioprinted and dispensed material, such as tissue models.

In the field of bioprinting, manufacturing cell structures or scaffolding are traditionally produced in single standalone devices, wherein one device may for instance handle 3D printing of scaffolding, a second device may add layers of live cells on the previously printed scaffold, and a third device may handle crosslinking (solidifying) within the construct. This requires manual handling of sample holders, such as well plates or petri dishes, where the cell or scaffold structure is placed during the 3D printing process. When handling sample holders there is a risk that the cell or scaffold structures may be damaged or shifted due to shear stress, temperature change or mechanical handling that alters position or composition of the samples. Manually handling living cells, such as human or mammal cells, also confers a risk of the material being contaminated or otherwise affected by environmental conditions. The inventors have thus identified a need for a system and method that overcomes these deficiencies.

SUMMARY OF THE INVENTION

It is an object of the present disclosure to provide a system and a method that reduces the risk of mechanical stresses or environmental effects on a construct being manufactured.

It is another object of the present disclosure to provide improved systems and methods, thereby providing the user with improved flexibility and improved means for controlling the workflow. Furthermore, it is an object of the disclosure to provide systems and methods to allow for mass production of bioprmted, cellular models, such as tissue models, thereby necessitating a system equipped with necessary devices and organized in such way as to allow high throughput.

Moreover, it is an object of the present disclosure to mitigate, alleviate or eliminate one or more of the above-identified challenges of the prior art.

Embodiments of the present invention provide systems and methods related to bioprinting workflows. Exemplary aspects of the embodiments are described in more detail below, however, these examples are merely examples and variations of such are also within the scope of the invention.

According to a first aspect, a customizable bioprinting workflow system is disclosed, the system comprising at least two of: one or more 3D bioprinter; and/or one or more scanner; and/or one or more liquid dispenser; and/or one or more cell incubator; wherein the bioprinter, scanner, liquid dispenser and/or incubator are arranged in operable communication with one another, optionally as one or more modules. The system further comprises a transfer unit in operable communication with the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them, which transfer unit is configured to move one or more structure from one to another of the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them.

Bioprinted constructs printed in the system can be used as models for testing (e.g. cosmetics, skin care products, drugs, etc.), transplantation, and/or high throughput drug discovery, screening, and/or toxicity testing.

By arranging modules or stations for all of, or at least some of, the necessary tasks in a process, from bioprinting a first layer of tissue, monitoring the construct, scanning the product, adding single cells, adding cell media, washing and incubating and live imaging of the incubation process, and allowing the user to choose which steps to perform for a sample holder transported through the system, a great flexibility is provided, thereby supporting scaled production as well as the research and development work related to cell printing, dispensing and culturing.

According to this modular approach, limited user intervention or instruction is needed to prepare bioprinted constructs and perform testing (e.g. cosmetics, skin care products, drugs, etc.), transplantation, and/or high throughput drug discovery, screening, and/or toxicity testing. Due to the modular nature of this approach, multiple bioprinting and testing workflows can be performed by re-arranging, substituting, replacing, removing or adding one or more or multiple or duplicative processes/functionalities to a customizable workflow-based system of components.

In some aspects, the transfer unit is configured to move a sample holder from one to another of the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them, wherein the sample holder is adapted to support and hold said one or more structure.

In some aspects, the 3D bioprinter comprises means for dispensing cell(s) and/or matrix material into at least one sample holder.

In some aspects, the system comprises a single-cell dispenser.

In some aspects, a system comprises a detection or monitoring system, such as for detecting and/or monitoring at the cellular level, such as one or more imaging modality.

In some aspects, the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof, are configured to be arranged within one or more clean chamber.

In some aspects, the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof are configured to be arranged in any order relative to one another.

In some aspects, the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof are configured to be re-arranged relative to one another, such as after initial assembly of the system.

In some aspects, one or more additional bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof can be added to the system, such as after initial assembly of the system.

In some aspects, the system is configured to accept one or more of additional bioprinters, scanners, liquid dispensers, incubators, and/or detection or monitoring systems, or the module(s) thereof, such as after an initial assembly of the system.

In some aspects, the transfer unit comprises a conveyor belt or comprises an intelligent transfer unit, or comprises an intelligent transfer unit with a conveyor belt.

In some aspects, a system further comprises one or more robotic arms, which can be part of the transfer unit or additional, which robotic arms are configured to be capable of transferring one or more of the structures, such as one or more bioprinted structures or wellplates and/or slides, within one or more of the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof and/or between one or more of the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof and the transfer unit, and/or between two or more of the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof.

In some aspects, the transfer unit is configured to transport sample holders such as well plates, microplates, slides, bioprinted structures, and/or petri dishes.

In some aspects, the transfer unit and/or bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof are configured to be capable of maintaining an air temperature between 4 °C and 60 °C.

In some aspects, one or more of the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof is configured to be maintained at a first temperature, while one or more of a different bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof is configured to be maintained at a second temperature, such as for maintaining a scanner at about 22 °C and maintaining an incubator at about 37 °C.

In some aspects, the scanner is configured to be capable of generating a 3D point cloud in a time period on the order of seconds.

In some aspects, the scanner is configured to be capable of evaluating one or more features of a bioprinted structure in the xy-plane.

In some aspects, the scanner is configured to be capable of evaluating one or more features of a bioprinted structure in the zx-plane and/or zy-plane.

In some aspects, the scanner is configured to be capable of measuring step height, positioning, distance, angle, diameter, radius, flatness, roughness, and/or contour of one or more structural features of the bioprinted structure.

In some aspects, the scanner is configured to be capable of scanning materials comprising cells, tissue, polymers, plastics, metal, and/or glass.

In some aspects, the scanner is configured to be capable of scanning surfaces that are matte, glossy, transparent, mirror, and/or opaque.

In some aspects, the scanner is configured to be capable of contour comparison.

In some aspects, the scanner is configured to be capable of sampling surface topography and intensity at a sub-micron level. In some aspects, the scanner is configured to be capable of performing quality inspection of the bioprinted structures based on one or more parameters, such as parameters set by a user and/or provided by computer-readable instructions.

In some aspects, the scanner is configured to be capable of spotting visual defects including, spots, scratches, voids, air bubbles, and/or impurities in the printed structure.

In some aspects, the scanner is configured to be capable of movement along more than one axis.

In some aspects, the system further comprises an outfeed configured to be capable of discarding rejected bioprinted structures.

In some aspects, the system further comprises a feeder configured to be capable of feeding sample holders such as well plates, microplates, slides, and/or petri dishes into the bioprinter, wherein the feeder is configured to be operably connected with the transfer unit, transfer unit, and/or robotic arms.

In some aspects, the system further comprises a liquid dispenser configured to be capable of dispensing cell culture media.

In some aspects, the liquid dispenser comprises multiple cell culture media storage tanks.

In some aspects, the system further comprises wash and/or waste tanks to wash and/or rinse the liquid dispenser.

In some aspects, the module is configured to be capable of providing detection or monitoring at a cellular level is a microscope.

In some aspects, the transfer unit is configured to be capable of forward and/or backward and/or up and/or down movement along one or more or each of an x-, y-, and/or z-axis.

In some aspects, the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them, are assembled horizontally and/or vertically relative to another.

In some aspects, one or more of the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them, are assembled horizontally relative to at least one other of the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them, and vertically to at least one other of the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them.

In some aspects, one or more of the bioprmter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof has a scanner configured to be capable of identifying each sample holder, well plate, microplate, slide, and/or petri dish as it enters and/or exits the one or more bioprmter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof.

In some aspects, the identifying is performed by scanning a bar code, QR code, or sequence of numbers and/or letters.

In some aspects, a method is disclosed comprising: depositing bioink onto a printing surface to prepare one or more bioprmted structure; scanning the bioprinted structure to obtain one or more measurement; comparing the one or more measurement against a model; and repeating one or more of the depositing, scanning, and/or comparing until one or more final bioprinted structure is produced; wherein the bioprinted structure is transported to and/or from a bioprinter or bioprinter module to a scanner or scanner module using a transfer unit in operable communication with the bioprinter or bioprinter module and the scanner or scanner module.

