Stabilization of high-density cell bioinks

Abstract

Additive manufacturing methods, additive manufacturing systems, structure materials for additive manufacturing, and products thereof are provided. The structure material includes at least 1 mg / mL of a bio-compatible polymer, a solvent, at least 10 million cells / mL, and a polymerization inhibitor.

Description

IN THE UNITED STATES PATENT AND TRADEMARK OFFICEPCT APPLICATION FORSTABILIZATION OF HIGH-DENSITY CELL BIOINKSInventors: Brian Coffin, Adam Walter Feinberg, Phillip Campbell, Samuel MossRELATED APPLICATIONS

[0001] The present application claims priority to United States provisional patent application Serial No. 63 / 533,091, filed August 16, 2023, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Recently, 3 -dimensional (3D) bio-printing has emerged as a viable platform for engineering tissues, with exciting applications as platforms for drug discovery and disease modeling as well as new therapies for tissue regeneration. However, translation of these technologies from the laboratory into industry and the clinic has been limited, in large part due to the poor reproducibility of the 3D bio-printing process itself. There are challenges with 3D bio-printing.SUMMARY

[0003] In one general aspect, the present disclosure is directed to a structure material for additive manufacturing. The structure material comprises at least 1 mg / mL of a biocompatible polymer, a solvent, at least 10 million cells / mL, and a polymerization inhibitor.

[0004] In another general aspect, the present disclosure is directed to a structure material for additive manufacturing. The structure material comprises 30 mg / mL to 200 mg / mL of fibrinogen, water, at least 100 million eukaryotic cells / mL, a buffer, and 1 pM to 50 mM of bilvalirudin.

[0005] In yet another general aspect, the present disclosure is directed to an additive manufacturing method. The method comprises depositing, by a nozzle, a structure material into a support material based on a first computer model of an object, thereby forming a first portion of the object in the support material. The structure material comprises at least 1 mg / mL of a bio-compatible polymer, a solvent, at least 10 million cells / mL, and a polymerization inhibitor. The method comprises repeating, by the nozzle, the depositing of the structure material as necessary to additively form the object.

[0006] Various embodiments and implementations of the present invention provide many benefits and improvements relative to prior additive printing techniques, such as, for example, techniques related to embedded printing. For example, the methods according to the present disclosure can enhance filament resolution, prevent formation of agglomerates on the needle, control diffusion of structure material, control gelation of structure material, control reaction rate, enable a wider range of reactant concentrations to be utilized in the support material, and enable wider range of cell and / or polymer concentrations to be used in the structure material. These and other benefits that are potentially realizable through various implementations of the present invention will be apparent from the description that follows.

[0007] It will be understood that the invention disclosed and described in this specification is not limited to the aspects summarized in this Summary. The reader will appreciate the foregoing details, as well as others, upon considering the following detailed description of various non-limiting and non-exhaustive aspects according to this specification.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Features and advantages of the examples presented herein, and the manner of attaining them, will become more apparent, and the examples will be better understood, by reference to the following description taken in conjunction with the accompanying drawings, wherein:

[0009] FIG. 1 is a block diagram of an example of an additive manufacturing FRE system according to the present disclosure, the X-axis is coming out of the page.

[0010] FIG. 2 is flow chart of an example of an additive manufacturing FRE method according to the present disclosure.

[0011] FIG. 3 A is a window frame CAD model with top and isometric views and a geode preview model.

[0012] FIG. 3B is representative images of scaffolds fabricated with baseline bioink while embedded in microparticle support, in melted support material and after media exchange. The structure breaks apart partially after release and fractures during washing.

[0013] FIG. 4A is a graphical protocol for generation of a high cell density bioink where cells are (i) dissociated by a method appropriate for the cell line. Any pretreatments to the cells are applied at (i). (ii) A high cell density pellet in a syringe is then collected by transferring centrifuged cells to a 1 mL syringe and recompacted at 500 ref for 5 minutes in a custom syringe centrifugation adapter, (iii) The supernatant is decanted from the syringe and the remaining cell pellet is mixed with an equal volume of 120 mg / mL fibrinogen stock to createa bioink with bulk fibrinogen concentration of 60 mg / mL. Cells are counted at (iii) with a hemocytometer. The bioink cell density is enriched by centrifugation at 1000 ref for 5 minutes, (iv) To create bioinks with 100, 200, or 300xl06cells / mL, a known volume of supernatant is removed to reach the desired concentration and the remaining bioink is homogenized with a viscous pipette.

[0014] FIG. 4B is a graph of parallel plate rheological characterization of cellular bioink storage and loss moduli when prepared with 0, 100, 200, and 300xl06cells / mL.

[0015] FIG. 4C is a graph of terminal gel storage modulus illustrating bioink gel storage modulus observed between prepared bioinks.

[0016] FIG. 4D is images of an example of a bioink that developed distinct phases and an example of a bioink that did not develop distinct phases.

[0017] FIG. 5 is images of window frame models to test ability to maintain structural integrity of single filament width features after release of the print with high cell density bioinks. The released model had 100% of the single filament width features were intact after release from the support bath. The construct was fixed and stained with DAPI to visualize the cell nuclei density in the fabricated construct. The construct appeared to shrink slightly after fixation causing the filaments to appear wavy. The fluorescent image is a maximum intensity projection of a 250 pm z stack. Scale bars are 2 mm for first three panels and 1 mm in the fluorescent image.

[0018] FIG. 6A is a geode preview model of a single scaffold that was printed every 10 minutes for 2 hours. Syringe pressure is continuously recorded during the fabrication process to assess the effect of various treatments on bioink stability.

[0019] FIG. 6B is representative darkfield stereomicrographs of scaffolds printed at 0, 60, and 90 minutes after initiating the print. Scale bar = 2 mm.

[0020] FIG. 6C is a graph of scaffold area of tissues at various timepoints normalized to the first scaffold fabricated.

[0021] FIG. 6D is a graph of syringe extrusion pressure traces for C2C12 bioinks prepared with no treatment (control).

[0022] FIG. 6E is a graph of syringe extrusion pressure traces for C2C12 bioinks prepared with indirect thrombin inhibition with 50 mM EDTA.

[0023] FIG. 6F is a graph of syringe extrusion pressure traces for C2C12 bioinks prepared with direct thrombin inhibition with 5 pM bivalirudin stabilization treatments.

[0024] FIG. 6G is a graph of average extrusion pressure during printing for each treatment in FIGs. 6D-6E.

[0025] FIG. 7A is darkfield micrographs of rings fabricated from C2C12 bioinks with 100, 200, and 300xl06cells / mL.

[0026] FIG. 7B is a graph illustrating tracking of outer ferret diameters of C2C12 rings cultured in C2C12-GM for 8 days. Scale bars = 2 mm.

[0027] FIG. 7C is a graph illustrating tracking of inner ferret diameters of C2C12 rings cultured in C2C12-GM for 8 days. Scale bars = 2 mm.

[0028] FIG. 8A is a geode preview model and darkfield micrographs of embedded ring and grid models fabricated from C2C12, 3T3, primary embryonic chick muscle, and cardiac bioinks.

[0029] FIG. 8B is a graph of mean percent viable cells for tissues printed from bioinks of each cell type. Data shown as mean ± Standard Deviation. Statistics based on one way analysis of variance test with Tukey’s multiple comparisons test ( *p < 0.05).

[0030] FIG. 8C is max intensity projection images of ring models showing live and dead cells. Scale bars = 2 mm.

[0031] FIG. 9A is a graphical representation for fabrication of aligned skeletal muscle strips from C2C12 bioinks. Bioinks were prepared with 300x106 C2C12 cells / mL then engineered muscle strip models with four unique infill orientation angles (0°, 30°, 60° and ±45°) were created by modification of infill direction in slicing software. Aligned muscle strips were printed from the bioink and constrained on PDMS pillars for culture.

[0032] FIG. 9B is representative images of tissues after printing, at start of differentiation, and after culture. Scale bar = 1 mm.

[0033] FIG. 9C is max intensity projection images of 45x45 tissues after culture period stained for nuclei, actin, and myosin heavy chain. The lower and upper 60 pm of the stack were split into separate images to quantify myotubes in each printed filament. Scale bar = 50 pm

[0034] FIG. 9D is a box and violin plot of myotube orientation angle by stack slice depth. Upper and lower filaments were separated at 62.5 pm. Data plotted as means with 10-90th percentile error bars.

[0035] FIG. 9E is a graph of upper and lower filaments that have a significantly different (****p < 0.001) mean orientation angle by filament position analyzed by two-sided t-test.

[0036] FIG. 10A is a print path preview model of high infill density muscle strips with 0°, 90°, and rotating 0° - 90° infill directions and stereomicrographs of muscle strips after 1, 5, and 10 days of culture on PDMS posts. Scale bar = 2 mm.

