WO2018035282A1

WO2018035282A1 - Methods and libraries for use in inkjet bioprinting of biostructures

Info

Publication number: WO2018035282A1
Application number: PCT/US2017/047268
Authority: WO
Original assignee: Nano-Dimension Technologies, Ltd.; The IP Law Firm of Guy Levi, LLC
Priority date: 2016-08-17
Filing date: 2017-08-17
Publication date: 2018-02-22

Abstract

The disclosure relates to methods, systems and libraries for use in the inkjet printing of biostructures having predetermined two- (2D) and three- dimensional (3D) pattern of cells, extra cellular matrix, organic, inorganic components and their combination. Specifically, the disclosure relates to methods, systems and libraries for use in the direct inkjet printing of biostructures using drop-on-demand, having a predetermined three-dimensional structure with viable cells incorporated therein in a non-random 2D and 3D pattern, wherein the cells can be of different types.

Description

METHODS AND LIBRARIES FOR USE IN INKJET BIOPRINTING OF BIOSTRUCTURES

BACKGROUND

[0001] The disclosure is directed to methods, systems and libraries for use in inkjet printing of biostructures having predetermined two (2D)- and/or three-dimensional (3D) pattern cells and/or extra cellular matrix and/or other organic and inorganic components. Specifically, the disclosure is directed to methods and libraries implementable in computerized inkjet printing systems for the fabrication of biostructures using drop-on-demand, having a predetermined 3D structure that can be assembled from 2D patterns with Bruno incorporated therein in a non-random 2D and 3D pattern.

[0002] For various incentives, there is a need for alternatives to animal studies for development of novel pharmaceuticals, formulations, medical devices and combinations thereof, or development of pharmaceuticals, formulations, medical devices and combinations thereof for new applications or dosage, as well as screening of various molecules and medical devices in achieving therapeutic effectiveness. Provided herein are methods, systems and libraries for use in inkjet printing of different biostructures that correspond to the 3D tissue-tissue interfaces, structurally, mechanically and metabolically active microenvironments, electrical stimulation, chemical conditions and complex organ-level functions, for instance, breathing lung, beating heart, metabolic liver, flowing kidney, gut, reactive airway, contracting skeletal muscle, skin barrier, blood-brain barrier, reproductive/endocrine testis and self-renewing bone marrow, as well as, optional instrumentation for linking these biostructures for physiological and pharmacological analysis and diagnostics. The use of the disclosed biostructures integrated in macro/micro-physiological systems can shorten drug and medical device development and examination timeline, save animal lives, reduce failure rates, inform regulatory decision-making, and accelerate development of new therapeutics, including in the face of emerging infectious diseases, as well as chemical or biological attack.

[0003] Further, a number of pressing problems confront the healthcare industry. As of January 11, 2016, there were approximately 121,678 patients registered by United Network for Organ Sharing (UNOS) as needing a life-saving organ transplant in the U.S. alone. Each year more patients are added to the UNOS list than transplants are performed, resulting in a net increase in the number of patients waiting for a transplant and an increase in the waiting time. For example, the median waiting time for a kidney transplant is 3.6 years, with over 3,000 people added to the list every month and about 8,000 deaths occurring every year due to untimely donations. [0004] In addition, additive manufacturing approaches using extrusion for scaffold and tissue fabrication for bone tissue engineering are used, but rigorous thermal or chemical treatments to the scaffolds during the fabrication process may adversely affect the efficacy of the resulting treatment in-vivo, incompatibility of mechanical characteristics and reduce cell viability and/or functionality.

[0005] Therefore, the need exists for methods, systems and libraries for use in the inkjet printing of precision-fabricated biostructures.

SUMMARY

[0006] Disclosed, in various embodiments, are methods, systems and libraries for use in inkjet printing of biostructures having predetermined 2D and 3D pattern of cells and/or extra cellular matrix and/or other organic and inorganic components. In other embodiments, provided herein are methods for inkjet printing of biostructures having a predetermined 3D-structure with cells and/or extra cellular matrix and/or other organic and inorganic components incorporated therein in a non-random 2D and 3D pattern.

[0007] Using the methods, systems and libraries for use in inkjet printing and print-head combination described herein, in communication with a processor, a 3D computer aided design (CAD) model of the biostructure to be made can be assembled, from which a stereolithography (STL) or other suitable format file is generated within a CAD package. The file can then be processed and in effect virtually sliced in the Z-axis at a thickness matching the thickness of the systems' capabilities or other requirements. This creates a series of plan cross sections layer of the part and at any particular height, each having a simple 2D profile. Within each of the substantially 2D layer, the various cells and/or extra cellular matrix and/or other organic and inorganic componentscan be designated.

[0008] The fabrication of the desired biostructure is enabled by several main features: 1. bio- ink comprising a certain type, and or several types of cells and/or extra cellular matrix and/or other organic and inorganic components. 2. A computer software controlling the functionality of the printer, directing the fabrication of the 3D biostructure by a drop on demand digital printing 3. An inkjet printer equipped with a designated software and bio-inks, which deposits the bio-ink according to the directing of the software, to provide with the desired biostructure.

[0009] In an embodiment provided herein is method of bioprinting using a 3D inkjet printer comprising: providing a 3D inkjet printer, the printer having: a bitmap library to store printer operation parameters; a processor in communication with the bitmap library; a memory device storing a set of operational instructions for execution by the processor; a micromechanical inkjet print head or heads in communication with the processor and with the bitmap library; and a print head or, heads' interface circuit in communication with the bitmap library, the memory and the micromechanical inkjet print head or heads, the bitmap library configured to provide printer operation parameters specific for a layer or portion of a specific layer; pre-processing Computer-Aided Design/Computer-Aided Manufacturing (CAD/CAM) generated information associated with the bioprinted structure to be fabricated, thereby obtaining a plurality of vector data models and/or bitmaps, each vector data model and/or bitmap specific for a predetermined layer or their interface and/or cross section and/or a portion thereof; loading the plurality of bitmaps and/or vector data models processed in the step of preprocessing onto the bitmap library; and using the bitmap library, instructing the processor to print the predetermined layer, its interface and/or a portion thereof in a predetermined order.