In some aspects, a method further comprises: dispensing media with a liquid dispenser or liquid dispenser module, where in the dispensing is performed on, around or near the bioprinted structure, such as on the bioprinted structure and/or portion thereof and/or on the printing surface and/or a portion thereof; incubating the bioprinted structure and media with an incubator or incubator module; optionally repeating the dispensing and/or incubating until a desired cell growth or tissue maturation is reached; wherein the repeating of the dispensing and/or incubating is facilitated by the transfer unit, which is in further operable communication with the liquid dispenser or liquid dispenser module and the incubator or incubator module to provide transport therebetween.

In some aspects, a method further comprises: removing media from the printing surface and/or the bioprinted structure, and/or washing the printing surface and/or the bioprinted structure; and dispensing new media onto the printing surface and/or the bioprinted structure; such as by transporting the printing surface and/or the bioprinted structure to and/or from a washing function and/or washing module by way of the transfer unit, which is in further operable communication with the liquid dispenser or liquid dispenser module and the washing function/module.

In some aspects, the printing surface is in or on a sample holder such as a well plate, microplate, slide, or petri dish.

In some aspects, the scanning is performed by one or more 3D scanner, camera, or other imaging module. In some aspects, a method further comprises: transporting the bioprinted structure to the scanner or scanner module and/or to a detection or monitoring system using the transfer unit, which is in further operable communication with the detection or monitoring system; and detecting and/or monitoring one or more feature of the bioprinted structure using the scanner or scanner module and/or the detection of monitoring system.

In some aspects, the transporting is performed using the transfer unit which comprises an intelligent transfer unit, which is configured to be programmed by a user to perform a desired workflow and/or which is configured to follow a set of computer-readable instructions.

In some aspects, the transfer unit comprises one or more conveyor belt and/or one or more robotic arm.

Further, a method of bioprinting is disclosed using the system of any aspects disclosed herein, such as a method comprising depositing a bioink using the system.

In some aspects, a method is disclosed comprising: bioprinting one or more structure with a bioprinter/bioprinter module; transporting the structure to a scanner/scanner module by way of a transfer unit which enables workflow between the bioprinter/bioprinter module and the scanner/scanner module; scanning the structure to determine if there is compliance with one or more parameter(s) and/or model.

In some aspects, a method further comprises: transporting the structure to the vicinity of a liquid dispenser/liquid dispenser module by way of the transfer unit, which further enables workflow between the bioprinter/bioprinter module, the scanner/scanner module, and/or the liquid dispenser/liquid dispenser module; and dispensing cell media using the liquid dispenser/liquid dispenser module.

In some aspects, a method further comprises: transporting the structure to an incubator/incubator module by way of the transfer unit, which further enables workflow between the bioprinter/bioprinter module, the scanner/scanner module, the liquid dispenser/liquid dispenser module, and/or the incubator/incubator module; and incubating the structure.

In some aspects, a method further comprises: assaying the structure, such as before, during or after the bioprinting, scanning, dispensing, transporting, and/or incubating.

In some aspects, the bioprinting, scanning, dispensing, transporting, incubating and/or assaying are performed in any order relative to one another. In some aspects, a system as disclosed herein is configured to operate one or more methods as disclosed herein in whole or in part, or combinations thereof.

In a further aspect, the use of a system as described herein, is disclosed for performing one or more workflow function chosen from: 1) bioprmting one or more bioprinted structure; and/or 2) scanning the bioprinted structure and comparing with specifications and/or a model;

3) dispensing cell media on, around, and/or near the bioprinted structure; and/or 4) incubating the bioprinted structure.

The present disclosure will become apparent from the detailed description given below. The detailed description and specific examples disclose preferred embodiments of the disclosure by way of illustration only. Those skilled in the art understand from guidance in the detailed description that changes and modifications may be made within the scope of the disclosure.

Hence, it is to be understood that the herein disclosed disclosure is not limited to the particular component parts of the device described or steps of the methods described since such device and method may vary. It is also to be understood that the terminology used herein is for purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that, as used in the specification and the appended claim, the articles "a", "an", "the", and "said" are intended to mean that there are one or more of the elements unless the context explicitly dictates otherwise. Thus, for example, reference to "a unit" or "the unit" may include several devices, and the like. Furthermore, the words "comprising", "including", "containing" and similar wordings does not exclude other elements or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate certain aspects of embodiments of the present invention, and should not be used to limit the invention. Together with the written description, the drawings serve to explain certain principles of the invention.

FIGS. 1 A-B are illustrations showing example bioprinting workflow systems according to embodiments of the invention.

FIG. 2 is an illustration showing modules/components of a bioprinting workflow system according to embodiments of the invention. FIG. 3 is an illustration showing some modules/components of a bioprinting workflow system are directly connected and some components/modules are separated from the rest of the system according to embodiments of the invention.

FIG. 4 is an illustration showing components of a transfer unit according to embodiments of the invention.

FIG. 5 is an illustration showing components of a transfer unit according to embodiments of the invention.

FIG. 6 is an illustration showing components of a transfer unit according to embodiments of the invention.

FIG. 7 is an illustration showing a platform component of a transfer unit in which printing surfaces are manually slid across the platform.

FIG. 8 is an illustration showing the transfer unit platform contains customizable panels according to an embodiment of the invention.

FIGS. 9A-C are illustrations of the transfer unit assembly present on each module including an individual assembly (9A), two assemblies operably connected (9B), and the assembly installation for a module/component (9C).

FIGS. 10A-B are illustrations showing the moveable module/component coverings according to embodiments of the invention.

FIGS. 11A-B are illustrations showing infeed systems according to embodiments of the invention: bulk infeed (11 A) and well plate infeed (1 IB).

FIG. 12 is an illustration of a system component/module showing features according to embodiments of the invention.

FIG. 13 is an illustration showing a bioprinter component/module according to embodiments of the invention.

FIG. 14 is an illustration showing a scanner component/module according to embodiments of the invention.

DETAILED DESCRIPTION

Reference will now be made in detail to various exemplary embodiments of the invention. It is to be understood that the following discussion of exemplary embodiments is not intended as a limitation on the invention. Rather, the following discussion is provided to give the reader a more detailed understanding of certain aspects and features of the invention.

Any one or more methods in whole or part can be incorporated in any one or more of the systems to provide a desired workflow function, and any one or more of the systems can be used to perform any one or more of the methods in whole or in part. It is noted in particular that where a range of values is provided in this specification, each value between the upper and lower limits of that range is also specifically disclosed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range as well. Where temperatures are given in this specification, the values are understood to be the indicated temperature ±1 °C. For example, where a temperature of 4 °C is given, it is understood to mean any temperature between 3 °C and 5 °C inclusive. The term “about” is defined in this specification in the context of quantitative measurements to mean the indicated amount ±10%. For example, with a ±10% range, “about 1 mL” can mean 0.9-1.1 mL.

The Workflow System

The bioprinting workflow system and methods described herein all employ a modular approach, and the term “module” may be interpreted as any of the different units described, such as a 3D bioprmter, scanner, liquid dispenser, cell incubator, single-cell dispenser etc. In some aspects different units may be combined in a single module. Vice versa, any of the different units described may be interpreted as a module.

The modular approach thus includes two or more stations or unit, where different tasks are performed, so that a sample in a sample holder, such as a well plate, that accesses the modular workflow system will be exposed to different activities at each station, such as bioprinting a tissue (with or without cells), dispensing single cells or a plurality of cells, addition of cell media, incubation and live-cell imaging and optionally one or more washing steps. Typically, the sample holder is transported between each station (module) by means of a conveyor belt, a robotic means or the like. One or more than one sample holder can be transported simultaneously in the modular workflow, i.e. each module can process more than one substrate at a time, either simultaneously or multiple robotic arms working on the same substrate.

Embodiments of the present invention include a bioprinting workflow system configured to fully and/or partially automate the bioprinting workflow including, but not limited to, bioprinting, confirming a structure of a bioprinted object/structure, dispensing single cells into or onto a printed construct, addition/removal of cell media, incubation, monitoring of cell growth and/or tissue maturation. In embodiments, the inventive system comprises one or more of integrated photocuring toolheads, bulk liquid dispensers, and one or more positioning arm(s) which allow a user to bioprint constructs, crosslink, wash, and/or add media without user intervention or minimal user involvement. Nanoliter and picoliter dispensing capabilities allow the fabrication of constructs followed by dosing with growth factors, small molecules, and other therapies in preparation for characterization.