[0037] FIG. 10B is whole mount average intensity projection images of muscle strips stained for nuclei, actin, and MYL2. Second harmonic multiphoton imaging was utilized to visualize cell deposited collagen in the tissue. Scale bars = 2 mm, 50 pm (left, right).

[0038] Corresponding reference characters indicate corresponding parts throughout the several views. The exemplifications set out herein illustrate certain embodiments, in one form, and such exemplifications are not to be construed as limiting the scope of the appended claims in any manner.DETAILED DESCRIPTION

[0039] Various examples are described and illustrated herein to provide an overall understanding of the structure, function, and use of the disclosed installation apparatus, fasteners, and methods of fastening. The various examples described and illustrated herein are non-limiting and non-exhaustive. Thus, the invention is not limited by the description of the various non-limiting and non-exhaustive examples disclosed herein. Rather, the invention is defined solely by the claims. The features and characteristics illustrated and / or described in connection with various examples may be combined with the features and characteristics of other examples. Such modifications and variations are intended to be included within the scope of this specification. As such, the claims may be amended to recite any features or characteristics expressly or inherently described in, or otherwise expressly or inherently supported by, this specification. Further, Applicant reserves the right to amend the claims to affirmatively disclaim features or characteristics that may be present in the prior art. The various examples disclosed and described in this specification can comprise, consist of, or consist essentially of the features and characteristics as variously described herein.

[0040] As used herein, “additive manufacturing” means a process of joining materials to make objects from 3D model data, usually layer upon layer, as opposed to subtractive manufacturing methodologies. For example, additive manufacturing can comprise fused deposition modeling (FDM) and Freeform Reversible Embedding (FRE). FDM can comprise extruding a material by heating it to a temperature above its melting temperature and depositing the extruded material in a pattern to form a layer of an object. Subsequent layers can be deposited on top of the previous layer as necessary to form an object.

[0041] FRE is similar to FDM, but instead of depositing a material on top of previous depositions or supports, FRE embeds structure material near other embedded deposits inside a support material and relies on the triggered assembly or reorganization of the material using targeted heating, photopolymerization, crosslinking, slow reaction kinetics, application ofbinders, and / or other curing technique. For example, the support material may provide divalent cations for crosslinking, such that when the structure material contact the support material, the structure material begins to cure.

[0042] For additive manufacturing techniques such as FDM, support materials are usually as stiff as the printed material, printed as part of the previous layer, and placed only underneath or neighboring the print layers to prevent deformations. In FRE, the support material can surround the extrusion nozzle and the print material can be deposited inside the support. The support material can be a non-newtonian fluid that allows for deposition of various materials while maintaining a buoyant, physical support for already embedded deposits of print material. When two embedded deposits of print material with a predetermined distance inside of the support material, they can fuse. After printing, the support material can be removed from the deposited print material to form a fully assembled object from the deposited print material.

[0043] In FRE, an object can be printed in any direction in 3D space and is not limited to layer-by-layer printing. For example, a structure can also be printed layer by layer in an X-Y plane, or a non-X-Y plane, such as the X-Z plane, or in a plane at any angle offset from the X-Y Plane. An object can also be printed utilizing FRE in a non-planar fashion, such as, for example, in a curved path such as a helix. Utilizing FRE can enable printing of objects with mechanical properties that are different in the plane of printing versus orthogonal to the plane of printing or other angle to the plane of printing. Additional details regarding the FRE process can be found in U.S. Patent No. 10,150,258, titled ADDITIVE MANUFACTURING OF EMBEDDED MATERIALS, filed January 29, 2016, which is hereby incorporated by reference herein.

[0044] As the demand for donor tissue and organs continues to outpace the supply, clinicians are turning to regenerative medicine and tissue engineering strategies to create tissue de novo. 3D bioprinting has emerged as a way to build these tissues using robotic control to precisely pattern cells and biological hydrogels in a layer-by-layer process. However, this technology has been slowed by the difficulty of printing these soft, deformable materials into complex 3D architectures that recapitulate anatomic structure from the micro to macro length scale. Embedded 3D bioprinting, such as FRE is a FRE of Suspended Hydrogels (FRESH), can addresses this challenge by providing a sacrificial support that prevents tissue deformation during bioprinting to produce highly relevant constructs such as heart valves, cardiac tissue scaffolds, and perfusable vascular networks. These sophisticated bioprinted constructs could prove very useful as clinical diagnostic tools or direct tissue replacements. However, for anapproved therapy, ensuring accuracy of the manufactured product may be desired. To continue to move bioprinting techniques such as FRESH towards clinical application, the present disclosure provides improved printing capabilities to allow for high-fidelity reproduction of patient-specific organic shapes, and / or improved quality control of geometric fidelity. In embedded 3D bioprinting techniques such as FRESH and related approaches, a challenge can be controlling the gelation of the structure material during extrusion.

[0045] High cell density bioinks are desirable for tissue engineering of cellularized tissue constructs for a wider range of applications including disease models for pharmaceutical discovery and regenerative medicine. Specifically, cellularized bioinks are desirable for 3D bioprinting, where the ability to precisely place specific cell types in 3D space is required to build tissues. However, to date the majority of cell laden bioinks have a cell density that is low (<50 million cells / mL) compared to native tissue (>100 million cells / mL). Fibrinogen- cell bioinks have not previously been utilized to produce high cell density tissues with bioprinting because they do not have the desirable characteristics, such as, for example, viscosity and yield stress properties required by many bioprinting processes.

[0046] The present disclosure provides methods, systems, and materials that can enhance quality control, and process reliability during the FRE process, other 3D bio-printing process, or other additive manufacturing process. The structure material according to the present disclosure can comprise at least 1 mg / mL of a bio-compatible polymer, a solvent, at least 10 million cells / mL, and a polymerization inhibitor. The structure material can be used in an additive manufacturing method comprising depositing the structure material into a support material based on a first computer model of an object, thereby forming a first portion of the object in the support material. The method comprises repeating the depositing of the structure material as necessary to additively form the object. The methods, systems, and materials according to the present disclosure can enhance filament resolution, prevent formation of agglomerates on the needle, control diffusion of structure material, control gelation of structure material, control of reaction rate, enable a wider range of reactant concentrations to be utilized in the support material, and / or enable a wider range of cells and / or polymer concentrations to be utilized in the structure material. It is also believed the present disclosure applies to other additive manufacturing techniques in addition to FRE.

[0047] Referring to FIG. 1, a block diagram illustrating an example of an additive manufacturing system 100 for additive manufacturing, such as, for example, FRE, according to the present disclosure is provided. The system 100 comprises an extruder assembly 102, a computer system 104, and a material deposition region 106. In various examples, a detector,additional extruders, and / or nozzles may be added to the additive manufacturing system to increase the printing capabilities of the additive manufacturing system. The system 100 can be capable to print structure material in the support material 108 in the material deposition region 106 using the extruder assembly 102 to form an object 114.

[0048] The structure material can comprise a yield stress material that transitions between a fluid (e.g., liquid) state to a solid or semi-solid state by application of a pressure. For example, the structure material can be in a solid or semi-solid state in the extruder assembly 102, a pressure can be applied to the structure material to transition the structure material to a fluid state such that the structure material can flow through the nozzle 110 and can be deposited into the support material 108. After leaving the nozzle 110, the applied pressure to the structure material is removed and the structure material can transition into a solid or semisolid state and thereby resist deformation while in the material deposition region 106.

[0049] The structure material can comprise a bio-compatible polymer, a solvent, cells, and a polymerization inhibitor. The structure material can comprise other additives as desired.

[0050] The biocompatible polymer can comprise a synthetic hydrogel, a naturally derived hydrogel, a polysaccharide solution, a mammalian derived material, a polymer network, or a combination thereof. For example, the bio-compatible polymer can comprise a collagen material, an alginate material, a decelluarized extracellular matrix material, a fibrinogen material, a fibrin material, a hyaluronic acid material, a protein material, a polysaccharide hydrogel material, a synthetic gel material, an elastomeric polymer material, a rigid polymer material, a Matrigel, or a combination thereof. In various examples, the polymer can comprise a fibrinogen material. In certain examples, the polymer can comprise a fibrin material.

[0051] The structure material can comprise at least 1 mg / mL of the biocompatible polymer, such as, for example, at least 10 mg / mL, at least 20 mg / mL, at least 25 mg / mL, at least 30 mg / mL, or at least 50 mg / mL of the biocompatible polymer. For example, the structure material can comprise 1 mg / mL to 200 mg / mL of the of the bio-compatible polymer, such as, for example, 10 mg / mL to 200 mg / mL, 20 mg / mL to 200 mg / mL, 25 mg / mL to 200 mg / mL, 30 mg / mL to 200 mg / mL, or 20 mg / mL to 75 mg / mL of the biocompatible polymer.