[00010] The files used to recreate the Computer-Aided Design/Computer-Aided Manufacturing (CAD/CAM) generated information libraries of the substantially 2D layer(s) or portions thereof associated with the bioprinted structure to be fabricated for use in the inkjet printer can be converted from MRI's DICOM files (e.g., *.dcm, *.ima), as well as computer tomography (CT) DICOM files e.g., *.SEQ. It is further noted that other sources that can be converted in any way to a raster representation. A DICOM file, as used in the methods and systems described herein, which can also be generally referred to as a diagnostic patient study, a diagnostic image, or a high-resolution digital pathology image, can include a header and one or more additional diagnostic images. The header, which can also be referred to as diagnostic data, can include patient data and exam data. Further, extracting the header from a DICOM file can leave one or more diagnostic images. In an embodiment, the DICOM header data, as well as the image(s) are converted to files suitable for printing using the 3D inkjet printer systems described herein.

[00011] In an embodiment provided herein is an inkjet bioprinting method for forming biostructures having predetermined 3D pattern of cells and/or extra cellular matrix and/or other organic and inorganic components therein, the method comprising: providing an inkjet printing system comprising: a first print head having: at least one aperture, a first bio-ink reservoir, and a first pump configured to supply the first bio-ink through the aperture, wherein the first bio-ink is a composition comprising cells and/or extra cellular matrix and/or other organic and inorganic components, and/or a bio-compatible polymer, monomer, or oligomer; a second print head having: at least one aperture, a second bio-ink reservoir, and a second pump configured to supply the second bio-ink through the aperture, wherein the second bio-ink is a composition comprising a bio-compatible compounds dispersed within a medium, and/or a polymer, monomer, or oligomer; a third print head having: at least one aperture, a third bio-ink reservoir, and a third pump configured to supply the third bio-ink through the aperture, wherein the third bio-ink comprises cells and/or extra cellular matrix and/or other organic and inorganic components; and a conveyor, operably coupled to the first print head, the second print head and to the third print head; following the uploading of a first 2D image corresponding to a first printed layer, using the first inkjet print head, forming a first layer of cells' pattern or a portion thereof; using the third print head, functionalizing the first layer containing the cells pattern or portion thereof; using the second inkjet print head, forming a first layer of predetermined pattern or portion thereof of the second bio-ink composition on the functionalized layer of cells pattern; and using the third print head, functionalizing the first layer of predetermined pattern of the second bio-ink. In certain circumstances the 2D images uploaded from the libraries stored (e.g., remotely) are used to form a portion of the first layer and can be used as well to define the printing of the second bio-ink on a portion of the first layer, such that the first bio-ink and the second bio-ink are alternated followed in certain embodiment, by the third bio-ink. In other embodiments, a printed layer can be adapted to functionalize neighboring layers, improve connection between neighboring layers, create structures composed of neighboring layers, trigger compounds of neighboring layers.

[00012] In another embodiment, the methods described further comprising subsequent to the step of functionalizing the first layer of predetermined pattern of the second bio-ink: following uploading the substantially 2D image representing the second layer or portion thereof, using the first inkjet print head, forming a second layer of cells' pattern (or portion thereof) on and/or adjacent to the first layer of cells' pattern and/or the first layer of predetermined pattern of the second bio-ink composition; and functionalizing the second layer of cells pattern, wherein the cells in the second layer are different than the cells in the first layer. Portions of the second layer can be formed similar to the layers in the first layer.

[00013] In another embodiment, the methods, systems and libraries for use in the direct inkjet printing of biostructures described herein further comprise, using the second inkjet print head, forming a second layer of predetermined pattern of the second bio-ink on and/or adjacent to the first layer of cells' pattern and/or the first layer of predetermined pattern of the second bio-ink composition; and functionalizing the second layer of predetermined pattern of the second bio-ink, wherein a triggering compounds in the second layer of predetermined pattern of the second bio-ink, is different than the triggering compounds in the first layer of predetermined pattern of the second bio-ink.

[00014] In an embodiment, the number of print heads and bio-inks used will depend on the complexity of the biostructure sought to be printed and as defined in the substantially 2D images (e.g., raster images and/or vector data models). For example, a first bio-ink can comprise a dispersing medium, and/or cell growth media and buffers, including amino acids broths and saline (e.g., Minimum Essential Medium Eagle, Cell Applications Dermal Microvascular Endothelial Cells (CADMEC™) or ISOYEAST™) with a first polymer, monomer or oligomer without any cells suspended in it, which can be used to form the scaffolding for the biostructure. The first print head may be associated with a dedicated functionalizing print head which can be used to stiffen the polymer to a desired degree. A second print head with a second bio-ink can be used, wherein the second bio- ink can comprise dispersing medium with cells which has been triggered to undergo a specific manipulation and/or alternatively, cells and either the same or different biocompatible polymer, monomer or oligomer, associated with another functionalizing print head. A third print head can be used with a third bio-ink comprising a dispersing medium with triggering compounds therein and either the same or different biocompatible polymer, monomer or oligomer. Another (fourth) print head can be used with a fourth bio-ink, comprising dispersing medium with viable cells, associated with a dedicated functionalizing print head. Adding and/or removing of the various print head can be done based on the printed biostructure.

[00015] In the methods, systems and libraries for use in the direct inkjet printing of biostructures provided, a first scaffold layer or portion thereof, as defined in the images provided, can be printed, functionalized using a first method (e.g., photopolymerization), followed alternatively by printing of a pattern of other tissue or organ cells using another print-head and then followed by printing of a pattern of extracellular matrix (ECM) compositions; for example, manipulation triggering compounds, growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), or epidermal growth factor (EGF) , allowing the cells to multiply or undergo a specific manipulation or additionally or alternatively, retaining native growth of the tissue or organ; and then, using a dedicated print head, functionalizing the patterns defined by the 2D images (e.g., using spray nozzles, it is noted that the functionalization may not necessarily be applied through ink-jet print heads of KC1, and/or CaCl₂). Under certain circumstances and as required by the biostructure printed, the scaffold precursor for the support of the biostructure (for example; tissue/organ-on-a-chip, tissue scaffold or stand-alone tissue organ printed); may be different from the scaffold precursor in the bio-ink which also comprises the cells, wherein, for example one scaffold support is photo-cured and another bio-ink scaffold support is chemically cured, e.g., using KC1 as well as another bio-ink scaffold support that can be adapted to be biologically cured. Other print heads can then be used with additional bio-inks, each which can provide different cells and/or extra cellular matrix and/or other organic and inorganic components, and based on the 2D images stored in the libraries, be used to create a predetermined, selectable biostructure, which, for example, emulates a single organ, a tissue, or an interface between tissues and organs (e.g., BBB). It is understood, that the order of printing and/or of functionalizing, described herein can be altered and the images providing the various layers or portions thereof can be combined to create various 3D structures using scaffolding material within the cells layers and in combination with the cells' layers, and that the scaffold precursor materials may also change from one type of bio- ink to another.