Advantages of using a modular customizable bioprinting workflow system as described herein include efficiency of a bioprinting process and less risk of contamination of a bioprinted product. As a sample or construct is kept in a single sample holder throughout the process, and a transfer unit eliminates the need for a user to manually move the sample holder between different modules, there is less risk of contaminating the construct during the process. This is especially important for bioprinting which uses biological cells and other material that is sensitive to contamination, temperature changes, varying humidity and other environmental variables. Furthermore, not having to move the construct between different sample holders also minimizes the risk of loss of material and increases accuracy of the different steps, as the constmct remains arranged in a defined position on the sample holder.

Embodiments of the invention comprise a variety of modules and/or components and/or workstations that are configured to be connected to complete the workflow (FIGS. 1 A-B). The modules, components, and/or workstations can include: one or more bioprinter, one or more scanner, such as a camera or other imaging modality configured to collect 2D and/or 3D images, one or more liquid dispenser, and/or one or more cell incubator (FIG. 2) In embodiments, the modules/components/workstations are connected. In other embodiments, some of the modules, components, and/or workstations are directly connected while others are not. For example, in an embodiment, the bioprmter, scanner, and liquid dispenser (or bulk dispenser) are connected, while the incubator is separated from the system (FIG. 3). In embodiments, whether or not the individual modules/components are connected, a workflow from one module/component to another or back again is enabled by a transport unit and/or an intelligent transport system to move the subject of the workflow, such as a bioprinted construct, from one module/component to the next in a manner that minimizes mvolvement/interaction from a user. In embodiments, the modules/components are configured to be arranged horizontally, vertically, or both relative to other modules/components within the system. In embodiments, the system is fully customizable. For example, the modules, components, and/or workstations are configured to be arranged in any order relative to one another, and are configured to be re-arranged relative to one another. In some aspects, the modules can be arranged along a longitudinal substrate direction with mutual distances therebetween. In this case, spacers between the modules can be used for arranging additional processing stations and/or monitoring devices. Alternatively, the modules can be arranged directly adjacent to each other. With this variant, advantages in terms of compact apparatus structure are obtained. Additional modules, components, and/or workstations may be added (i.e. operably connected), such as after initial assembly of the system, or as needed to add tasks to the workflow. Modules/components/workstations within the system may also be removed, such as after initial assembly, or as needed to remove tasks from the workflow. The modules/components/workstations may use the same or different power sources. A workflow and system configured to perform a desired workflow can employ multiple systems, such as transferring the subject of the workflow from one station comprising a first set of modules/components to another station comprising a second set of modules/components. For example, a first station can comprise multiple bioprinters, while a second station comprises multiple liquid dispensers, while a third station comprises multiple incubators, and the subject of the workflow, such as a bioprinted construct can be transported from one station to another and back and/or forth between one or more of the stations to perform a desired workflow. The transporting is performed by a transfer unit, such as an intelligent transfer unit configured for operable assembly and/or connection with the modules/components/stations.

In embodiments the modules and/or components and/or workstations comprise housings that are universal in shape and/or size and/or are otherwise compatible with other modules/components/workstations. In other embodiments the housings are of different heights, widths, and/or shapes, but the modules/components/workstations remain compatible with one another. In embodiments, the system comprises 2 or more modules/components/workstations, such as 3, 4, 5, 6, 7, 8, 9, or 10 modules/components/workstations.

In embodiments, the systems further comprise a transfer unit to move printing surfaces/structures from one module or component/workstation to the next (FIG. 4). In embodiments, the transfer unit is configured to be capable of being programmed by a user to perform the desired workflow and/or can be configured to run computer-executable instructions. The transfer unit is configured to be capable of forward and backward and/or up and down movement along one or more or each of an x, y, and z-axis. In some embodiments, the transfer unit comprises one or more conveyor 40, such as a belt conveyor, pallet conveyor, accumulation conveyor, magnetic conveyor, vibrating conveyor, pneumatic/vacuum conveyor, walking beam conveyor, one or more robotic positioning arm 42 (e.g. cartesian, six-axis, and/or SCARA robots), or combinations thereof.

Advantageously, a conveyor belt has a longitudinal shape, thus facilitating the movement along a longitudinal substrate direction. The at least one conveyor belt may comprise an endless conveyor belt circulating along the path of the movable means for transportation. In this case, the conveyor device preferably comprises a motor with a roller driving the conveyor belt. For collecting the sample holder(s), it is separated from the conveyor belt, which is returned for further use to the modules for further use. The conveyor belt can be made from e. g. a continuous, integral band material or chain elements (plastic chain link conveyor). Alternatively or additionally, the at least one conveyor belt may comprise a flexible strip-shaped substrate sheet, which is moved on a roller bed along a longitudinal substrate direction.

According to a further preferred embodiment of the invention, the conveyor device is adapted for a continuous operation. The term "continuous operation" of the conveyor device covers both of a non-interrupted, continuous movement and alternatively a continued, but step-wise movement of the carrier substrate past the deposition modules, thus allowing an increasing production speed. The modules can be operated continuously as well, so that the tissue model is continuously manufactured

If, according to a further preferred aspect, the means for transportation comprises multiple conveyor belts being connected via at least one intersection, wherein each of the conveyor belts provides a carrier substrate branch and the modules are arranged along the carrier substrate branches, further advantages in terms of increasing production speed and additional degrees of freedom in adjusting the tissue model composition are obtained. As an example, an initial conveyor belt can be provided for depositing a basis layer of a tissue model. Subsequently, the initial conveyor belt can be split into multiple branches wherein in each branch further food layers are deposited with different another tissue model composition. The skilled person would be aware of robotic arms to be used, and, for example, such robotic arms can be found on www.gmobs.com. The skilled person would also be aware of suitable incubators and live cell imaging systems to be used, and, for example, such incubators and live cell imaging systems can be found on www.cellink.com.

In some embodiments the transfer unit comprises a platform 50 connecting one or more of the modules/components (FIGS. 5-6). In embodiments, carriers/holders 54 are configured to transport the printing surfaces 60 between modules/components. In embodiments the platform is substantially glass, plastic, or metal and/or the carriers/holders 54 are magnetic. The carriers/holders 54 and/or printing surfaces 60 can be transported in and out of the modules/components using robotic arms/positioners which can optionally be fitted with magnets. In other embodiments the transfer unit comprises a platform 50 for transporting holders and/or print surfaces in which the holders 54 and/or print surfaces 60 are manually slid across the platform 50 by one or more arms 72 attached on/near the platform (FIG. 7).

In embodiments, the platform is customizable and multiple methods of transport can be implemented. In embodiments, the platform is customized through addition of different types of panels 80 (FIG. 8). For example, part of the platform can comprise a belt conveyor, then the platform switches to a glass surface where magnetic holders are moved across the platform using magnets below the surface.

In embodiments, modules/components/workstations are designed to slide into a transfer unit platform assembly (FIG. 9A-C). Individual platform assemblies (FIG. 9A) are configured to be combined (FIG. 9B) to connect multiple modules/components.

The transfer unit is configured to be capable of transferring bioprinted structures within one or more of the modules/components and/or between one or more modules/components and the transfer unit, and/or between two modules/components. In embodiments, the robotic positioning arm is included for plate/lid positioning and to provide the function of adding, removing, or exchanging one or more objects, such as lids and/or well plates (or any substrate on which material is bioprinted, such as slides) and for positioning them within the workflows. The robotic positioning arm is configured to be capable of gripping, releasing, pushing, pulling, lifting, positioning and/or placing one or more of the same or different of such objects. The robotic positioning arm 42 can include grippers 62, such as pneumatic grippers, electric grippers, and/or under pressure grippers (FIG. 6). The conveyor is configured to be capable of providing at least bidirectional movement and/or can include lifts.

The transfer unit is configured to transport a variety of printing surfaces. In embodiments the transfer unit is configured to transport well plates, microplates, slides, and/or petri dishes, as well as custom-sized printing surfaces. The custom-sized printing surfaces can beany shape, such as square, rectangular, trapezoidal, triangular, circular, and/or irregularly shaped.