[0052] In various examples, the structure material can comprise at least 1 mg / mL of the fibrinogen, such as, for example, at least 10 mg / mL, at least 20 mg / mL, at least 25 mg / mL, at least 30 mg / mL, or at least 50 mg / mL of fibrinogen. For example, the structure material can comprise 1 mg / mL to 200 mg / mL of the of fibrinogen, such as, for example, 10 mg / mL to200 mg / mL, 20 mg / mL to 200 mg / mL, 25 mg / mL to 200 mg / mL, 30 mg / mL to 200 mg / mL, or 20 mg / mL to 75 mg / mL of fibrinogen.

[0053] The solvent can be aqueous and suitable for use with cells. For example, the solvent can be water.

[0054] The cells can be eukaryotic cells derived from an animal. The cells can be for various tissues, such as, for example, dermal, muscle, liver, kidney, and / or pancreatic tissues.

[0055] The structure material can comprise at least 10 million cells / mL, at least 20 million cells / mL, at least 30 million cells / mL, at least 40 million cells / mL, at least 50 million cells / mL, at least 75 million cells / mL, at least 100 million cells / mL, at least 150 millions cells / mL, or at least 200 million cells / mL. The use of the polymerization inhibitor can enable higher concentrations of cells to be used during printing while maintaining properties of the structure material that are suitable for printing over a period of time.

[0056] For example, the polymerization inhibitor can be a bioactive component that can inhibit cell agglomeration and / or adhesion at high cell densities. The polymerization inhibitor can stabilize interactions between cells and the bio-compatible polymer in the structure material prior to and / or during deposition, which can enable higher concentrations of cells and / or biocompatible polymer to be used. The polymerization inhibitor can reduce unwanted gelation, which can enhance the ability to print structurally complex and / or functional tissue constructs.

[0057] The polymerization inhibitor can be various small molecules and / or biomolecules that directly and / or indirectly interfere with the curing and / or gelation of the biocompatible polymer. For example, the polymerization inhibitor can comprise bivalirudin, hirudin, argatroban, dabigatran etexilate, ximelagatran, heparin, L-ascorbic acid, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'- tetraacetic acid (EGTA), hydroquinone, sodium citrate, or a combination thereof.

[0058] In various examples where thrombin is present in the cells and / or support material, the polymerization inhibitor can be an indirect and / or direct thrombin inhibitor. The polymerization inhibitor can be applied to harvested cells prior to combining with the biocompatible polymer as thrombin may be present during cell culture and preparation of cells for structure material. Thereafter, the cells can be combined with the remaining components to form the structure material. In various examples, EDTA can be used at the polymerization inhibitor and EDTA can indirectly inhibit polymerization of fibrinogen by binding calcium ions to interfere with fibrin formation, thereby prolonging the pot life of structure materials.

[0059] In various examples, the polymerization inhibitor can comprise bivalirudin. Bivalirudin can directly inhibit the interaction of thrombin with fibrinogen.

[0060] The structure material can comprise a pot life of at least 0.5 hours, such as, for example, at least 1 hour, at least 1.5 hours, at least 2 hours, or at least 3 hours. The enhanced pot life can enable the use of high cell density structure materials in additive manufacturing.

[0061] The structure material can comprise 1 pM to 50 mM of the polymerization inhibitor, such as, for example 2 pM to 50 mM, 1 pM to 20 mM, 2 pM to 20 mM, or 2 pM to 10 mM of the polymerization inhibitor.

[0062] In various examples, the structure material can comprise 1 pM to 50 mM of bivalirudin, such as, for example 2 pM to 50 mM, 1 pM to 20 mM, 2 pM to 20 mM, or 2 pM to 10 mM of bivalirudin.

[0063] The structure material can comprise additional additive as desired, such as, for example, a buffer, a vitamin, a mineral, glucose, an antibiotic, a rheological modifier, or a combination thereof.

[0064] The structure material can exhibit a viscosity in a range of 1 cP to 5,000 cP at 25 degrees Celsius as measured using a discovery hybrid rheometer 2 (DHR-2, TA Instruments) having a 40 mm 1 degree cone geometry, temperature-controlled stage, and solvent trap,, such as, for example, 1 cP to 4,000 cP, 1 cP to 3,000 cP, 1 cP to 2,000 cP, or 1 cP to 1,000 cP as measured using a discovery hybrid rheometer 2 (DHR-2, TA Instruments) having a 40 mm 1 degree cone geometry, temperature-controlled stage, and solvent trap,. The viscosity and / or extrusion pressure to print the structure material can be controlled with the polymerization inhibitor. The control of the viscosity and / or extrusion pressure to print the structure material can control a shear force that is applied to the cells during extrusion thereby enhancing cell viability and / or function.

[0065] The support material 108 can mechanically support at least a portion of the embedded structure material (i.e., object 114), maintain the intended geometry of the embedded structure material, and inhibit deformation of the structure material during the FRE additive manufacturing process. For example, the embedded structure material can be held in position within the support material 108 until the structure material is cured. The support material 108 can be stationary at an applied stress level below a threshold stress level and can flow at an applied stress level at or above the threshold stress level during the FRE additive manufacturing process.

[0066] The support material 108 can be a viscoplastic material with Bingham plastic-like rheological behavior. The support material 108 may demonstrate a significant shear thinningbehavior such that the support material 108 acts like a solid material during deposition of the structure material and then acts like a fluid when the nozzle 110 is moved through the support material 108 such that the movement of the nozzle 110 does not disturb deposited structure material. A drop in viscosity of the support material 108 under dynamic loading can make the support material 108 suitable for FRE. For example, in FRE, the dynamic loading can be caused by the force of the nozzle 110 through the support material 108, affecting the support material 108 in a number of ways. The extruder assembly 102 can be configured to change the support material 108 by imposing a mechanical load via shear, pressure, or vibration. The extruder assembly 102 can be configured to irradiate or heat the support material 108 to thin it. In various examples, the support material 108 can reduce viscosity under vibration, heating, or irradiation that occurs locally to the extruder assembly 102.

[0067] The support material 108 can comprise other materials with viscoplastic behavior, such as Herschel-Bulkley fluid. Bingham plastics and Herschel-Bulkley fluids are viscoplastic materials included in the “shear-thinning” or “yield-stress fluid” category.Below a specific shear stress, these materials appear as a solid material. Above a threshold shear force, these materials behave as a fluid. A Bingham plastic may not necessarily “shear thin,” but rather may act much like a Newtonian fluid once it begins to flow. In contrast, the Herschel-Buckley fluid undergoes shear thinning once it begins to flow.

[0068] The support material 108 can comprise at least two phases. For example, the support material 108 can comprise particles (e.g., microparticles) dispersed in a diluent. In various examples, the support material 108 can be a hydrogel. The at least two phases can enable the Bingham plastic-like rheological behavior.

[0069] The diluent can be aqueous or non-aqueous depending on the desired properties of the support material.

[0070] The particles can comprise gelatin, alginate, calcium chloride, soy lectithin, Carbopol, acrylamide, agarose, alginate, a cell spheroid, a cell organoid, gellan gum, hyaluronic acid, laponite nanoclay, nanoclay, pluronic F127, poly(ethylene oxide), oxidized bacterial cellulose, xanthan gum methacrylate, fumed silica, hyaluronic acid-norbomenefibronectin & hyaluronic acid, K-Carrageenan, decellularizaed extracellular matrix, other suitable particle forming compound, or a combination thereof. For example, the particles can comprise gelatin.

[0071] The support material 108 can comprise a reactant. The reactant can be capable to increase a viscosity of the structure material and / or cure the structure material. For example, the reactant can catalyst a reaction of the structure material. The reactant can be a catalyst,such as, for example, an enzyme. For example, the reactant can comprise thrombin, a photo initiator, a pH buffer, calcium, magnesium, sodium nitrate, a salt solution, transglutaminase, or a combination thereof.

[0072] The photo initiator can comprise be suitable for methacrylated polymers. For example, the photo initiator can comprise Irgacure-2959, lithium phenyl-2,4,6- trimethylbenzoylphosphi-nate (LAP), eosin Y, or a combination thereof.

[0073] The pH buffer can cure acidified materials. The pH buffer can comprise 2-[4-(2- hydroxyethyl)piperazin-l-yl]ethane-l -sulfonic acid (HEPES).

[0074] The calcium and / or magnesium can cure alginate and / or other ionic materials.

[0075] The sodium nitrate can cure silk fibroin.