[00016] In yet another embodiment, the biostructure is a tissue or organ being lung, heart, liver, kidney, gut, airway, skeletal muscle, skin, blood-brain barrier, reproductive/endocrine testis bone marrow, or cartilage and the like.

DETAILED DESCRIPTION

[00017] Provided herein are embodiments of methods, systems and libraries for use in inkjet printing of biostructures having predetermined, non-random 2D and 3D pattern of cells and/or extra cellular matrix and/or other organic and inorganic components. The cells and/or extra cellular matrix and/or other organic and inorganic components can comprise, for example, fibroblasts, chondrocytes, hepatocytes, osteocytes, endothelial cells, muscle cells, mesothelial cells, pericyte cells, macrophage cells, monocyte cells, plasma cells, mast cells, adipocyte cells or a cell population cultured from a specific cell type), cell structures, cells which can be manipulated to multiply and/or change its properties using a trigger, proteins, extracellular matrix (ECM), polysaccharides, enzymes, growth factors (including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), bone morphogenic protein (BMP), glucoseaminoglycan (GAG), transforming growth factor (TGF), insulin-like growth factor (IGF), or epidermal growth factor (EGF)), PEGilated-methacrylate, PEG-dimethacrylate (PEGDMA), carrageenan, poly(lactic) acid, poly(lactic-co-glycolic acid), their methacrylate conjugates, co-polymers, interpenetrating networks, antigens, signaling compound, therapeutically effective compounds, antimicrobial compounds, immunosuppressing compounds, bio-compatible monomers, bio-compatible polymers, bio-compatible oligomers, spheroids, organoids and other biological molecules and structures, biocompatible dispersing media, means for functionalizing or a composition comprising one or more of the foregoing, all with or without combination with inorganic structures.

[00018] The inkjet printing embodiments described provide biostructures having a predetermined 3D structure with cells and/or extra cellular matrix and/or other organic and inorganic components incorporated (imbedded) therein in a non-random 2D and 3D pattern.

[00019] Accordingly and in an embodiment, provided herein is an inkjet bioprinting method for forming a composite biostructure (e.g., lab-on-a-chip, tissue-on-a-chip, tissue scaffold) having predetermined 2D or 3D pattern of cells and/or extra cellular matrix and/or other organic and inorganic components, the method comprising: providing an inkjet printing system in communication with a processing unit having a memory with a library of images corresponding to layers of the biostructures or portions thereof, the system further comprising: a first print head having: at least one aperture, a first bio-ink reservoir, and a first pump configured to supply the first bio-ink through the aperture, wherein the first bio-ink is a composition comprising cells and/or extra cellular matrix and/or other organic and inorganic components; a second print head having: at least one aperture, a second bio-ink reservoir, and a second pump configured to supply the second bio-ink through the aperture, wherein the second bio-ink is a composition comprising cells and/or extra cellular matrix and/or other organic and inorganic components suspended in a bio-compatible dispersing medium, and/or a polymer, monomer, or oligomer; a third print head having: at least one aperture, a third bio-ink reservoir, and a third pump configured to supply the third bio-ink through the aperture, wherein the third bio-ink comprises means for functionalizing thecells and/or extra cellular matrix and/or other organic and inorganic components; and a conveyor, operably coupled to the first print head, the second print head and to the third print head; uploading a first image corresponding to a first layer of the biostructure; based on the first image, using the first inkjet print head, forming a first patterned layer of cells and/or extra cellular matrix and/or other organic and inorganic components or their combination, or portion thereof; using the third print head, performing post treatment, for example, functionalizing the first patterned layer of cells and/or extra cellular matrix and/or other organic and inorganic components or their combination or its portion; based on the first image, using the second inkjet print head, forming a first layer of predetermined pattern of the second bio-ink composition on the treated first patterned layer or portion thereof of the first bio-ink; and using the third print head, treating the first layer of predetermined pattern of the second bio-ink. The steps described above do not necessarily need to take place in the order listed, and can be shuffled and take place in parts over a period where partial biostructure is taken out of the printer, allowed to undergo other treatments and be returned. For example, printed cells may be allowed to undergo several monolayer expansion cycles before the functionalizing or the introduction of the manipulation triggering compounds are printed and only then would functionalizing take place.

[00020] The biostructures described herein may be, for example "Organ-on-a-Chip", "Tissue- on-a-Chip", "Lab-on-a-Chip", structures containing viable cells culture(s), tissue scaffold, stand-alone tissue, structure that emulates an organ, a tissue, or an interface between tissues and organs, bones, cartilage, as well as; an implantable tissue substitute including but not limited to a bone cartilage, liver, epithelial, nerve, vascular and the like, or other tissue substitute for either a portion of a tissue or an entire tissue or organ. The biostructure, or its corresponding matrix, may have dimensions which may be customized for a particular patient and which may be based on medical imaging data and may further include geometric features not present in the medical imaging data. The biostructure may be used in culturing cells outside the body of a patient and to form lab-on-a-chip.

[00021] The term "tissue-on-a chip" as used herein refers in an embodiment to the biostructure described herein wherein the cells configured to form the tissue analog are bioprinted into a given structure to form the biostructure in a shape of a chip or another discrete 3D object.

[00022] The term "bioprinting" refers to a process of making specific type or several types of native or manipulated cells and/or extra cellular matrix and/or other organic and inorganic components configurations using inkjet printer having drop-on-demand capabilities.

[00023] The term "lab-on-a-chip" refers in an embodiment to framework that creates microsystems incorporating one or more steps of an assay into a single biostructure. For example, integrated microfluidic devices fabricated using the methods and systems described herein can be used to perform rapid and reproducible measurements on small sample volumes. Microfluidic s allows in an embodiment to carry out experiments and diagnostics that cannot be performed simply by miniaturizing and mechanizing conventional laboratory procedures using robotics and microplates. For example, in cell-based studies, the transition from 384- to 1,536-wells plates is proving challenging, largely because edge effects and uncontrolled evaporation from very small wells result in poorly defined culture conditions. Conventional handling of very small fluidic volumes is difficult, and subject to both variability and high fixed losses. The fabrication of many copies of an analytic device, small reagent volumes, and diminished labor associated with use of automated micro- fabricated devices provided herein, can address these shortcomings. Moreover, the utilization of 3D printing for the formation of 3D complex structures with high surface area (such as network, helix or lung alveolus) can dramatically reduce the losses and significantly increase the yield, efficiency and effectiveness of diagnostics procedures.