In embodiments of the invention, one or more of the modules/components are housed in one or more clean chamber(s) which provide an essentially airtight enclosure under positive pressure with filtered air. Non-limiting examples of clean chambers can be found in International Patent Application Publication No. WO/2017/040675, which is hereby incorporated herein by reference in its entirety. In embodiments, individual modules/components can be housed in separate clean chambers or together in one clean chamber. In other embodiments, two or more modules/components are present in one clean chamber and one or more modules/components are present in a separate clean chamber. In embodiments, some modules/components are present within clean chamber(s) and some are present outside clean chamber(s). In embodiments, the module/component housing comprises the clean chamber. In embodiments, the separate clean chambers are connected to the transfer unit and slots are provided to move samples from one module/component to the next while maintaining positive air pressure in each module/component. In embodiments, a clean chamber provides an essentially airtight enclosure for at least a sample holder, wherein the airtight enclosure is configured to provide positive pressure inside the clean chamber, and filtered air is provided inside the airtight enclosure.

In embodiments, the clean chamber technology is disposed in the housing of the system/module/component. In embodiments, unfiltered air enters the clean chamber system from the top, side, back, bottom, and/or front of the system housing. The unfiltered air is directed, using a fan or blower, through a high efficiency filter, such as a HEPA filter, and into the interior of the system/module/component and exits through one or more vents. The one or more vents are disposed in the housing in a manner to provide a clean flow of air for a work surface.

In embodiments, the modules/components can have moveable coverings (FIG. 10 A). In some embodiments, some modules/components have moveable coverings and others do not (FIG. 10B). In embodiments, these moveable coverings may provide a substantially airtight seal and/or are otherwise configured as appropriate for a clean chamber. In embodiments of the invention, the system includes machine vision guided infeed of printing surfaces in or on sample holders such as well plates, microplates, slides, and/or petri dishes into the bioprinter and/or onto/from the transfer unit. The system may also include one or more bulk feeder (FIG. 11A), reel feeder, flexible feeder, well plate feeder (FIG. 11B), or customized feeder, each of which is configured to be capable of feeding one or more or multiple well plates, microplates, slides, and/or petri dishes into the bioprinter and/or other modules/components.

In embodiments of the invention, the system is temperature controlled and able to maintain a temperature, within ±1 °C, between -20 °C and 120 °C, such as 0 °C, 4 °C, 10 °C, 15 °C, 20 °C, 25 °C, 28 °C, 30 °C, 32 °C, 35°C, 37 °C, 39 °C, 45 °C, 50 °C, 75 °C, 100 °C, or 115 °C. Temperature of the system can be maintained the same throughout, or can be different in each module/component. For example, the cell incubator can be maintained at 37 °C while the bioprinter is maintained at 4 °C.

The system can also include one or more environmental controls such as particle filters, sterile filters, humidity control, and/or dew point control. In some embodiments, the system comprises software to report summaries and graphs showing environmental parameters over a selected time period.

In some aspects, the system may comprise a control unit, controlling the operation of the system. The control unit is connected to a user interface, which may be a PC, a touchscreen, a tablet or smartphone or the like, so that a user, via instructions, can control the modular workflow, and for example choose to inactivate (exclude) or activate (include) one or more modules of the system/apparatus, and/or rearrange order of modules, and/or choose parameters or duration of each modules, depending on the specific tissue(s) and applications to be studied/used. In embodiments, the system comprises a user interface for workflow programming, viewing workflow status (e.g. location, progress, and/or completion status for printing surfaces), viewing/troubleshootmg component/module and/or instrument errors, and/or performing maintenance on the system or component. In some embodiments, a separate user interface is present for control of one or more or each module/component of the system. In embodiments, the user interface(s) 120 is/are able to be detached/reattached to the system (FIG. 12).

In some embodiments, one or more of the modules/components/workstations comprise a UV light source capable of providing decontamination and/or sterilization. In some embodiments, one or more of the modules/components/workstations comprise a light source capable of cross-linking bioprmted structures during and/or after printing at wavelengths between 300 and 800 nm, such as 365 nm, 405 nm, 425 nm, and/or 480 nm. In other embodiments, cross-linking is performed via ionic crosslmking, stereocomplex crosslinking, thermal crosslinking, enzymatic gelation, or click chemistry. Crosslinking steps involving the addition of liquids can be performed by one or more liquid dispenser or liquid dispenser module containing a liquid dispenser.

In embodiments of the invention, the systems comprise one or more identifying scanner. The scanner is configured to be capable of identifying which step(s) of the bioprinting process a structure has completed, identifying which step(s) of the bioprinting process a structure has yet to complete, and sending/transferring it to the next appropriate module/component/workstation. In one embodiment, the identifying is performed by scanning a unique identifier on the printing surface, such as a bar code, QR code, or sequence of numbers and/or letters. In some embodiments scanners are present at various points along the transfer unit. The scanners can additionally be present in each module/component, or one scanner can be used for the entire system.

In embodiments, the system, one or more module(s), and/or transfer unit comprises one or more pressure sensors to sense the presence of one or more printing surface(s), for example, within one or more printing surface holder, within one or more module, and/or at one or more location on the transfer unit.

Bioprinter

Embodiments of the present invention include one or more bioprinter or module comprising a bioprinter. In one module of the system, typically the first module, a 3D bioprinting device is included, thereby allowing for bioprintmg a first layer of tissue in the sample holder. The 3D bioprinter prints the tissue in accordance with chosen parameters and chemicals, i.e. with a predetermined size and shape, and one or more bioinks and additional constituents suitable for the bioprmted tissue. The bioprmted tissue may comprise cells, but can also be without cells. Bioinks to be used and bioprinter devices to be used are known to a skilled person in the art, and will depend on the tissue application under study. For example, suitable bioinks and bioprinter devices may be found on www.cellink.com. Preferably, extrusion-based and light-based bioprinters are used. The bioprinters can be used in the system for developing/fabricating cell culture models and for high throughput testing using such models and provide improvements in the field of bioprinting, which field includes such technologies as described in, for example, W02020/165322, W02019/109127, WO2019/246623, WO2017/109394, WO2017/040675, WO2015/148646, US9,315,043, US8,931,880, US2020/0139623, US2020/0070421, US2019/0344500, US2019/0016052, US2018/0326665, US2018/0281280, US2016/0344500, US2016/0288414, US2016/0243618, US2015/0375453, US2015/0105891, and US2011/024699, which references are each incorporated by reference herein in their entireties.

In general, the method for bioprinting of tissue (with cells) or scaffolds (without cells) comprises combining one or more bioink, (with or without human or mammalian cells), and human tissue-specific extracellular matrix (ECM) material, wherein the bioprinting is performed under physiological conditions. The bioprinted tissue or scaffold can be in the form of a grid, drop, tissue- specific shapes like hepatic lobule for liver etc., or the like. In embodiments, the bioprinted tissue, construct or scaffold can have a printed size in the interval from 0.1 mm to 50 cm in diameter and/or length or width.

Bioprinters dispense bioinks and/or support material(s) through one or more printer heads. In embodiments, the printer head(s) comprises one or more cartridge capable of holding one or more bioink and/or support material. In embodiments, the bioprinter comprises a print stage on which the bioprinting takes place.

In embodiments, the bioprinters can comprise one or more of the following features/functionalities, including clean chamber technology, semi-automation, automation, PDCs (piezo dispense capillaries or pico dispense capillaries), NDCs (nano dispense capillaries), i-DOT source wells, spheroid printheads, BIO X iPH technologies, i-DOT printheads, sciDROP PICO printheads, computer vision, imaging modules, cooling solutions, sciDROP NANO printheads, and/or cell dispensing and dosing in a single unit.

Bioinks capable of being printed using embodiments of the bioprinter can include any one or more of hydrogel-based bioinks, polysaccharides, protein -based bioinks, dECM-based bioinks (e.g., decellularized bioinks), and/or synthetic polymer-based bioinks, including bioinks comprising one or more of alginate, gelatin, collagen, fibrin/fibrinogen, gellan gum, hyaluronic acid (HA), agarose, chitosan, silk, silk fibroin, decellularized extracellular matrix (dECM), poly(ethylene glycol) (PEG), PEG diacrylate (PEGDA), and Pluronics, gelatin-alginate composites, functionalized gelatin (GelMA), fibrinogen, fibrin and alginate, alginate and fibroblasts, cell aggregate based bioinks, and/or pellet-based bioinks. Examples of such bioinks are double network bioinks, biogum and botanical gum hydrogel bioinks, RGD conjugated polysaccharide bioinks with or without fibrin, and bioinks comprising cellulose nanofibrils with extracellular matrix components, such as any bioink described in W02020/077118, US2021/0001009, US2019/0160203, and/or US2019/0209738, which are each incorporated by reference herein in their entireties. Bioinks and/or media can include samples comprising aqueous solutions (e.g. oligonucleotides) and organic solvents, samples containing organic solvents like DMSO, DMF etc. and protein mixtures (e g. lysates, allergens etc ), samples containing protein solutions and organic solvents like methanol, isopropanol, acetonitrile etc., protein solutions and solgel samples.