[0076] In various examples, the reactant can comprise thrombin. For example, the support material 108 can comprise 0.001 to 10,000 units of thrombin per mL, such as, for example, 0.01 to 10,000 units of thrombin per mL, 0.1 to 10,000 units of thrombin per mL, 1 to 10,000 units of thrombin per mL, 0.01 to 20 units of thrombin per mL, 0.01 to 10 units of thrombin per mL, 0.1 to 20 units 10 units of thrombin per mL, 0.1 to 10 units of thrombin per mL, 1 unit to 20 units of thrombin per mL, 1 unit to 10 units of thrombin per mL, 2 units to 10 units of thrombin per mL, 3 units to 10 units of thrombin per mL, 4 units to 10 units of thrombin per mL, 5 units to 20 units of thrombin per mL, or 5 units to 10 units of thrombin per mL.

[0077] Depending on the printing technique, the support material 108 can be clear or opaque. The support material 108 can further comprise other components, such as, for example, a surfactant, a thickening agent, or a combination thereof. In various examples, the support material 108 does not comprise a thickening agent.

[0078] The structure material and / or support material 108 can comprise a contrast agent.

[0079] The extruder assembly 102 may be a syringe-based extruder, which can include a reservoir 112 (e.g., a barrel of a syringe) for receiving and storing structure material or support material, and a nozzle 110 (e.g., a needle) which can be in fluid communication with the reservoir 112 and can receive the structure material or the support material from the reservoir 112. For example, the reservoir 112 can comprise structure material and the structure material can be extruded through the nozzle 110 can be configured to deposit the extruded structure material in the support material 108 disposed in the material deposition region 106.

[0080] In various examples, the first extruder assembly 102 and / or additional components can comprise a gantry or other robotic device to support and / or move the extruder assembly 102 relative to the material deposition region 106. Optionally, the extruder assembly 102 cancomprise a motor assembly or other movement assembly configured to translate and / or rotate the gantry and / or robotic device. In various examples, the extruder assembly comprises an actuator (e.g., a motor) configured to depress a plunger into the reservoir 112 to extrude material through the nozzle 110 into material deposition region 106 as nozzle 110 is translated through the material deposition region 106 to additively form an object 114.

[0081] The computer system 104 is in signal / data communication with the extruder assembly 102 (such as via a wired and / or wireless data bus or link). The computer system 104 can be configured through programming to control the operation of the extruder assembly 102. The computer system 104 can also receive data from and send data (e.g. control data) to the extruder assembly 102a. The components in the additive manufacturing system 100 may be in communication with the computer system 104 via any suitable type of data bus (e.g., parallel or bit serial connections).

[0082] The computer system 104 comprises one or more processors 120 operatively coupled to one or more non-transitory memories 122 (only one processor 120 and one memory 122 are shown in FIG. 1 for simplicity). The processor 120 may comprise one or multiple processing cores. The memory 122 can comprise primary storage (e.g., main memory that is directly accessible by the processor 120, such as RAM, ROM processor registers or processor cache); secondary storage (e.g., SSDs or HDDs that are not directly accessible by the processor); and / or off-line storage. The memory 122 stores computer instructions (e.g., software) that are executed by the processor 120. The processor 120 can be configured (through execution of the software stored in the memory 122) to control operation of the extruder assembly 102 to thereby control the deposition of the structure material through the nozzle 110. For example, the processor 120 can control the flow rate of material through the nozzle 110 (e.g., by the actuation rate of a plunger in the extruder assembly 102) and / or the pose of the extruder assembly 102 relative to the material deposition region 106.

[0083] The memory 122 can store a digital or electronic computer model 124 of the object 114 to be manufactured by the additive manufacturing process. The computer model 124 can be loaded locally into the memory 122 or can be downloaded from another device (e.g., another computer device, cloud) that is in data communication with the computer system 104. To that end, the computer system 104 may comprise a network interface controller (NIC) that connects the computer system 104 to a computer network. The computer model 124 can be in a variety of different digital or electronic formats, such as an STL file, a OBJ file, a FBS file, a COLLADA file, a 3DS file, an IGES file, a STEP file, a VRML / X3D file, a point cloud, or another 3D model file format type. The computer model 124 can be generated fromimage data of a biological structure, an engineered structure, a computationally derived structure, or a combination thereof. In various examples, the biological structure can be generated from the image data of a patient.

[0084] The optional detector can comprise a light-based camera, a brightfield microscope, a fluorescence microscope, CT scanner, a MRI scanner, an OCT scan, a laser scan, an ultrasound scan, or a combination thereof.

[0085] The processor 120 can be configured to separate (e.g., slice (utilizing Slic3r, Cura, Simplify3D, Skeinforge, KISSlicer software, etc.)) the computer model 124 into different segments 126, each segment containing a portion of the computer model 124. In various examples, the processor 120 can be configured to convert the computer model 124 to a different 3D model file format prior to separating.

[0086] Each segment 126 can be a layer, 114a and 114b, of the object 114 to be deposited, a portion of a layer, 114a and 114b, of the object 114 to be deposited, or other geometry of the object 114. The segments 126 can be created based on a design of the computer model 124. For example, a segment of segments 126 can comprise an overlapping region, an overhang region, an infill region, a perimeter region, another region of the object 114, or a combination thereof. Each segment 126 may or may not be in the X-Y plan and a segment can be in a non-X-Y plane, such as the X-Z plane, the Y-Z plane, other plane offset from the X-Y plane, or a non-planar segment, such as, for example, a curve. Utilizing various segments 126 for different regions of the object 114 can enable variations of machine path instructions and / or print parameters for each segment 126. Therefore, the machine path instructions and / or print parameters can be selected to suit the particular geometry to be printed in the respective segment 126.

[0087] From the segments 126, the processor 120 can be configured to create machine path instructions (e.g., G-code instructions) 132 for the segments 126 based on the design of the portion of the computer model 124 in the respective segment 126. The machine path instructions 132 can be stored in the memory 122. The machine path instructions 132 can comprise print parameters 134 and can be executed by the processor 120 to cause the processor 120 to control the operation (e.g., pose, extrusion, suction, cure) of the extruder assembly 102 or other device (e.g., structure material removal device such as a suction tube).

[0088] The nozzle 110 can be configured to deposit a structure material into the support material 108 by applying a force to the structure material in the reservoir 112 such that the structure material can flow from the reservoir 112 through the nozzle 110. The structure material can comprise a yield stress, a thixotropic property, an increased viscosity, or acombination thereof. In examples where the structure material comprises a yield stress, the force applied can be at least the yield stress. In certain examples, applying the force to the structure material can cause the structure material to flow through the nozzle 110. For example, with an increase viscosity, the force can overcome the increased viscosity and cause the material to flow through the nozzle 110. In examples wherein the structure material comprises a thixotropic property, the thixotropic property can cause the time scale to start flow of the structure material to be longer than the printing process.

[0089] In various examples, a plunger can be translated through the reservoir 112. In various examples, the force can be pneumatically applied or the deposition can be controlled by a cavity pump. The application of the force can cause the material in the reservoir 112 to change form a solid or semi-solid state into fluid state (e.g., liquid), so that the material can be deposited into the material deposition region 106. The structure material can be suspended in the support material 108 at a location where the structure material was deposited by the nozzle 110 within the support material 108. Since the processor 120 can control the extruder assembly 102 and nozzle 110, the deposition of the structure material by the nozzle 110 can be based on the machine path instructions 132 and associated print parameters 134 as executed by the processor 120.

[0090] The extruder assembly 102 can move the nozzle 110 in two-dimensions when depositing structure material similar to FDM or in three-dimensions when depositing material, i.e., simultaneously in the X, Y, and Z directions. Further, the extruder assembly 102, nozzle 110, and / or material deposition region 106 can be rotatable. The machine pathing instructions 132 can be defined according to both Cartesian and polar coordinates, which can allow for the production of objects having complex geometries or very specific mechanical properties. 3D movement of the nozzle 110 during deposition of the structure material can enable, for example, additive manufacture of a helical spring in one constant motion. In various examples, other complex geometries are achievable with robotic arm assemblies capable of simultaneously controlling movement with six degrees of freedom (i.e., in any Cartesian or rotational direction).

[0091] The depositing of the structure material 108 can be repeated as necessary to additively form an object. For example, the processor 120 can control the nozzle 110 to deposit the structure material in layers, such as layers 114a and 114b, in order to additively form the object 114 in the support material 108 based on the computer model 124, another plane, and / or non-planar movement. In some examples, layer 114a can be deposited prior to layer 114b. Layer 114a may not be partially and / or fully cured prior to deposition of layer 114b.Thus, the processor 120 can control the nozzle 110a to deposit layer 114b proximal to (e.g., adjacent, in contact with, directly on top of) the layer 114a such that the deposition of the layer 114b contacts the layer 114a. For example, deposition of layer 114b can change the shape of at least a portion of layer 114a. The changed shape of at least a portion of layer 114a can be one that cannot be achieved by simple extruding out of the nozzle 110.Changing the shape of the layer 114a by deposition of layer 114b can increase contact surface area between the layers, 114a and 114b, decrease void space between the layers, 114a and 114b, improve adhesion between the layers, 114a and 114b, or a combination thereof.