[00024] The term "forming" (and its variants "formed", etc.) refers in an embodiment to pumping, injecting, pouring, releasing, displacing, spotting, circulating, nebulizing, spaying, inkjetting, jetting, or otherwise placing a fluid or material in contact with another material (e.g., the substrate, or another layer) using any suitable inkjet printing method. In an embodiment, "forming" refers to the assembly of the structure itself from its underlying 2D layer images, which, in another embodiment are derived from various raster images (e.g., *.dcm) and/or vector data models.

[00025] As used herein, the term "scaffold", or "scaffolding" refers in an embodiment to an engineered platform having a predetermined 3D structure, which serves as a 3D physical substrate for cells and/or extra cellular matrix and/or other organic and inorganic components. As indicated, the scaffold can also be a composite scaffold. A "composite scaffold" refers to a scaffold platform which is engineered of two or more tissue types which together comprise a "heterogeneous tissue". For example, the systems and methods described herein can be used to form a composite scaffold comprising a first 3D, chondrocytes-embedded biostructure for supporting formation of a first tissue type thereupon and a second 3D, osteocytes-embedded biostructure for supporting formation of a second tissue type thereupon.

[00026] As used herein, the term "print head" refers in an embodiment to any device, module or system or technique that deposits, transfers or creates material on a surface in a controlled accretive or additive manner, or parts thereof.

[00027] The methods, systems and libraries for use in inkjet printing or forming the 3D biostructure can comprise a step of optionally providing a substrate (e.g., a film). The print head depositing the first bio-ink can be configured to provide the ink droplet(s) upon demand, in other words, as a function of various process parameters such as conveyor speed, desired cells layer thickness, layer type (e.g., cells and/or extra cellular matrix and/or other organic and inorganic components) and the like. The substrate can also be a relatively rigid material, for example, glass or crystal (e.g., sapphire) or various waiters. Alternatively, the substrate may be a flexible (e.g., rollable) substrate (or film) and can be, for example, poly(ethylenenaphthalate) (PEN), polyimide (e.g. KAPTONE^® by DuPont), silicon films etc. Moreover, the substrate does not need necessarily to be solid and printing can take place into a vessel containing a liquid into which the biostructure is printed.

[00028] Likewise, other functional heads may be located before, between or after the first print head, the second print head and/or the second print head. These may include a source of electromagnetic radiation used as means of functionalizing polymers (which, in certain circumstances, can be biocompatible polymers, copolymers and their combination), monomers and/or oligomers used in the bio-inks and be configured to emit electromagnetic radiation at a predetermined wavelength (□), for example, between 190 nm and about 450nm, e.g. 420 nm which in an embodiment, can be used to accelerate and/or modulate and/or facilitate a photo-polymerizable monomer/oligomer/polymer or otherwise. Other functional heads can be heating elements, additional printing heads with various inks (e.g., print heads for accurately dispensing different ionic solution for chemical functionalizing, for example by spraying, dispensing, nebulizing, aerosolizing, jetting and the like) and a combination of the foregoing. In addition, all printing heads and the method of forming 3D biostructure can be configured to take place in a housing having controlled atmosphere therein.

[00029] Other similar functional and treating steps (and therefore means for affecting these steps) may be taken before or after the first or second print heads (e.g., for performing post treatment or otherwise treating the predetermined pattern of the second bio-ink comprising the manipulation triggering compounds). These steps may include (but not limited to): a heating step (affected by a heating element, induction, or hot air); exposing to electromagnetic radiation source (using e.g., a UV light source, and/or a xenon lamp); drying (e.g., using vacuum region, or heating element); cross linking (e.g., selectively initiated through the addition of a cross-linking agent to, for example a biocompatible polymer solutions); or a combination thereof. In an embodiment, the 3D structure is formed by the support matrix exterior to the bio-structure as well as by scaffold within the interior of the biostructure.

[00030] Accordingly, in an embodiment, the steps of using the first print head and depositing the first bio-ink onto the substrate, thereby forming a first patterned layer of the first bio-ink comprising a composition consisting of the cells, extra cellular matrix, organic, inorganic components and their combination in the second patterned layer or its portion is different than the first patterned layer and/or the step of using the second print head - depositing the second bio-ink onto and/or adjacent to the first layer of cells), is preceded, followed or takes place concurrently with a step of heating, electromagnetic radiation (EMR) exposure, drying, cross linking, annealing, depositing ionic solution or a combination of steps comprising one or more of the foregoing.

[00031] Formulating the first inkjet composition, may take into account the requirements, if any, imposed by the deposition tool and the surface characteristics (e.g., hydrophilic or hydrophobic, and the surface energy of and optionally provided substrate). Using inkjet with a piezo printing head, the viscosity of either the first bio-ink and/or the second bio-ink (measured at printing temperature) can be, for example, not lower than about 5 cP, e.g., not lower than about 8 cP, or not lower than about 10 cP, and not higher than about 30 cP, e.g., not higher than about 20 cP, or not higher than about 15 cP. The first bio-ink, can be configured (e.g., formulated) to have a dynamic surface tension (referring to a surface tension when an inkjet bio-ink droplet is formed at the print-head aperture) of between about 25 mN/m and about 35 mN/m, for example between about 29 mN/m and about 31 mN/m measured by maximum bubble pressure tensiometry at a surface age of 50 milliseconds (ms.) and at 25°C. The dynamic surface tension can be formulated to provide a contact angle with the substrate of between about 1 degree and about 166 degrees, for example, between about 20 degrees and about 130 degrees, or between about 35 degrees and about 90 degrees. Likewise, contact angles between layers and between various bio-inks can be formulated to provide either wetting or form discrete drops on the surface as needed.

[00032] In an embodiment, the first bio-ink composition used to form the patterned layer of cells and/or extra cellular matrix and/or other organic and inorganic components, and/or the second bio-ink comprising a composition consisting of, for example; bio-compatible polymers, such as PEGilated-methacrylate, PEG-dimethacrylate (PEGDMA), carrageenan, poly(lactic) acid, poly(lactic-co-glycolic acid), their methacrylate conjugates, co-polymers, interpenetrating networks or a composition comprising one or more of the foregoing. Under certain circumstances, it may be advantageous to add polymers or antigen compounds that will cause an immune response. These compounds may also be incorporated in the first and/or second bio-ink.