Dispensing to prepare bioprinted constructs and/or to dispense cell media is capable of being performed with a resolution of down to 1 pL, or down to 10 pL, such as with a dispensing resolution in the range of from about 1-10 pL, or from 10 pL to 1 nL, or from 1 nL to 10 nL, or below 10 nL, or from 10 nL to 1,000 nL, or any range in between. Embodiments can comprise dispensing/printing with piezo dispense capillary capability (or otherwise referred to as pico- or nano-dispense capillary) with a fixed drop volume for example ranging from 50-800 pL drops, such as from 100-150 pL, or from 150-220 pL, or from 220-300 pL, or form 300-360 pL, or from 360-440 pL, or from 440520 pL, or from 520-600 pL, or from 600-800 pL, or from 100 pL to 1.0 mL, or from 1-100 nL drops, such as from 1-10 nL drops, or from 5-50 nL drops, etc.

In embodiments, the bioprinter, or module comprising the bioprinter, and/or a liquid dispenser or liquid dispenser module, are capable of delivering liquids in bulk, such as for delivery of cell media to larger printing surfaces, for example, 6 well plates, petri dishes, and custom-sized print surfaces, in amounts in the range of 1 mL to 250 mL, such as 1.5 mL, 2 mL, 3 mL, 4, mL, 5 mL, 10 mL, 25 mL, 50 mL, 75 mL, 100 mL, or 200 mL. Smaller volumes can be dispensed for smaller printing surfaces, for example, 12, 24, 48, 96, 384, or 1536 well plates, in the range of for example 1 pL to 2 mL, such as 2 pL, 3 pL, 4 pL, 5 pL, 10 pL, 15 pL, 20 pL, 25 pL, 50 pL, 75 pL, 100 pL, 125 pL, 150 pL, 250 pL, 500 pL, 750 pL, 1 mL, 1.1 mL, 1.25 mL, 1.5 mL, and 1.75 mL. Scanner

In embodiments, the present invention includes one or more quality control scanner, or module comprising a scanner, configured to be capable of scanning a bioprinted structure to provide quality control (e.g., comparing the bioprinted structure to a model and determining if the structure is within acceptable limits) and/or dictate work flow. In some embodiments, the scanner is an optical scanner. In some embodiments, the scanner is present within one or more of the other existing modules/components to provide quality control and/or dictate work flow. In some embodiments, a system comprises a single scanner or module/component comprising a scanner. In other embodiments multiple scanners and/or modules/components comprising a scanner are present and can be located in various locations relative to the other modules/components depending on the desired workflow application. For example, a scanner can be located at a position relative to a bioprinter to accept a bioprinted structure from the bioprinter for analysis, then if further adjustments are needed to be made to the bioprinted structure, the structure can be transported back to the biprmter or to another bioprmter in the system for revision and/or can be transported back to the scanner or another scanner to verify quality of the build. In embodiments, one bioprinter and one scanner can be used, or two or multiple bioprinters and scanners can be used.

In embodiments, the quality control scanner is a 3D scanner configured to be capable of generating a 3D point cloud in a time period on the order of seconds.

The quality control scanner is configured to be capable of evaluating features in the xy- and/or zx-p lanes. In embodiments, the scanner is configured to be capable of measuring and/or comparing to a model: step height, positioning, distance, angle, diameter, radius, flatness, roughness, and/or contour. The scanner is configured to be capable of scanning materials comprising cells, tissue, polymers, plastic, metal, and/or glass. In embodiments, the scanner is configured to be capable of sampling surfaces that are matte, glossy, transparent, mirror, and/or opaque. In embodiments the scanner is configured to be capable of sampling surface topography and intensity up to 2.5 kHz at a sub-micron level.

The scanner is configured to be capable of performing quality control inspection of bioprinted structures based on one or more parameters (as described above) as set by a user and/or provided on computer-readable instructions The parameters can be individually set by the user and/or based on a model in computer-readable form. The scanner is additionally configured to be capable of spotting visual defects including spots, scratches, voids, air bubbles, and/or impurities in a bioprinted structure.

In embodiments, the module/component/workstation comprising the scanner comprises a motorized stage, pick and place robot, and/or integrated statistical process control. In embodiments, the statistical process control feature is capable of accepting or rejecting a bioprinted construct based on its adherence to its model or specifications. In another embodiment, the statistical process control feature is capable of monitoring the system (or module/component) to detect variations before they result in major errors. The scanner is additionally configured to be capable of determining completeness of a printed structure. In some embodiments, the scanner is configured to be capable of scanning a bioprinted structure, comparing it to a model, and rejecting the structure. In embodiments, the rejected structure can be routed back to the bioprinter or another bioprinter for additional printing, may be reported as rejected to a system user, or may be removed from the system automatically. In some embodiments, the rejected structure is removed from the workflow queue by a pick and place robot and placed in a designated area for rejected structures.

The scanner is configured to be capable of movement along axes in the x-, y-, and/or z- directions and rotating up to 360° in any one or more of the x-, y- and/or z-directions.

In embodiments, the optical profile length of the scanner is in the millimeter range, such as 4.3 mm, 11.26 mm, or 16.4 mm. In embodiments, the pixel size (X, Y) is in the range of about 1- 50 micrometers, such as 2.10 pm, 5.5 pm, 8.0 pm, 10 pm, 25 pm, or 36 pm. In embodiments, the resolution (Z) is about 0.1 to 1 micrometers, such as 0.11 pm, 0.66 pm, or 0.98 pm. In embodiments, the stand-off distance is in the range of about 5 to about 75 mm, such as 8 mm, 20.58 mm, or 59.00 mm. In embodiments, the Z-range is about 1-10 mm, such as 1.2 mm, 3 mm, or 5.50 mm. In embodiments the measuring speed is about 100-10,000 Hz, such as 300 Hz, 500 Hz, or 5,000 Hz. In embodiments, the number of points per profile is in the range of about 1024- 4096 points, such as 2048 points. In embodiments, the maximum slope of objects is about 30 degrees, such as 13.5 degrees, 15.0 degrees, or 20.0 degrees.

Single-cell Dispenser

In another module of the system, typically the second module, a single-cell dispensing device is arranged, thereby allowing for single cells to be dispensed in the sample holder, such as to the tissue printed m a previous (bioprinting) module of the system. The cell(s) dispensed would be chosen in accordance with the application under study. The skilled person would be aware of single-cell dispensing devices to be used, such as those that can be found at www.sciemon.com and www.cytena.com.

Liquid Dispenser

In yet another module of the apparatus, typically a third module of the system, a device for dispensing cell media would be included, thereby providing a suitable environment for the tissue and cells under study, i.e. provided in the sample holder. In this module, cell media of chosen type and quantity would be added to the sample of the sample holder.

Non-limiting examples of liquid dispensers can be found in International Patent Application Publication No. WO2020/165322 and U.S. Patent No. 10,286,415, each of which is hereby incorporated herein by reference in its entirety.

In some embodiments, the system comprises multiple liquid dispensers configured to be capable of different functions such as dispensing bioink, cell media, crosslinking solution(s), and/or chemical and/or biological reagent(s) (or solutions of chemical or biological reagents) such as small molecules, peptides, antibodies, antibody-drug conjugates, proteins, growth factors, etc. Liquids dispensed can include aqueous solutions (e.g. oligonucleotides) and organic solvents, samples containing organic solvents like DMSO, DMF etc. and protein mixtures (e.g. lysates, allergens etc.), samples containing protein solutions and organic solvents like methanol, isopropanol, acetonitrile etc., protein solutions and solgel samples.

In some embodiments, the chemical and/or biological reagent is stored as a solid, semi-solid, powder, gel, or stock solution and mixed with a liquid just prior to dispensing. In embodiments, the mixing is performed within the liquid dispenser module/component.