[0092] The material deposition region 106 can be configured for mechanically supporting and / or holding the support material 108 during FRE additive manufacturing. For example, the material deposition region 106 can comprise a vessel in which the support material 108 is disposed and a platform on which the vessel is supported. The material deposition region can comprise a motor and / or actuator that can move the platform in 3D space as needed.

[0093] The structure material can be curable and after curing, the structure material can be considered cured. The object 114 can be at least partially cured in the support material 108 after deposition of the structure material. In various examples, the structure material can be at least partially cured prior to removing the support material 108. In some examples, the structure material may not be cured until after removing the support material 108. As used in this specification, the terms “cure” and “curing” refer to the chemical crosslinking of components in the structure material. Accordingly, the terms “cure” and “curing” do not encompass solely physical drying of structure material through solvent or carrier evaporation. In this regard, the term “cured,” as used in this specification, refers to the condition of the structure material in which a component of the structure material forming the object 114 has chemically reacted to form new covalent bonds in the structure material e.g., new covalent bonds formed between a polymeric resin and a curing agent), new ionic bonds, new hydrogen bonds, new Vander walls bonds, or combinations thereof.

[0094] For example, curing of the object 114 can comprise cross-linking. The object 114 can be treated through various cross-linking techniques to selectively increase the rigidity of the overall object 114 or portions thereof. Cross-linking can be induced by various mechanisms such as, for example, photo mechanisms (e.g., exposing the structure material to UV light), ionic mechanism, enzymatic mechanism, pH mechanisms (e.g., exposing the structure material to a different pH) or thermally driven mechanisms (e.g., cooling, heating). In various examples, the support material 108 can include a cross-linking agent or pH suitable for curing the structure material as it is deposited into the support material 108.

[0095] The mechanical properties of the object 114 can be controlled by controlling the amount of curing that occurs within the object 114. For example, the machine pathing instructions 132 can be modified to control the amount of crosslinking that occurs within the object 114. For example, the extruder assembly 102 and / or other assembly can comprise a UV light and can selectively subject the embedded structure material to the UV light as desired.

[0096] The object 114 can be at least partially removed from the support material 108. Removing the support material 108 may include heating the support material 108, cooling the support material 108, removing cations to disrupt crosslinking of the support material 108, physically removing the support material 108, vibration, irradiation with ultraviolet, infrared, or visible light, application of a constant or oscillating electric or magnetic field, other mechanism, or a combination thereof. For example, the support material can comprise a thermoreversible material and removing the support material can comprise heating the support material to a threshold temperature at which the support material transitions from a solid or semi-solid state to a liquid state.

[0097] The methods for additive manufacturing herein, such as those illustrated in described in FIG. 2 below, can be implemented in whole or in part as computer-executable instructions stored in the memory 122 of the computer system 104 that, when executed by a processor 120 of the computer system 104, cause the computer system 104 to perform the enumerated steps. The computer instructions can be implemented as one or more software modules 116 stored in the memory 122 that are each programmed to cause the processor 120 to execute one or more discrete steps of the processes described herein or other functions. For example, the software modules 116 can comprise a separation module programmed to convert the computer model 124 into segments; a conversion module programmed to convert the segments 126 into computer instructions (e.g., G-code) for controlling the movement of the extruder assembly 102 to fabricate the object 114; an imaging module for controlling imaging parameters and movement of a detector; a modeling module programmed to receive, store, create, and / or modify part files of objects to be fabricated; and a robotic control module programmed to control the extruder assembly 102 according to the instructions generated by the conversion module to fabricate the object 114. Various other modules can be implemented in addition to or in lieu of the aforementioned modules. In certain examples, the processes described herein can be executed across multiple computer systems that are communicably connected together in a network, a computer system communicably connectedto a cloud computing system configured to execute one or more of the described steps, and so on.

[0098] Referring to FIG. 2, a flow chart illustrating an additive manufacturing method according to certain implementations of the present disclosure is provided. The method can comprise receiving, by the processor 120, a computer model 124 of an object 114 at step 202. At step 204, the processor 120, executing the separation module software, can separate (e.g., slice) the computer model into different part segments and the processor 120, executing the conversion module, can create machine path instructions (e.g., G-code instructions) based on the design computer model. The machine path instructions can be stored in memory 122.The support material 108 can be placed in the material deposition region 106 prior to printing of the object.

[0099] The method can comprise, at step 208, depositing a structure material, by the nozzle 110, into the support material 108 such as, for example, a first portion (e.g., the first layer 114a) of the object 114 can be deposited in the support material 108a. The structure material and the support material together are also referred to herein as an assembly.

[0100] The depositing of structure material at step 208 can be repeated over as many iterations as necessary to additively form the object 114. Each iteration can deposit portions (e.g., the second layer 114b) of the structure material and the iterations can be repeated until additive formation of the object 114 is complete (if not aborted earlier).

[0101] Thereafter, at step 210, the structure material can be at least partially cured after depositing and then, at step 212, the support material can be at least partially removed from the object 114. The curing can occur prior to, during, after, or a combination thereof, removal of the support material at step 212.

[0102] The methods for additive manufacturing and systems for additive manufacturing described herein can be used to create various products. The products can be various product types, such as, for example, a soft structure, a bioprosthetic, a scaffold, a medical device, an implantable device, a gasket, a tube, a seal, an aerospace part, an automotive part, a building component, or other structures that may be additively manufactured. In various examples, the product (e.g., object 114) can be surgically fit into a patient.

[0103] EXAMPLES

[0104] Various aspects, benefits and features that are potentially realizable through implementation of the present invention will be more fully understood by reference to the following examples, which provide illustrative non-limiting aspects of the invention. It isunderstood that the invention described in this specification is not necessarily limited to the examples described in this section.

[0105] Development of High Cell Density Fibrinogen Bioink for FRESH

[0106] Introduction

[0107] Cardiovascular disease is simultaneously the leading cause of death in the United States and one of the costliest to treat affecting more than 41% of the adult population with direct annual costs exceeding $318 billion. Cardiovascular disease which progresses to endstage heart failure affects more than 64 million persons worldwide and the only cure is whole heart transplant. However, many patients in need of a heart transplant are unable to receive one due to lack of donor supply, high donor heart nonutilization, existing patient comorbidities, or other risk factors. To address the gap between supply and demand, researchers have turned their attention to the biofabrication of de novo tissue from human induced pluripotent stem cell (hiPSC) derived cardiomyocytes. Cardiac tissue models have been used to enhance the scientific communities understanding of cardiac development and disease progression, pharmaceutical product development, and to develop curative regenerative medicine therapies. These cardiac models have proved particularly useful as they respond to electromechanical stimulation in addition to pharmaceutical treatments such as ace inhibitors, beta blockers, calcium channel blockers, digoxin and diuretics. These positive results coupled with advancement of bioprinting technology has resulted in a growing interest in the fabrication of models with structure, cell density and function more representative of the native heart.

[0108] To date extrusion bioprinting has demonstrated the potential to fabricate large anatomically accurate structures with high fidelity from acellular bioinks including aortic valves, perusable vascular networks, ascending aortic arch, and a human scale anatomical heart model from patient imaging data. High cell density tissues have been fabricated utilizing robotic assembly of aggregates, co-deposition of cellular bioinks with acellular structural supportive bioinks, or casting of aggregate slurries into a mold then bioprinting a fugitive vasculature within the tissue. While these advancements are promising, none of the existing biofabrication processes and associated bioink formulations can fabricate cardiac muscle with sufficient cell density, structural integrity, matrix composition and precise anatomical structure to repair or replace components of the heart.

[0109] High cell density fibrinogen bioinks have been previously used in our lab to fabricate contractile ventricle models but these bioinks lacked sufficient strength to print standalone tissues. In the ventricle model, a cardiac bioink was prepared by combining humanembryonic stem cell (hESC) derived cardiomyocytes (CM), ventricular cardiac fibroblasts and 20 mg / mL fibrinogen. The ventricle model was fabricated by extruding the cardiac bioink between collagen shells. The collagen shells provided structural support which prevented the ventricle model from collapsing after release from the support bath and subsequent culture. The storage modulus of the cell laden bioink as prepared was insufficient to maintain the geometry of the printed tissue after release from the gelatin support bath. Eliminating the dependence on collagen to provide structural integrity to printed tissues is desirable because it can eliminate the use of two extruders for structure material during fabrication and enables finer structural features to be fabricated. Eliminating a secondary structural material from printed muscle may have the added benefit of increasing the force output of printed muscle tissues as there is less resistance to deformation.