[00033] The term "biocompatible polymer" refers to any polymer which when in contact with the cells, tissues or body fluid of an organism; does not induce adverse effects such as immunological reactions and/or rejections and the like. In addition, the first and/or second bio-ink used in the methods and systems described herein can be a biodegradable polymer, referring in an embodiment to any polymer which can be degraded in the physiological environment such as by proteases. Examples of biodegradable polymers are; collagen, fibrin, hyaluronic acid, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polydioxanone (PDO), trimethylene carbonate (TMC), polyethyleneglycol (PEG), alginate, chitosan or mixtures thereof.

[00034] In an embodiment, the carrageenan used in the first and/or second bio ink, can be kappa carrageenan (κ-CA) and functionalizing comprises heating and using the third print head - depositing ionic solution comprising KC1, CaCl₂, and their combination. Likewise, the first bio-ink composition comprises poly(ethylenoxide)-dimethacrylate (PEODMA) and functionalizing comprises exposure to EMR. The poly(ethylenoxide)- can be, for example Poly(ethylenglycol). The chemistry and properties can be controlled by varying for example molecular weight, chemical composition, the amount and type of functionalizing agent used and the degree of functionalization, which modifies their mass transport properties, physico-chemical properties and biological properties. Also, poly(ethyleneglycol)-diacrylate (PEG-DA) hydrogels have been shown to be compatible in vivo with porcine islet cells and poly(ethylene glycol) -dimethacrylate (PEG-DMA) hydrogels to be compatible with chondrocytes. By varying the suspension polymer used in the first and/or second bio-ink; biocompatible, or bio-mimetic polymer, the viscosity of the 3D pattern of the embedded cells can be maintained and configured to either prevent colonization and/or manipulation, or alternatively optimize and accelerate manipulation . The biocompatible polymer can also be Chitosan or PEGilated Chitosan where the PEGilated Chitosan is conjugated to methyl methacrylate, or dimethyl methacrylate (e.g. CEG-MA and or CEG-DMA).

[00035] As indicated, the first and/or second bio-inks can be used to form 3D cell-embedded biostructure with compositions from PEG-DMA monomers suspended in aqueous solution and be gelled by radical polymerization in the presence of a photoinitiator. The polymerization reaction starts when the solution is exposed to UV light. Each PEG-DMA monomer has two methacrylate groups which can react with up to two other methacrylate groups to make covalent bonds in other words, cross linking forming a covalently crosslinked branched network. Accordingly, by varying PEG chain length, the concentration of the photoinitiator (e.g., Phenylglyoxylate, benzophenone) duration and wavelength of the EMR exposure, the final compressive modulus of the biostructure support layer, or the predetermined cells' suspending layer can be optimized.

[00036] In an embodiment, the methods, systems and libraries for use in the continuous or semi- continuous inkjet printing of the 3D biostructure can be patterned by expelling droplets of the liquid bio-ink provided herein from an orifice one-at-a-time, as the print-head (or the substrate) is maneuvered, for example in two (X-Y) (it should be understood that the print head can also move in the Z axis) dimensions at a predetermined distance above the substrate or any subsequent layer. The height of the print head can be changed with the number of layers, maintaining for example a fixed distance. Each droplet can be configured to take a predetermined trajectory to the substrate on command by, for example a pressure impulse, via a deformable piezo-crystal in an embodiment, from within a well operably coupled to the orifice. The printing of the first inkjet bio ink can be additive and can accommodate a greater number of layers. The ink-jet print heads provided used in the methods described herein can provide a minimum layer film thickness equal to or less than about 15 μιη-ΙΟ,ΟΟΟ μηι. The velocity of the substrate can depend, for example, on the number of print heads used in the process, the desired 3D structure of the 3 Dbio structure printed, the functionalizing and/or treatment/post-treatment time of the bio-ink(s), the evaporation rate of the ink solvents, the distance between the print head containing the first bio-ink and the second print head comprising the second bio-ink, the desired spatial distribution of the cells in the embedded portion, whatever the biostructure is or a combination of factors comprising one or more of the foregoing.

[00037] In an embodiment, the apparent viscosity of the first bio-ink composition, or the second bio-ink, can each be (before treatment) between about 0.1 and about 30 cP-s (mPa-s) at the printing temperature, for example the final ink formulation can have a viscosity of 8-12 cP-s at the working temperature, which can be controlled. For example, cells' dispersion, solution, emulsion, suspension, hydrogel or liquid composition comprising the foregoing, or the second bio-ink comprising suspended cells can each be between about 5 cP-s and about 25 cP-s, or between about 7 cP-s and about 20 cP-s, specifically, between about 8 cP-s and about 15 cP-s.

[00038] In an embodiment, the volume of each droplet of the bio-ink, and/or the bio-ink, can range of about 5 pL to about 450 picoLiter (pL), for example between about 50 pL and about 150 pL, depended on the printer parameters and the properties of the ink. The waveform to expel a single droplet can be a 10V to about 170 V pulse, or about 16V to about 90V, and can be expelled at frequencies between about 1 kHz and about 500 kHz.

[00039] In addition, to the extent used; polymer concentration (e.g., PEGDMA, Chitosan), although being the same in the first and second bio-ink (in other embodiment, the suspending polymer can be different), can vary in concentration between the bio-inks or the treatment inks, thus providing different physico-chemical characteristic to the hydrogels formed. In an embodiment, after curing (in other words, solidifying the matrix), the scaffold support pattern formed can exhibit compressive modulus (in other words, the ratio between the load and strain needed to achieve irreversible deformation of the gel), used in the systems and methods for direct printing of a composite biostructure having predetermined 3D pattern of cells and scaffold described herein, can be no less than 0.5 MPa, for example, between 0.08 and 1.5 MPa or between about 0.6 MPa and 1.0 MPa. Other compressive moduli can be used for biostructures, for example, for screening of various compounds' therapeutic effect on tissues and organs (e.g. tissue-on-a-chip)