In embodiments, the liquid dispensers comprise a single dispensing head/nozzle. In other embodiments, the dispensers comprise multiple dispensing heads/nozzles that can dispense liquid individually (such as in a single well of a well plate) or simultaneously (such as in multiple wells of a well plate). The number of dispensing heads can range from 1-1536, such as 6, 12, 24, 48, 96, 384, or 1536 heads. In embodiments, the multiple dispensing heads are arranged in a single row. In other embodiments, the multiple dispensing heads are arranged in a grid, such as in manner to dispense liquid into multiple wells present in multiple rows of a well plate or an entire well plate at once. In embodiments, the dispensing heads are substantially plastic, ceramic, metal, and/or rubber, or composites or combinations thereof.

In some embodiments, the liquid dispenser or module comprising the liquid dispenser is configured to be capable of fitting the dispensing head(s) with disposable tips (such as pipette tips). In embodiments, the liquid dispenser is configured to be capable of both dispensing liquids and drawing liquids into the dispensing head(s), such as to remove cell media from well plates, wash the wells, and/or fill the wells with fresh media or solutions of chemical and/or biological analytes for testing. In embodiments, the liquid dispenser, or module comprising the liquid dispenser, comprises a waste tank for disposing of a used liquid and/or washing station for washing the dispensing heads between samples and/or between dispensing different liquids. In some embodiments the wash step involves dispensing all liquid from the dispensing head and “washing” the dispensing head by dispensing a small amount of the next liquid to be dispensed into the waste tank. In other embodiments, the liquid dispenser, or module comprising the liquid dispenser, contains means for collection of one or more liquid(s). For example, cells can be printed on a surface and incubated with one or more analyte(s) to be tested. The liquid can be removed from the printing surface and stored in a collection container (such as a vial, well plate, bottle, etc.) for analysis. For example, the analysis can include identification of metabolites.

In embodiments, the liquid dispenser, or module/workstation comprising the liquid dispenser, comprises one or more containers (such as bottles, reservoirs, and/or tanks) configured to be capable of holding the liquid to be dispensed. The number of containers can range from 1 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 containers.

In some embodiments the liquid dispenser is configured to be capable of controlling the temperature of the liquid to be dispensed in the range of 0 °C to 120 °C, such as 2 °C, 4 °C, 10 °C, 15 °C, 20 °C, 25 °C, 28 °C, 30 °C, 32 °C, 35°C, 37 °C, 39 °C, 45 °C, 50 °C, 75 °C, 100 °C, or 115 °C. The liquid can be kept at the desired temperature in the storage tanks or heated and/or cooled to the desired temperature at any time during the workflow process, such as during storage or during delivery of the fluid, and/or before or after delivery. In embodiments, the liquid is dispensed at a temperature that is different from or the same as the temperature within the system, such as at ambient temperature. In embodiments, the temperature of the fluid, whether in storage or before, during or after delivery, can be higher than or lower than the temperature of the environment within one or more of the units, modules, components, stations, and/or workstations of the system. In some embodiments the liquid dispenser, or module comprising the liquid dispenser, comprises multiple cell culture media tanks/bottles which may contain the same or different cell culture media.

In embodiments, the liquid dispenser comprises wash and/or waste tanks to clean the dispenser between samples and/or between dispensing different fluids, such as different types of cell culture media.

In embodiments, the liquid dispenser, and/or any module/workstation comprising a liquid dispenser (such as a bioprinter or wash station), is configured to be capable of delivering liquids in bulk, such as for delivery of cell media to larger printing surfaces, for example, 6 well plates, petri dishes, and custom-sized print surfaces, in amounts in the range of 1 mL to 250 mL, such as 1.5 mL, 2 mL, 3 mL, 4, mL, 5 mL, 10 mL, 25 mL, 50 mL, 75 mL, 100 mL, or 200 mL. Smaller volumes can be dispensed for smaller printing surfaces, for example, 12, 24, 48, 96, 384, or 1536 well plates, in the range of 1 pL to 2 mL, such as 2 pL, 3 pL, 4 pL, 5 pL, 10 pL, 15 pL, 20 pL, 25 pL, 50 pL, 75 pL, 100 pL, 125 pL, 150 pL, 250 pL, 500 pL, 750 pL, 1 mL, 1.1 mL, 1.25 mL, 1.5 mL, and 1.75 mL.

In embodiments, the liquid dispenser, and/or any module/workstation comprising a liquid dispenser, is additionally capable of dispensing volumes of liquid in the picoliter and nanoliter ranges, such as for dosing the printing surface with one or more analyte to be tested. Dispensing is capable of being performed with a resolution of down to 1 pL, or down to 10 pL, such as with a dispensing resolution in the range of from about 1-10 pL, or from 10 pL to 1 nL, or from 1 nh to 10 nL, or below 10 nL, or from 10 nL to 1 ,000 nL, or any range in between. Embodiments can comprise dispensing/printing with piezo dispense capillary capability (or otherwise referred to as pico- or nano-dispense capillary) with a fixed drop volume for example ranging from 50-800 pL drops, such as from 100-150 pL, or from 150-220 pL, or from 220-300 pL, or form 300-360 pL, or from 360-440 pL, or from 440 520 pL, or from 520-600 pL, or from 600-800 pL, or from 100 pL to 1.0 mL, or from 1-100 nL drops, such as from 1-10 nL drops, or from 5-50 nL drops, etc.

In embodiments, one or more liquid dispenser is present and configured to be capable of providing a liquid capable of cross-linking one or more bioink, such as calcium chloride solution, during and/or after printing.

In embodiments, the liquid dispenser, or module comprising the liquid dispenser, further comprises a degasser capable of degassing liquids to be dispensed. In embodiments the liquid dispenser is configured to be capable of dispensing up to 150 drops per second, such as 10 drops, 50 drops, 75 drops, or 100 drops. In embodiments, the drops can be dispensed over a maximum area of about 128 mm x about 434 mm. In embodiments, drops can be placed with an accuracy of ±0.01 mm and/or repeatability of ±0.005 mm.

In embodiments, the liquid dispenser comprises one or more pumps, such as 2, 3, 4, or 5 pumps.

Cell incubator

The system further comprises an incubator, or module comprising an incubator, configured to be capable of holding printing surfaces in or on sample holders such as well plates, microplates, slides, and/or petri dishes. In embodiments of the invention, the incubator has removable shelves that are customizable based on the size of the printing surfaces to be stored in the incubator. The incubator is configured to be capable of processing a bioprinted structure at a desired temperature for a desired period of time. In embodiments, the construct can be transferred to the incubator at a specified point in the workflow process, the incubator can be adjusted to reach and maintain a desired temperature for a desired period of time to incubate the construct and/or cell media to obtain a desired growth result. Once the desired growth and/or processing has been achieved or a desired period of time has elapsed, the temperature maintained by the incubator can be adjusted to another temperature so as to complete the desired processing. The construct can be transferred to another module/component of the workflow once the incubation is complete.

In embodiments, the incubator is configured to be capable of maintaining relative humidity as selected by a user, such as in the range of 50% to 99%.

Optional Additional Modules/Components

Optional additional modules/components/workstations include a module/component configured to be capable of providing detection and/or monitoring at the cellular level. A non-limiting example of this module/component type is the compact fluorescence microscope described in W02020/157077, which reference is hereby incorporated by reference in its entirety.

Additional components can include one or more well plate reader. Examples

The following examples are intended to be illustrative only. One or more method steps in the following examples may be added to, omitted from, or combined with method steps of separate example, likewise any one or more components/modules of a system can be combined. In embodiments, to obtain a system for a desired workflow, the components, functionalities, or functional modules can be operably connected to one another or a transfer unit, can be linked together via a transfer unit, or in operable communication with a transfer unit.