[0110] Here we sought to develop a high cell density cardiac bioink from materials native to the natural healing process: fibrinogen, cardiomyocytes, and cardiac fibroblasts. We developed a fibrinogen bioink formation utilizing C2C12 mouse myoblasts and a treatment to inhibit interactions between the cells and the fibrinogen components. After developing a bioink formulation, we fabricated tissues with freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting, which allows for the printing of soft materials, like fibrinogen, into high cell density cardiac tissue. Our objective was to understand how cell density affects tissue remodeling and assess how infill direction affects myocyte directionality in an engineered muscle strip model.[OHl] Materials and Methods

[0112] Experimental design

[0113] The objective of the experiments described in this work were to develop a bioink formulation for the direct manufacture of high cell density cardiac muscle tissue utilizing FRESH 3D bioprinting. To achieve this goal, we first utilized C2C12 myoblasts as a development muscle cell line to investigate; how increasing cell concentration affects the bioink storage modulus, stabilization of interactions between the cells and fibrinogen in the bioink, how cell concentration affects remodeling of engineered models, and how printing parameters can be utilized to direct myotube orientation within a printed muscle model. We also tested the applicability of the developed C2C12 bioink stabilization treatment to other cell types and muscle cells isolated by means other than trypsinization. Finally, we applied the method to the fabrication of high cell density aligned cardiac muscle strips.

[0114] C2C12 culture and bioink preparation

[0115] Acellular fibrinogen stock was prepared by dissolving bovine fibrinogen (Sigma, 341573) at 120 mg / mL in sterile lx phosphate buffered saline (PBS) without calcium or magnesium in a cell culture incubator overnight. C2C12 fibrinogen bioinks were prepared using a process similar to those developed. Briefly, C2C12 mouse myoblast cells (ATCC, CRL-1772) were cultured in growth media (C2C12-GM) consisting of Dulbeccos Modification of Eagles Medium (DMEM) with high glucose (Coming, 15-013-CM) supplemented with 2 mM L-glutamine (ThermoFisher, 25030081), 100 U / mL penicillin, 100 pg / mL streptomycin (ThermoFisher, 15140122), and 10% (v / v) fetal bovine serum (VWR, 89510-186). Cells were passaged for bioink preparation at 90% confluency with Trypsin- EDTA (0.25%) (ThermoFisher, 25200056), collected in C2C12-GM, centrifuged at 200 ref for 5 minutes, resuspended in 1 mL DMEM with 25 mM HEPES (Corning, 15-018) and transferred to a 1 mL BD syringe. The syringe was placed in a custom adapter, loaded into a swing bucket centrifuge, and cells were isolated at 500 ref for 5 minutes. The supernatant was decanted, and cell pellet was mixed 1: 1 (v / v) with 120 mg / mL fibrinogen stock solution with a viscous pipette to achieve a concentration of 60 mg / mL fibrinogen in the bioink. The cells were counted on a hemocytometer after a 4000x dilution in lx PBS. The bioink cell density was enriched by centrifugation at 1000 ref for 5 minutes, the supernatant was removed to reach the desired bioink cell density. The final bioink was mixed with a viscous pipette and transferred to a 500 pL gastight syringe (Hamilton, 81222) for printing (FIG. 4A).

[0116] Rheological characterization of bioink gel strength

[0117] Rheological characterization of the fibrinogen bioinks was conducted utilizing a protocol published by Zuidema et al with a Discovery Hybrid Rheometer 2 (TA Instruments) equipped with a temperature-controlled stage and 40 mm parallel plate geometry. Briefly, the linear viscoelastic region was identified by performing oscillatory frequency and amplitude sweeps on acellular 60 mg / mL fibrinogen samples to determine the appropriate parameters for subsequent tests. All further samples were tested at 5% strain and 1 Hz frequency which falls within the linear viscoelastic regime for the material. Initially the stage was prechilled to 4°C prior to loading the rheometer stage with 2 U thrombin and 350 pL of 60 mg / mL fibrinogen bioink containing 0, 100, 200, or 300xl06C2C12 cells / mL was loaded onto the rheometer stage. The geometry was lowered onto the sample, trimmed, and then the gel storage and loss moduli were recorded for 1 hour at 37°C. All experiments were performed in triplicate utilizing the same batch of fibrinogen stock to prepare the bioinks. Terminal gel storage modulus was reported for each replicate then plotted and analyzed in GraphPad Prism 9.

[0118] Bioprinter setup

[0119] The bioprinter used in this study was built using a four linear ball screw stages (Fuyu, FSL40XYZ-L) mounted on aluminum extrusion tubing in a xyz gantry configuration. The printer utilizes three open source Replistruder 5 syringe pumps mounted to an aluminum mounting plate with independently adjustable z heights. The x, y, and z axes and extruders are controlled using a Duet 2 motion controller with a wireless graphical user interface.

[0120] Model design and slicing.

[0121] Fusion360 (Autodesk) computer aided design (CAD) software was used to design all models in this study. Briefly, models were designed and exported as 3D manufacturing format (,3mf) files and imported into Simplify3D to generate print pathing files (.geode). Print parameters such as printing speed, percent infill, infill direction, layer height, syringe diameter, and filament size are defined. All samples were printed with 150 pm blunt tip extrusion needles (Jensen, JG30-HPX), 60 pm layer height, and 500 pL glass syringe (Hamilton, 1700 series).

[0122] FRESH support bath generation for cell printing.

[0123] FRESH v2.0 gelatin microparticle support was produced as previously described using a complex coacervation method. Briefly, 3.0% (w / v) Gelatin Type B (Fisher Chemical, G7), 0.125% (w / v) Pluronic® F-127 (Sigma, P2443), and 0.3% (w / v) gum arabic (Sigma, G9752) were spread on a 150 mm petri dish and exposed to high intensity UV light for 15 minutes. Reagents were then dissolved in 50% (v / v) solution of sterile deionized (DI) water preheated to 70°C and absolute ethanol (200 proof) at room temperature in a sterile 1 L beaker on a magnetic stir plate in a biosafety cabinet. The solution was adjusted to a 5.65 pH by dropwise addition of 1 N hydrochloric acid (HC1). The beaker was sealed with plastic wrap to minimize evaporation and stirred overnight at -500 RPM. The solution was distributed evenly to 250 mL containers and centrifuged at 300 ref for 3 min to isolate the gelatin microparticle slurry. The slurry was washed three times in sterile DI water with centrifuging at 500 ref for 3 minutes. Slurry was stored at 30% solids (v / v) in sterile DI water at 4°C for up to 6 months prior to use.

[0124] On the day of printing, gelatin microparticles were washed three times in lx DMEM with 25 mM HEPES (Corning, 15-018). On the third wash the solution was supplemented with 0.1 U / mL thrombin (Sigma, T4648). Washed slurry was evenly distributed among 10 mL BD syringes, degassed for 20 minutes, centrifuged in 1750 ref for 5 minutes, decanted, and transferred to the desired print container.

[0125] Extrusion pressure measurement.

[0126] Syringe extrusion pressure was measured utilizing an inline pressure sensor (FlowPlus 16) connected to a National Instruments USB-6009 data acquisition system run through a Labview program developed in house. To measure the extrusion pressure over time a machine toolpath file was created for a 6x6x1.2 mm model for a 500 pL syringe and 30 gg (160 pm) blunt tip dispensing needle with Simplify3D. A custom printing script was created to print a single model every 10 minutes for 2 in a well plate and mechanically removing debris from the extrusion needle between prints was written in a text editor. Three conditions were evaluated for bioink stabilization by washing cells with: no treatment (negative control), indirect thrombin inhibition with 50 mM ethylenediaminetetraacetic acid (EDTA) in lx PBS, and direct thrombin inhibition with 5 pM bivalirudin (Cayman Chemical, 23035) in DMEM lx with 25 mM HEPES. Raw extrusion pressure data was smoothed by averaging 5 second of data to decrease signal noise. All treatments were completed in triplicate and pressure traces from each trial were plotted until the time of print failure or completion of the experiment.

[0127] 3T3 fibroblast culture.

[0128] NIH / 3T3 fibroblasts were purchased and cultured according to distributor provided instructions (ATCC, CRL-1658). Briefly, cells were thawed, plated at 5000 cells / cm2, and cultured in C2C12-GM. Cells were passaged at 70% confluency by aspirating spent media, rinsing flask with lx PBS, and dissociating cells with 0.25% trypsin-EDTA (ThermoFisher, 25200056). Trypsin was neutralized by addition of C2C12-GM and cells were collected by centrifugation at 200 ref for 5 minutes to be replated or prepared into a bioink.