[00040] As indicated, provided herein is an inkjet bioprinting method for forming a composite biostructure having predetermined 2D and/or 3D pattern of cells and/or extra cellular matrix and/or other organic and inorganic components therein, the method comprising: providing an inkjet printing system in communication with a microprocessor coupled to a non-volatile memory having thereon an executable set of instructions configured to perform the method, as well as to a database with a library of 2D images of the various layers to be printed, the system comprising: a first print head having: at least one aperture, a first bio-ink reservoir, and a first pump configured to supply the first bio-ink through the aperture, wherein the first bio-ink is a composition comprising cells and/or extra cellular matrix and/or other organic and inorganic components which, in certain embodiments can be suspended in a bio-compatible dispersing medium, a bio-compatible polymer, monomer, or oligomer and a dispersing medium; a second print head having: at least one aperture, a second bio-ink reservoir, and a second pump configured to supply the second bio-ink through the aperture, wherein the second bio-ink is a composition comprising extra cellular matrix and/or other organic and inorganic components; a third print head having: at least one aperture, a third bio-ink reservoir, and a third pump configured to supply the third bio-ink through the aperture, wherein the third bio-ink comprises means for treating the cells and/or extra cellular matrix and/or other organic and inorganic components; and a conveyor, operably coupled to the first print head, the second print head and to the third print head; following the uploading of a 2D image of a first layer or portion thereof, using the first inkjet print head, forming a first patterned layer cells and/or extra cellular matrix and/or other organic and inorganic components or a portion thereof; using the third print head, treating the first patterned layer of cells and/or extra cellular matrix and/or other organic and inorganic components; using the second inkjet print head, forming a first layer of predetermined pattern of the second bio-ink composition or portion thereof; and using the third print head treating the first patterned layer of the second bio-ink; using the first inkjet print head, forming a second patterned layer of cells and/or extra cellular matrix and/or other organic and inorganic components and/or adjacent to the first patterned layer of the second bio-ink composition or a portion of the first patterned layer of the second bio-ink; and treating the second patterned layer of cells and/or extra cellular matrix and/or other organic and inorganic components and in addition, using the second inkjet print head, forming a second layer of predetermined pattern of the second bio-ink on and/or adjacent to the first patterned layer of the second bio-ink composition; and treating the second patterned layer of the second bio-ink or a portion thereof.

[00041] As indicated, other functional print heads can be incorporated into the systems and methods for direct printing of a composite biostructure having predetermined 2D and/or 3D pattern of cells and/or extra cellular matrix and/or other organic and inorganic components and biostructure described herein. Thus, in an embodiment, the inkjet printing system further comprises a third print head having: at least one aperture, a reservoir for an ionic composition, and a third pump configured to supply the ionic composition through the aperture, the method comprises, in the step of treating the first bio-ink and/or treating the second-bio-ink, using the third inkjet print head; depositing the ionic composition on the layer of cells and/or extra cellular matrix and/or other organic and inorganic components and/or the predetermined pattern of the second bio-ink, wherein the ionic composition is an ionic solution comprising; for example, KC1, CaCl₂, or a combination thereof. Accordingly and in an embodiment functionalization may take place through depositing the functionalizing by means of, for example, ink jet print heads, spray dyes, nebulizing nozzles, injectors etc. Furthermore, functionalizing means may not necessarily be an integral part of the printer used and can take place externally to the printer.

[00042] Likewise, the predetermined 3D pattern of the second bio-ink, embedded in the layer of cells and/or extra cellular matrix and/or other organic and inorganic components, or otherwise in the biostructures described herein, is non-random. In other words, the biostructure has a substantial variation in the spatial distribution and/or density of the cells and/or extra cellular matrix and/or other organic and inorganic components, forming a predetermined 2D (gleaned for example, from MRI and/or CT images converted to raster and/or vector data models and converted to ink-jet printing instructions) and/or 3D pattern of cells and/or extra cellular matrix and/or other organic and inorganic components, or otherwise in the biostructures. For example, the predetermined 3D pattern of the second bio-ink can be configured to; accelerate cell adhesion, retain native growth of the cells, and/or organ and/or tissue and other similar functions. Furthermore, the 3D scaffolding support and the cell- laden scaffold can be configured to about a complementary portion of a tissue or organ surface, or in another embodiment, have a surface configured to abut the interface between two tissues or organs (for example in circumstances requiring a composite scaffold).

[00043] Moreover, the second bio-ink, can further comprise other additives that affect colonization, proliferation or other manipulation of the cells, retain native growth of the cells, and/or organ and/or tissue and other similar functions. Accordingly and in an embodiment, the second bio- ink used in the methods, systems and libraries for use in inkjet bioprinting of a composite

biostructure having predetermined 3D pattern Of cells and/or extra cellular matrix and/or other organic and inorganic components, or otherwise in the biostructures described herein, can further comprise fibroblasts, chondrocytes, hepatocytes, osteocytes, endothelial cells, muscle cells, mesothelial cells, pericyte cells, macrophage cells, monocyte cells, plasma cells, mast cells, adipocyte cells or a cell population cultured from a specific cell type), cell structures, cells which can be manipulated to multiply and/or change its properties using a trigger, proteins, extracellular matrix (ECM), polysaccharides, enzymes, growth factors (including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), bone morphogenic protein (BMP), glucoseaminoglycan (GAG), transforming growth factor (TGF), insulin-like growth factor (IGF), or epidermal growth factor (EGF)), PEGilated-methacrylate, PEG- dimethacrylate (PEGDMA), carrageenan, poly(lactic) acid, poly(lactic-co-glycolic acid), their methacrylate conjugates, co-polymers, interpenetrating networks, antigens, signaling compound, therapeutically effective compounds, antimicrobial compounds, immunosuppressing compounds, bio-compatible monomers, bio-compatible polymers, bio-compatible oligomers, spheroids, organoids and other biological molecules and structures, bio-compatible dispersing media, means for functionalizing or a composition comprising one or more of the foregoing and the like.