Example systems component/module combinations, optionally operatively connected to and/or in operable communication with, a transfer unit, include but are not limited to any of the following functions:

System 1 : bioprinter and incubator, and optionally liquid dispenser

System 2: bioprinter, scanner, and optionally incubator

System 2: bioprinter, scanner, and liquid dispenser

System 3 : bioprinter, scanner, liquid dispenser, well plate reader

System 4: liquid dispenser, well plate reader, and incubator

System 5 : first bioprinter, scanner, second bioprinter

System 6: liquid dispenser, incubator, microscope

System 7: any combination of one or more of Systems 1-6, or one or more components/modules thereof

Example 1. Build and Confirm

An example bioprinting workflow system comprises a bioprmter and a scanner. The components are linked together by, connected with, or in operable communication with a transfer unit. Attached to the bioprinter is an infeed system for use with well plates (FIG. 11B). A user uploads a model to the system for printing. A robot arm moves a 24 well plate from the infeed system to a printing platform 130 within the bioprmter (FIG. 13). A robot arm removes the lid from the well plate. The bioprmter builds a structure based on the model into each of the 24 wells. A robot arm replaces the well plate lid and moves the 24 well plate to the transfer unit where it is placed in a magnetic sample holder on the transfer unit platform. Using magnets beneath the platform, the sample holder is transported along the platform to the scanner (FIG. 14). A robot arm removes the well plate from the sample holder, places it on a scanning platform 140, and removes the lid. The scanning platform 140 moves each printed structure under the scanner, and the scanner uses its optical scanner 142 to scan each printed structure. After scanning is completed, a robot arm replaces the lid and transfers the well plate to the transfer unit.

Example 2. Build. Confirm and Culture

An example bioprinting workflow system comprises: a bioprinter, a scanner, a liquid dispenser, and a cell incubator, linked together by, connected with, or in operable communication with a transfer unit. The method of Example 1, further comprising the following steps. The well plate is then transferred to the liquid dispenser. A robotic arm removes the well plate lid. The liquid dispenser dispenses cell media (type and amount set by a user) into each well. A robotic arm replaces the well plate lid. The well plate is transported back to the transfer unit, which then moves the well plate to a cell incubator. A robotic arm places the well plate in the cell incubator for a period of time specified by the user.

Example 3. Build. Confirm. Culture and Test/Assav

An example bioprinting workflow system comprises: a bioprinter, a scanner, a liquid dispenser, and a cell incubator, linked together by, connected with, or in operable communication with a transfer unit. The method of example 2, further comprising the following steps. A robotic arm removes the well plate from the cell incubator and places it in a sample holder on the transfer unit. The well plate is taken back to the liquid dispenser. The robotic arm removes the well plate lid. The liquid dispenser removes the used cell media, dispenses a user selected wash liquid, removes the wash liquid, and dispenses a liquid comprising one or more analyte to be tested. The well plate is returned to the cell incubator using the transfer unit. After a user set incubation period, the well plate is removed from the incubator and returned to the liquid dispenser as previously described. The liquid comprising one or more analyte to be tested/assayed is removed and optionally saved (for example, for testing for metabolites). The liquid dispenser completes a wash step as previously described, dispenses fresh cell media, and returns the well plate to the incubator.

Example 4. Build Scan Build and Confirm

An example bioprinting workflow system comprises: two or more bioprinters, two or more scanners, and at least one cell incubator, which are linked together by, operably connected to, or in operable communication with, a transfer unit. A printing surface enters the bioprinter from the transfer unit. The bioprinter prints a structure on the printing surface based on a user selected model. The printing surface is transferred to the scanner as previously described. The scanner scans the structure and accepts or rejects the structure. Rejected structures are transferred to a rejected structure outfeed. Accepted structures are moved to the second bioprinter which finishes printing the structure. The printing surface is transferred to the second scanner where it is evaluated against the model. Accepted structures are transferred to a cell incubator.

Example 5 Scan and Incubate

A bioprinting workflow system comprises a scanner and a cell incubator which are linked together by, operably connected to, or in operable communication with a transfer unit. A bioprinted structure is introduced into the system transfer unit. The transfer unit transports the bioprinted structure to the scanner. The scanner collects one or more measurement associated with the bioprinted structure. The bioprinted structure is then placed back onto the transfer unit using one or more robotic arms. The transfer unit transfers the bioprinted structure to the incubator where it is incubated for a set period of time. The scanning and incubating steps are repeated until a desired tissue or cell maturation is achieved. Once the desired tissue or cell maturation is achieved, the system user is notified.

According to embodiments, any one or more of the printing, dispensing, building, performing builds and/or assays, moving, positioning, controlling, controlling of temperature, the control system(s), operating, and/or performing a workflow can be performed manually and/or can be automated, for example, in connection with and/or automated by using software and/or programming to perform any one or more of these functions. For purposes of this disclosure, the terms “code”, “software”, “program”, “application”, “software code”, “software module”, “module” and “software program” are used interchangeably to mean software instructions that are executable by a processor

The present disclosure provides for a computer program comprising computer-executable instructions, which when the program is executed by a computer, cause the computer to carry out any one or more of the processes, methods, and/or algorithms according to the above. The computer-executable instructions can be programmed in any suitable programming language, including JavaScript, C, C#, C++, Java, Python, Perl, Ruby, Swift, Visual Basic, and Objective C. Also provided herein is a non-transitory computer-readable medium (or media) comprising computer-executable instructions, which when executed by a computer, cause the computer to carry out any of the processes, methods, and/or algorithms according to the above. As used in the context of this specification, a “non-transitory computer-readable medium (or media)” may include any kind of computer memory, including magnetic storage media, optical storage media, nonvolatile memory storage media, and volatile memory. Non-limiting examples of non-transitory computer-readable storage media include floppy disks, magnetic tape, conventional hard disks, CD-ROM, DVD-ROM, BLU-RAY, Flash ROM, memory cards, optical drives, solid state drives, flash drives, erasable programmable read only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), non-volatile ROM, and RAM. The non-transitory computer readable media can include one or more sets of computer-executable instructions for providing an operating system as well as for implementing the processes, methods, and/or algorithms of the invention.

The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is intended that the specification and examples be considered as exemplary in nature and that variations that do not depart from the essence of the invention fall within the scope of the invention. Further, all of the references cited in this disclosure are each individually incorporated by reference herein in their entireties and as such are intended to provide an efficient way of supplementing the enabling disclosure of this invention as well as provide background detailing the level of ordinary skill in the art.

The present invention has been described with reference to particular embodiments having various features. In light of the disclosure provided above, it will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. One skilled in the art will recognize that the disclosed features may be used singularly, in any combination, or omitted based on the requirements and specifications of a given application or design When an embodiment refers to “comprising” certain features, it is to be understood that the embodiments can alternatively “consist of’ or “consist essentially of’ any one or more of the features. Any of the methods disclosed herein can be used with any of the compositions disclosed herein or with any other compositions. Likewise, any of the disclosed compositions can be used with any of the methods disclosed herein or with any other methods. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention.

Claims

1. A customizable bioprinting workflow system comprising at least two of: one or more 3D bioprinter; and/or one or more scanner; and/or one or more liquid dispenser; and/or one or more cell incubator; wherein the bioprmter, scanner, liquid dispenser and/or incubator are arranged in operable communication with one another, optionally as one or more modules; and the system further comprising a transfer unit m operable communication with the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them, which transfer unit is configured to move one or more structure from one to another of the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them.

2. The system of claim 1 , wherein the transfer unit is configured to move a sample holder from one to another of the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them, wherein the sample holder is adapted to support and hold said one or more structure.

3. The system of any previous claim, wherein the 3D bioprinter comprises means for dispensing cell(s) and/or matrix material into at least one sample holder.

4. The system of any previous claim, further comprising a detection or monitoring system, such as for detecting and/or monitoring at the cellular level, such as one or more imaging modality.

5. The system of any previous claim, wherein the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof, are configured to be arranged within one or more clean chamber.

6. The system of any previous claim, wherein the bioprmter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof, are configured to comprise a clean chamber.

7. The system of claim 5 or 6, wherein said clean chamber provides an essentially airtight enclosure for at least a sample holder, said airtight enclosure being configured to provide positive pressure with filtered air within said clean chamber.

8 The system of any previous claim, wherein the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof are configured to be arranged in any order relative to one another.

9. The system of any previous claim, wherein the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof are configured to be re-arranged relative to one another, such as after initial assembly of the system.

10. The system of any previous claim, wherein one or more additional bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof can be added to the system, such as after initial assembly of the system.

11. The system of any previous claim, wherein the system is configured to accept one or more of additional bioprinters, scanners, liquid dispensers, incubators, and/or detection or monitoring systems, or the module(s) thereof, such as after an initial assembly of the system.