[0129] Primary embryonic chick muscle isolation.

[0130] Chicken eggs were purchased from a local farm and maintained in a humidified egg incubator at 37.5° C at 40-60% relative humidity. Eggs were candled to verify fertilization and viability of the eggs on day 8. Skeletal muscle was harvested from viable embryos on day 10 following published methods. Briefly, the egg was cleaned with 70% EtOH and transferred to a biosafety cabinet where the embryo was extracted from the egg and sacrificed by cervical transection. Muscle tissue was collected from the breast, thigh, and wing then washed three times in ice cold lx PBS. Cells were isolated from the muscle tissue by mechanical digestion with tweezers and scalpels followed by pipetting with a 1000 pL micropipette. Digested cells were filtered with a 100 pm cell strainer. Harvested cells were isolated by centrifugation, resuspended in chick growth media (chick-GM), and pre-plated on tissue culture flasks for 45 minutes to reduce fibroblasts content. Chick-GM is composed of Hams F-10 media, supplemented with 10% horse serum, 100 U / mL penicillin, 100 pg / mL streptomycin, and 1.26 mM CaCh. Nonattached cells were collected after 45 minutes,isolated by centrifugation, washed in chick-GM supplemented with 5 pM bivalirudin and prepared into a high cell density bioink as previously described.

[0131] Cardiomyocyte differentiation and purification

[0132] The WTC-11 (Coriell, GM25256) and WTC-11 with a mono-allelic mEGFP taggled MYL2 (Coriell, AICS-0060-027) hiPSC lines were maintained in E8 media (ThermoFisher, A1517001) on 12 pg / cm2Geltrex (ThermoFisher, A1413302) coated six well plates. Cells were seeded for differentiation onto 6 well plates coated with Matrigel growth factor reduced basement membrane matrix (Corning, 356231) in E8 media with 2 pM thiazovivin (Selleck, S1459). The media was refreshed with E8 media daily for 2 days and differentiation was initiated by replacing media with hiPSC cardiac differentiation media (CDM) supplemented with 6 pM CHIR99021 (LC Laboratories, C-6556) for 48 hours. hiPSC CDM is composed of RPMI 1640 (ThermoFisher, 21870076) media supplemented with B-27 supplement (lx) without insulin (ThermoFisher, A1895601), 2 mM L-glutamine (ThermoFisher, 25030081), 0.1% (v / v) Mycozap™ Plus-CL (Lonza, VZA-2012). On day 2, spent media was removed, the wells were rinsed with lx PBS, and replaced with 3 mL of CDM supplemented with 5 pM IWP 2 (Tocris, 3533) for 48 hours. On day 6 and 8 spent media was aspirated and replaced with cardiac maintenance media composed of RPMI 1640 (ThermoFisher, 21870076) media supplemented with B-27 supplement (lx) (ThermoFisher, 17504044), 2 mM L-glutamine, 0.1% (v / v) Mycozap™ Plus-CL. On day 10 media was exchanged every third day with cardiac maintenance media (CMM) composed of RPMI 1640 supplemented with B-27 supplement (0.5x), 2 mM L-glutamine, 50 ng / mL ascorbic acid (Sigma, A8960), 450 pM monothioglycerol (Sigma, M6145), and 0.1% (v / v) Mycozap™ Plus-CL. Cells were digested overnight in hanks balanced salt solution with 0.6 mg / mL collagenase 2 (Worthington, LS004176) and reseeded the following day onto Matrigel coated T75 flasks in CMM supplemented with 2 pM thiazovivin for purification. The following day cardiomyocyte purification by metabolic selection was initiated by treating the cells with chemically defined media 3 components with DL-lactate (CDM3L) for 4 days with media exchanges every other day30. Cells were transitioned back to CMM by increasing the percentage of CMM in 25% increments each day. Cells were maintained in CMM with exchanges every third day and used within one month following purification. Cells were collected by treating cells with 1 U / mL dispase in DMEM / F12 (Stemcell Technologies, 07923) for 30 minutes, aspirating dispase, digesting in TrypLE™ Express (ThermoFisher, 12604021) for 45 minutes, neutralization with lx DMEM, and filtering the cells with a 100 pm cell strainer.

[0133] Cardiac fibroblast culture

[0134] Normal ventricular human cardiac fibroblasts (NHCFv) (Lonza, CC-2904) were thawed and plated according to the manufacturer’s instructions. Fibroblasts were cultured in cardiac fibroblast growth medium-3 (FGM-3) (Lonza, CC-4526) until reaching 75% confluence. Fibroblasts were passaged by lifting cells with trypsin-EDTA and neutralizing the reaction with FGM-3. Fibroblasts were used between passage 3 and 8 for these studies.

[0135] Printability and viability assessment

[0136] Bioinks were prepared from isolated cells according to the procedure appropriate for the cell line and treated with 5 pM bivalirudin during the collection process. Bioinks were used to print grid and ring (minimum samples = 2) tissues into FRESH gelatin microparticle support washed in DMEM lx with 25 mM HEPES and 0.1 U / mL thrombin. Darkfield stereomicrographs of the printed tissues were captured on a Leica stereomicroscope equipped with a Prime 95B sCMOS camera while embedded in gelatin microparticle support. Prints were released in a cell culture incubator at 37°C for 30 minutes, washed 3x in prewarmed growth media, and stained for viability assessment with 2 pM calcein AM and 4 pM ethidium homodimer (Invitrogen, L3224) in PBS with calcium and magnesium. One grid and two rings were imaged from each cellular bioink on a Nikon AIR multiphoton confocal microscope with Nikon imaging software (NIS) elements. Cell viability analysis was performed in Imaris (Bitplane, 9.5.1) using the spots detection function to identify cell stained for the live and dead signal. For consistency, the spot size was set at 20 pm for all samples. Each tissue was divided into four regions of interest by quadrant and calculated for each region of the tissue. Percent viability for each bioink was reported as the number of live cells / total cell number for region of interest. Graphical and statistical analysis was performed in Prism 9 (Graphpad).

[0137] C2C12 compaction rings

[0138] Compaction rings to assess tissue remodeling were printed from bioinks containing 100, 200, or 300xl06C2C12 cells / mL in FRESH gelatin microparticle support. Printed tissues were allowed to gel for at least 15 minutes at room temperature then transferred to a 37°C cell culture incubator to be released. Prints were then transferred to a low attachment 12 well plate and cultured in C2C12-GM for 8 days. Darkfield stereomicrographs were taken of the tissues every day. The inner and outer mean ferret diameter of the tissues was measured in ImageJ and plotted in Prism 9.

[0139] Immunofluorescent staining of aligned muscle strips

[0140] Skeletal and cardiac muscle tissues were fixed in 4% formaldehyde (Electron Microscopy Sciences, 15710) in lx PBS with 1 :200 Triton X-100 (Sigma, T8787) for 1 hour followed and washed three times in lx PBS for 30 min each. The tissues were blocked overnight at 4°C on an orbital shaker in lx PBS supplemented with 3% goat serum, 0.05% tween 20, and 0.1% Triton-X 100. Blocked tissues were washed on an orbital shaker at room temperature in lx PBS three times for 30 min each prior to staining. Printed C2C12 tissues were stained with mouse anti-myosin heavy chain primary antibody (ThermoFisher, MA5- 11748) at 1 : 100 dilution for 3 days on an orbital shaker at 4°C then washed three times for 30 minutes each in lx PBS at room temperature on an orbital shaker. C2C12 tissues were then stained with 1 :200 DAPI (VWR, D1306, 5 mg / mL stock), 3:200 phalloidin 488 (ThermoFisher, A12379), and 1 :200 goat anti mouse conjugated to AlexaFlour 555 (ThermoFisher, A21422) diluted in lx PBS at 4°C for 3 days. Cardiac tissues were fabricated from a line genetically modified to express GFP with MYL2. Cardiac tissues were stained with 3:200 phalloidin 555 (ThermoFisher, A34055) and 1 :400 TO-PRO™-3 Iodide 642 / 666 (ThermoFisher, T3605) in lx PBS for 3 days at 4°C. Stained tissues were washed three times in lx PBS for 30 minutes on an orbital sharker at room temperature and stored at 4°C prior to imaging.

[0141] High magnification z-stack images of the cardiac tissues were captured on a Nikon AIR MP+ multiphoton confocal microscope using a 16x objective for collagen deposition by second harmonic multiphoton imaging, nuclei, f-actin, and MYL2 expression. After several high magnification images were captured, the tissues were serially dehydrated in 25%, 50%, 75%, and 100% ethanol for one hour per wash. Absolute ethanol was exchanged and the tissue was incubated overnight to fully dehydrate the tissue. Dehydrated tissues were optically cleared in a 1 : 1 (v / v) solution of benzyl alcohol and benzyl benzoate (BABB) for one hour. Large 3D (300 pm in Z) tile scans of the cleared tissues were captured with a 4x CFI Plan Apo Lambda Air (0.2 NA, 20 mm WD) objective. Images were captured rendered in Imaris.