[00044] In an embodiment, the systems and methods described herein can also be used for fabrication of biostructure comprising microfluidic channels and similar platforms, whereby the controlled microenvironment of microfluidic platforms can be very useful in the study of various types of biostructures and the impact of various molecules, compounds and physico-chemical conditions in cell proliferation and manipulation. Manipulating the temporospatial chemical environment of the culture can facilitate behavior of various cells, to be controlled. Using the biostructure fabricated using the methods described herein, screening of treatments can be achieved. Likewise, the direct, high precision and resolution fabrication described herein can allow for the direct printing of channels, conduits and wells in the biostructure support layer. [00045] Accordingly, and in an embodiment, the methods provided herein are used to print biostructures, comprising a step of printing a plurality of spatially concentrated cells forming wells' array (referring to an array of two or more wells fabricated in the biostructure) and further comprising direct printing of microfluidic conduits between at least two wells in the array. The term "well" or microwell, refers in an embodiment to a micro-scale chamber (optionally enclosed); able to accommodate a plurality of cells. A well can be, for example cylindrical in shape and has diameter and depth dimensions of between about 100 and 1500 microns, 10 and 500 microns, respectively. The term "microfluidic conduit" refers in an embodiment to a micron- scale enclosed or open channel used for connecting a functional portion in the biostructure (e.g., lab-on-a-chip) with a well, or a functional portion associated with the well. A microfluidic conduit can have a quadrilateral, e.g., square cross- section, with side and depth dimensions of between about 10 and 500 microns, and 10 and 500 microns, respectively. Fluids flowing in the microfluidic conduits may exhibit microfluidic behavior.

[00046] The term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[00047] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. "Combination" is inclusive of blends, mixtures, alloys, reaction products, and the like. The terms "a", "an" and "the" herein do not denote a limitation of quantity, and are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The suffix "(s)" as used herein is intended to include both the singular and the plural of the term that it modifies, thereby including one or more of that term (e.g., the image(s) includes one or more images). Reference throughout the specification to "one embodiment", "another embodiment", "an embodiment", and so forth, when present, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

[00048] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. Furthermore, the terms "first," "second," and the like, herein do not denote any order, quantity, or importance, but rather are used to denote one element from another.

[00049] Likewise, the term "about" means that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is "about" or "approximate" whether or not expressly stated to be such.

[00050] In an embodiment, provided herein is a inkjet bioprinting method for forming a biostructure having predetermined 3D pattern of cells and/or extra cellular matrix and/or other organic and inorganic components, or otherwise in the biostructures therein, the method comprising: providing an inkjet printing system comprising: a microprocessor in communication with: a non-volatile memory having thereon a microprocessor-readable medium with set of executable instructions configured, when executed to cause a processor to execute the method of inkjet bioprinting; and a library images corresponding to a 2D layer of cells, extra cellular matrix, organic, inorganic components and their combination within the biostructure having predetermined 3D pattern of the cells, extra cellular matrix, organic, inorganic components and their combination therein; a first print head in communication with the microprocessor having: at least one aperture, a first bio-ink reservoir, and a first pump configured to supply the first bio-ink through the aperture, wherein the first bio-ink is a composition consisting of cells, extra cellular matrix, organic, inorganic components and their combination; a second print head in communication with the microprocessor having: at least one aperture, a second bio-ink reservoir, and a second pump configured to supply the second bio-ink through the aperture, wherein the second bio-ink is a composition consisting of cells, extra cellular matrix, organic, inorganic components and their combination; a third print head in communication with the microprocessor having: at least one aperture, a third bio-ink reservoir, and a third pump configured to supply the third bio-ink through the aperture, wherein the third bio-ink comprises means for treating the cells, extra cellular matrix, organic, inorganic components and their combination; and a conveyor in communication with the microprocessor, operably coupled to the first print head, the second print head and to the third print head; uploading from the library a first image corresponding to a first two-dimensional (2D) layer of cells, extra cellular matrix, organic, inorganic components and their combination within the biostructure having predetermined 3D pattern of cells, extra cellular matrix, organic, inorganic components and their combination; using the first inkjet print head, forming a first patterned layer of the first bio-ink; using the third print head, treating the first patterned layer of the first bio-ink; using the second inkjet print head, forming a first layer of predetermined pattern of the second bio-ink; and using the third print head, treating the first patterned layer of the second bio-ink, wherein (i) the step of treating the first patterned layer of the first bio-ink and/or the step of treating the first patterned layer of the second bio-ink comprises depositing ionic solution, cross linking, exposure to electromagnetic radiation, or a combination comprising one or more of the foregoing, wherein (ii) the first bio-ink composition, the second bio-ink composition or both comprises: PEGilated-methacrylate, chitosan, carrageenan, poly(lactic) acid, poly(lactic-co-glycolic acid), their methacrylate conjugates, co-polymers, interpenetrating networks or a composition comprising one or more of the foregoing (iii), the first bio-ink comprises one or more of endothelial cells, muscle cells, fibroblast cells, mesothelial cells, pericyte cells, macrophage cells, monocyte cells, plasma cells, mast cells, adipocyte cells, chondrocyte cells, cells population manipulated from a specific type of cell to another, or cells composition comprising one or more of the foregoing cells; a bio-compatible polymer; and optionally a photoinitiator, (iv) the first bio-ink, the second bio-ink composition or both is a suspension, an emulsion, a duplex emulsion, or a liquid composition comprising one of the foregoing, wherein (v) the carrageenan is kappa carrageenan and treating comprises heating and using the third print head; depositing ionic solution comprising KCl, CaCb, or a composition comprising the foregoing, wherein (vi) the first bio-ink composition comprises PEGilated-methacrylate and functionalizing comprises exposure to electromagnetic radiation, wherein (vii) the biostructure exhibits compressive modulus is no less than 0.08 MPa, wherein (viii) the extracellular matrix composition of the second bio-ink comprises a composition configured to accelerate cell adhesion, promote cell colonization, proliferation, or manipulation, retain native growth of the cells, and/or organ and/or tissue, wherein (ix) the composition of the second bio-ink comprises epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), bone morphogenic protein (BMP), insulin-like growth factor (IGF), glucoseaminoglycan (GAG), Transforming growth factor (TGF) or extracellular matrix compound composition comprising the foregoing, the method further comprising (x) , subsequent to the step of treating the first layer of predetermined pattern of the second bio-ink: uploading from the library a second image corresponding to a second or subsequent 2D layer or portion thereof of cells within the biostructure having predetermined 3D pattern of cells; using the first inkjet print head, forming a second patterned layer or portion thereof of the first bio-ink, on and/or adjacent to the first patterned layer or portion thereof of the second bio- ink composition; and using the third print-head, treating the second patterned layer of the first bio-ink, and further comprising (xi) using the second inkjet print head, forming a second layer or portion thereof of predetermined pattern of the second bio-ink on and/or adjacent to the first patterned layer or a portion thereof of the first patterned layer of the second bio-ink composition; and treating the second patterned layer or portion thereof of the second bio-ink, wherein (xii) the cells, extra cellular matrix, organic, inorganic components and their combination in the second patterned layer or its portion is different than the first patterned layer, wherein (xiii) the biostructure is a lab-on-a-chip, a tissue-on-a-chip, organ-on-a-chip, organ, tissue, or a tissue scaffold, and wherein (xiv) the predetermined 3D pattern of the first bio-ink in the biostructure is non-random.