12. The system of any previous claim, wherein the transfer unit comprises a conveyor belt or comprises an intelligent transfer unit, or comprises an intelligent transfer unit with a conveyor belt.

13. The system of any previous claim, further comprising one or more robotic arms, which can be part of the transfer unit or additional, which robotic arms are configured to be capable of transferring one or more of the structures, such as one or more bioprinted structures or wellplates and/or slides, within one or more of the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof and/or between one or more of the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof and the transfer unit, and/or between two or more of the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof.

14. The system of any previous claim, wherein the transfer unit is configured to transport sample holders such as well plates, microplates, slides, bioprmted structures, and/or petn dishes.

15. The system of any previous claim, wherein the transfer unit and/or bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the modules thereof are configured to be capable of maintaining an air temperature between 4 °C and 60 °C.

16. The system of any previous claim, wherein one or more of the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof is configured to be maintained at a first temperature, while one or more of a different bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof is configured to be maintained at a second temperature, such as for maintaining a scanner at about 22 °C and maintaining an incubator at about 37 °C.

17. The system of any previous claim, wherein the scanner is configured to be capable of generating a 3D point cloud in a time period on the order of seconds.

18. The system of any previous claim, wherein the scanner is configured to be capable of evaluating one or more features of a bioprinted structure in the xy-plane.

19. The system of any previous claim, wherein the scanner is configured to be capable of evaluating one or more features of a bioprinted structure in the zx-plane and/or zy-plane.

20. The system of any previous claim, wherein the scanner is configured to be capable of measuring step height, positioning, distance, angle, diameter, radius, flatness, roughness, and/or contour of one or more structural features of the bioprinted structure.

21. The system of any previous claim, wherein the scanner is configured to be capable of scanning materials comprising cells, tissue, polymers, plastics, metal, and/or glass.

22. The system of any previous claim, wherein the scanner is configured to be capable of scanning surfaces that are matte, glossy, transparent, mirror, and/or opaque.

23. The system of any previous claim, wherein the scanner is configured to be capable of contour comparison.

24. The system of any previous claim, wherein the scanner is configured to be capable of sampling surface topography and intensity at a sub-micron level.

25. The system of any previous claim, wherein the scanner is configured to be capable of performing quality inspection of the bioprinted structures based on one or more parameters, such as parameters set by a user and/or provided by computer-readable instructions.

26. The system of any previous claim, wherein the scanner is configured to be capable of spotting visual defects including, spots, scratches, voids, air bubbles, and/or impurities in the printed structure.

27. The system of any previous claim, wherein the scanner is configured to be capable of movement along more than one axis.

28. The system of any previous claim, further comprising an outfeed configured to be capable of discarding rejected bioprmted structures.

29. The system of any previous claim, further comprising a feeder configured to be capable of feeding sample holders, such as well plates, microplates, slides, and/or petri dishes into the bioprinter, wherein the feeder is configured to be operably connected with the transfer unit, transfer unit, and/or robotic arms.

30. The system of any previous claim, further comprising a liquid dispenser configured to be capable of dispensing cell culture media.

31. The system of any previous claim, wherein the liquid dispenser comprises multiple cell culture media storage tanks.

32. The system of any previous claim, further comprising wash and/or waste tanks to wash and/or rinse the liquid dispenser.

33. The system of any previous claim, wherein the module configured to be capable of providing detection or monitoring at a cellular level is a microscope.

34. The system of any previous claim, wherein the transfer unit is configured to be capable of forward and backward or up and down movement along one or more or each of an x, y, and z-axis.

35. The system of any previous claim, wherein the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them, are assembled horizontally and/or vertically relative to another.

36. The system of any previous claim, wherein one or more of the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them, are assembled horizontally relative to at least one other of the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them, and vertically to at least one other of the bioprinter, scanner, liquid dispenser and/or incubator, or module(s) comprising them.

37. The system of any previous claim, wherein one or more of the bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof has a scanner configured to be capable of identifying each sample holder, well plate, microplate, slide, and/or petri dish as it enters and/or exits the one or more bioprinter, scanner, liquid dispenser, incubator, and/or detection or monitoring system, or the module(s) thereof.

38. The system of any previous claim, wherein the identifying is performed by scanning a bar code, QR code, or sequence of numbers and/or letters.

39. A method comprising: depositing bioink onto a printing surface to prepare one or more bioprinted structure; scanning the bioprinted structure to obtain one or more measurement; comparing the one or more measurement against a model; and repeating one or more of the depositing, scanning, and/or comparing until one or more final bioprinted structure is produced; wherein the bioprinted structure is transported to and/or from a bioprinter or bioprinter module to a scanner or scanner module using a transfer unit in operable communication with the bioprinter or bioprinter module and the scanner or scanner module.

40. The method of claim 39, further comprising: dispensing media with a liquid dispenser or liquid dispenser module, where in the dispensing is performed on, around or near the bioprinted structure, such as on the bioprinted structure and/or portion thereof and/or on the printing surface and/or a portion thereof; incubating the bioprinted structure and media with an incubator or incubator module; optionally repeating the dispensing and/or incubating until a desired cell growth or tissue maturation is reached; wherein the repeating of the dispensing and/or incubating is facilitated by the transfer unit, which is in further operable communication with the liquid dispenser or liquid dispenser module and the incubator or incubator module to provide transport therebetween.

41. The method of claims 39 or 40, further comprising: removing media from the printing surface and/or the bioprinted structure, and/or washing the printing surface and/or the bioprmted structure; and dispensing new media onto the printing surface and/or the bioprinted structure; such as by transporting the printing surface and/or the bioprinted structure to and/or from a washing function and/or washing module by way of the transfer unit, which is in further operable communication with the liquid dispenser or liquid dispenser module and the washing function/module.

42. The method of any of claims 39-41 , wherein the printing surface is in or on a sample holder such as a well plate, microplate, slide, or petri dish.

43. The method of any of claims 39-42, wherein the scanning is performed by one or more 3D scanner, camera, or other imaging module.

44. The method of any of claims 39-43, further comprising: transporting the bioprinted structure to the scanner or scanner module and/or to a detection or monitoring system using the transfer unit, which is in further operable communication with the detection or monitoring system; and detecting and/or monitoring one or more feature of the bioprmted structure using the scanner or scanner module and/or the detection of monitoring system.

45. The method of any of claims 39-44, wherein the transporting is performed using the transfer unit which comprises an intelligent transfer unit, which is configured to be programmed by a user to perform a desired workflow and/or which is configured to follow a set of computer- readable instructions.

46. The method of any of claims 39-45, wherein the transfer unit comprises one or more conveyor belt and/or one or more robotic arm.

47. A method of bioprintmg comprising using the system of any of claims 1-38.

48. A method comprising: bioprintmg one or more structure with a bioprmter/bioprinter module; transporting the structure to a scanner/scanner module by way of a transfer unit which enables workflow between the bioprinter/bioprinter module and the scanner/scanner module; scanning the structure to determine if there is compliance with one or more parameter(s) and/or model.

49. The method of claim 48, further comprising: transporting the structure to the vicinity of a liquid dispenser/liquid dispenser module by way of the transfer unit, which further enables workflow between the bioprinter/bioprinter module, the scanner/scanner module, and/or the liquid dispenser/liquid dispenser module; and dispensing cell media using the liquid dispenser/liquid dispenser module.

50. The method of claim 48 or 49, further comprising: transporting the structure to an incubator/incubator module by way of the transfer unit, which further enables workflow between the bioprinter/bioprinter module, the scanner/scanner module, the liquid dispenser/liquid dispenser module, and/or the incubator/incubator module; and incubating the structure.

51. The method of any of claims 48-50, further comprising: assaying the stmcture, such as before, during or after the bioprinting, scanning, dispensing, transporting, and/or incubating.

52. The method of any of claims 48-51, wherein the bioprinting, scanning, dispensing, transporting, incubating and/or assaying are performed in any order relative to one another.

53. The system of any of claims 1-38 configured to operate one or more methods in whole or in part, or combinations thereof, according to claims 39-52.

54. Use of a system of any of claims 1-38 or claim 53 for performing one or more workflow function chosen from:

1) bioprinting one or more bioprinted structure; and/or

2) scanning the bioprinted structure and comparing with specifications and/or a model;

3) dispensing cell media on, around, and/or near the bioprinted structure; and/or

4) incubating the bioprinted structure.