[00051] While in the foregoing specification the methods, systems and libraries for use in the inkjet printing of biostructures having predetermined two- (2D) and three- dimensional (3D) patterns of cells, extra cellular matrix, organic, inorganic components and their combination described herein have been described in relation to certain embodiments, and many details are set forth for purpose of illustration, it will be apparent to those skilled in the art that the disclosure is susceptible to additional embodiments and that certain of the details described in the specification and that are more fully delineated in the following claims, can be varied considerably without departing from the basic principles of this invention.

Claims

What is claimed:

1. An inkjet bioprinting method for forming a biostructure having predetermined 3D pattern of cells and/or extra cellular matrix and/or other organic and inorganic components, or otherwise in the biostructures therein, the method comprising: a. providing an inkjet printing system comprising: i. a microprocessor in communication with: a non-volatile memory having thereon a microprocessor-readable medium with set of executable instructions configured, when executed to cause a processor to execute the method of inkjet bioprinting; and a library images corresponding to a 2D layer of cells, extra cellular matrix, organic, inorganic components and their combination within the biostructure having predetermined 3D pattern of the cells, extra cellular matrix, organic, inorganic components and their combination therein; ii. a first print head in communication with the microprocessor having: at least one aperture, a first bio-ink reservoir, and a first pump configured to supply the first bio-ink through the aperture, wherein the first bio-ink is a composition consisting of cells, extra cellular matrix, organic, inorganic components and their combination; iii. a second print head in communication with the microprocessor having: at least one aperture, a second bio-ink reservoir, and a second pump configured to supply the second bio- ink through the aperture, wherein the second bio-ink is a composition consisting of cells, extra cellular matrix, organic, inorganic components and their combination; iv. a third print head in communication with the microprocessor having: at least one aperture, a third bio-ink reservoir, and a third pump configured to supply the third bio-ink through the aperture, wherein the third bio-ink comprises means for treating the cells, extra cellular matrix, organic, inorganic components and their combination; and v. a conveyor in communication with the microprocessor, operably coupled to the first print head, the second print head and to the third print head; b. uploading from the library a first image corresponding to a first two-dimensional (2D) layer of cells, extra cellular matrix, organic, inorganic components and their combination within the biostructure having predetermined 3D pattern of cells, extra cellular matrix, organic, inorganic components and their combination; c. using the first inkjet print head, forming a first patterned layer of the first bio-ink; d. using the third print head, treating the first patterned layer of the first bio-ink; e. using the second inkjet print head, forming a first layer of predetermined pattern of the second bio-ink; and f. using the third print head, treating the first patterned layer of the second bio-ink.

2. The method of claim 1, wherein the step of treating the first patterned layer of the first bio- ink and/or the step of treating the first patterned layer of the second bio-ink comprises depositing ionic solution, cross linking, exposure to electromagnetic radiation, or a combination comprising one or more of the foregoing.

3. The method of any one of claims lor 2, wherein the first bio-ink composition, the second bio- ink composition or both comprises: PEGilated-methacrylate, chitosan, carrageenan, poly(lactic) acid, poly(lactic-co-glycolic acid), their methacrylate conjugates, co-polymers, interpenetrating networks or a composition comprising one or more of the foregoing.

4. The method of claim 3, wherein the first bio-ink comprises one or more of endothelial cells, muscle cells, fibroblast cells, mesothelial cells, pericyte cells, macrophage cells, monocyte cells, plasma cells, mast cells, adipocyte cells, chondrocyte cells, cells population manipulated from a specific type of cell to another, or cells composition comprising one or more of the foregoing cells; a bio-compatible polymer; and optionally a photoinitiator.

5. The method of claim 4, wherein the first bio-ink, the second bio-ink composition or both is a suspension, an emulsion, a duplex emulsion, or a liquid composition comprising one of the foregoing.

6. The method of claim 3, wherein the carrageenan is kappa carrageenan and treating comprises heating and using the third print head; depositing ionic solution comprising KC1, CaCl₂, or a composition comprising the foregoing.

7. The method of claim 3, wherein the first bio-ink composition comprises PEGilated- methacrylate and functionalizing comprises exposure to electromagnetic radiation.

8. The method of claim 1, wherein the biostructure exhibits compressive modulus is no less than 0.08 MPa.

9. The method of claim 1, wherein the extracellular matrix composition of the second bio-ink comprises a composition configured to accelerate cell adhesion, promote cell colonization, proliferation, or manipulation, retain native growth of the cells, and/or organ and/or tissue.

10. The method of Claim 9, wherein the composition of the second bio-ink comprises epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), bone morphogenic protein (BMP), insulin-like growth factor (IGF), glucoseaminoglycan (GAG), Transforming growth factor (TGF) or extracellular matrix compound composition comprising the foregoing.

11. The method of claim 5, further comprising, subsequent to the step of treating the first layer of predetermined pattern of the second bio-ink: a. uploading from the library a second image corresponding to a second or subsequent 2D layer or portion thereof of cells within the biostructure having predetermined 3D pattern of cells; b. using the first inkjet print head, forming a second patterned layer or portion thereof of the first bio-ink, on and/or adjacent to the first patterned layer or portion thereof of the second bio-ink composition; and c. using the third print-head, treating the second patterned layer of the first bio-ink.

12. The method of claim 11, further comprising: a. using the second inkjet print head, forming a second layer or portion thereof of predetermined pattern of the second bio-ink on and/or adjacent to the first patterned layer or a portion thereof of the first patterned layer of the second bio-ink composition; and b. treating the second patterned layer or portion thereof of the second bio-ink.

13. The method of claim 12, wherein the cells, extra cellular matrix, organic, inorganic components and their combination in the second patterned layer or its portion is different than the first patterned layer.

14. The method of claim 1, wherein the biostructure is a lab-on-a-chip, a tissue-on-a-chip, organ- on-a-chip, organ, tissue, or a tissue scaffold.

15. The method of claim 14, wherein the predetermined 3D pattern of the first bio-ink in the biostructure is non-random.