[0142] Statistical analysis

[0143] GraphPad Prism 9 was used to plot all graphs and run statistical analyses. Data is presented as mean ± standard error of the mean and tested for normality using Shapiro-Wilk’s or Pearsons’ s normality tests unless otherwise noted. A one-way analysis of variance was run to evaluate differences in the terminal bioink storage moduli, average extrusion pressure and printed cell viability.

[0144] Results

[0145] Challenges of Printing Cellular Fibrinogen Bioinks with FRESH

[0146] FRESH offers the ability to fabricate scaffolds with filaments of cells encapsulated in a fibrin hydrogel matrix. However, utilizing methods developed by Lee et al. with 20 mg / mL fibrinogen, maximum cell concentration (~330xl06C2C12 cells / mL) and printed in support matrix supplemented with 1 U / mL thrombin (FIG. 3 A) produces scaffolds with insufficient structural integrity. To demonstrate the lack of structural integrity, we designed window frame constructs to test whether single extrusion width filaments can be printed with the baseline bioink formulation (e.g., structure material). These scaffolds reflect the designed structure but do not retain their shape after release from the support matrix (e.g., support material) and break apart during media exchange (FIG. 3B). These results show that while FRESH can successfully reproduce the intended structure features within the supported medium, the bioink formulation does not have a sufficient gel strength to maintain structural integrity in the absence of the support matrix.

[0147] Bioink formulation

[0148] The bioink formulation and preparation method we developed was adapted from previously published methods but utilizes a higher concentration of fibrinogen in order to maintain the structural integrity of the printed tissue. Initially we sought to understand how bioink cell concentration alters the strength of the bioink as many reports exist for other hydrogel systems such as collagen or Matrigel that increasing the bioink cell concentration decreases the gel strength of the material. However, these studies did not exceed lOOxlO6cells / mL and never considered how the gelation mechanism may impact the results. We prepared bioinks with 0, 100, 200, and 300xl06C2C12 cells / mL to assess how the gel strength changes as a function of cell density. The storage and loss moduli gelation time is not affected by the bioink cell density (FIG. 4B) and we do not find statistically significant differences between the terminal bioink storage moduli with respect to the cell density (FIG. 4C). Initial attempts to print structures with 300xl06C2C12 cells / mL bioinks often failed due to development of distinct phases in the bioink which clogged the needle preventing extrusion (FIG. 4D). The reformulated bioink was capable of fabricating self-supporting structures when the homogeneity was maintained. We were able to fabricate a window frame model 10 x 10 x 1 mm with an outer wall of 2 mm with ~6 mm long with a cross section of 160 x 160 pm (FIG. 5). To resolve this issue, we evaluated treatments to inhibit cell-cell and cell-matrix interactions in the bioink to stabilize the material prior to extrusion.

[0149] The effect of stabilization treatments on bioink printability was assessed by measuring the extrusion pressure during printing and by measuring the area of printed constructs. Asimple model was created in CAD with the dimensions of 6 x 6 x 1.2 mm (FIG. 6A) and was printed from bioinks with 300xl06C2C12 cells / mL. Prior to preparing the bioink cells were treated with either a control (no treatment), washed with 50 mM EDTA, or 5 pM bivalirudin. After printing, images were taken of the embedded scaffolds to determine if the constructs were reproducible across the target printing window of 2 hours (FIG. 6B). No control bioinks could produce scaffolds of the desired dimensions after 40 minutes. The EDTA and bivalirudin treatments improved the accuracy and repeatability of scaffold fabrication. Bivalirudin treated bioinks were the most consistent across the period of interest with area normalized to the first scaffold produced -90% (FIG. 6C). Extrusion pressure was sampled and recorded at a frequency of 20 Hz with Lab View (National Instruments) program developed in house. The recorded data was smoothed by boxcar averaging 5 seconds of data to remove noise. Non-treated cells were unable to be fabricated after gelation was observed in the bioink and clogged the needle. Sustained extrusion pressure spikes correspond to agglomeration of the bioink and failure of the print which was observed for every non-treated sample (FIG. 6D).

[0150] These sustained pressure spikes fell to baseline pressure after the luer-lock adapter needle adapter backed off to relieve the syringe pressure. After 60 minutes of printing EDTA treated bioinks, minor pressure spikes were observed at timepoint which correspond to the start of each print (FIG. 6E). These pressure spikes largely resolved themselves over the course of 2-3 minutes of printing. Bioinks prepared with bivalirudin treated cells either exhibited no or minor transient pressure spikes for all replicates at the start of each print which resolved within seconds of printing the individual scaffold (FIG. 6F). No bivalirudin treated bioinks agglomerated and all scaffolds were able to be fabricated. Mean extrusion pressure of non-treated control bioinks was 489 kPa which was significantly greater than either EDTA (57.7 kPa) or bivalirudin (27.85 kPa) treated bioinks (FIG. 6G). Bivalirudin treated cells produced the most stable bioink, reproducible scaffold dimensions over time, and had the lowest average extrusion pressure. Bivalirudin was used to treat cells prior to bioink preparation for all subsequent experiments.

[0151] Impact of cell density on tissue compaction

[0152] To understand how the bioink cell density affects printed tissue remodeling, we fabricated ring models with C2C12 bioinks containing 100, 200, 300xl06cells / mL and cultured the tissues for 8 days in C2C12-GM. Representative darkfield stereomicrographs demonstrate that all tissues were printed with equivalent dimensions at day 0 and compact over time in culture (FIG. 7A). The inner and outer ferret diameters of the tissues weremeasured daily to quantify tissue compaction with respect to time and bioink cell density (FIGs. 7B and 7C). We did not observe significant differences in compaction rate or final scaffold dimensions with respect to the bioink cell density. In all subsequent experiments the highest cell density bioinks would be used and tissues cultured >2 days were constrained on silicone posts to preserve the structure of the printed tissue throughout the culture period.

[0153] Application to additional cell lines

[0154] We tested the efficacy of bivalirudin to stabilize bioinks prepared from cells isolated by methods other than trypsinization by preparing three additional high cell density bioinks from 3T3 fibroblasts, primary embryonic chick muscle, and cardiac bioinks. Cardiac bioinks are composed of 98% purified hiPSC-CMs and 2% NHCFv. These cells were isolated by trypsinization, mechanical digestion, and TrypLE respectively. Cells were lifted according to the methods previously described then washed in cell specific growth media supplemented with 5 pM bivalirudin prior to bioink preparation. Pretreating cells with bivalirudin stabilized each bioink prepared as no agglomeration was observed by eye and 2 grid and 2 ring tissues were successfully printed from each bioink. Representative stereomicrographs of the ring and grid tissues were taken prior to releasing the tissues from the FRESH support (FIG. 8A). The viability of printed cells was >85% for all preparations. Cardiac bioinks had a significantly lower (*p < 0.05) cell viability than embryonic chick muscle bioinks (FIG. 8B).Representative immunofluorescent images of ring models show homogenous distribution of viable cells throughout the printed tissue (FIG. 8C).

[0155] Controlling muscle orientation in engineered strip models

[0156] To determine if infill angle could control the alignment of muscle fibers in printed muscle tissues, CAD models were designed with dimensions of 11 x 3 x 1 mm (length x width x height) and sliced with infill angles of 0°, 30°, 60° and ±45° (45x45). These models were printed from C2C12 bioinks with 300xl06cells / mL and cultured on PDMS pillars (FIG. 9A). C2C12 muscle strips were cultured for 5 days in growth media followed by 7 days in C2C12 differentiation media to induce myotube formation in the printed tissue. Tissues were successfully fabricated with aligned infill and all tissues compacted over time in culture. After 10 days in culture sufficient tension was generated by the cells to pull the tissue through the PDMS pillar (FIG. 9B). After 11 days in culture, tissues with 0°, 30°, and 60° infill directions have robust myotube formation but the myotube orientation relative to the long axis of the tissue was unable to be quantified. Tissues fabricated with alternating layers of +45° and -45° infill had the most discernable differences in myotube direction with respect to infill direction. Max intensity z-stack image projections, 120 pm total thickness, reveal twodistinct myotube vectors. The image stack was split into two 60 pm thick sub stacks which correspond to the layer height used to print the tissue (FIG. 9C). Myotube orientation angle was measured in ImageJ in each 5 pm thick image slice and shows significant differences in orientation between the upper and lower filaments (FIG. 9D). The myotube orientation angles are not normally distributed likely due to the overlap of upper and lower filaments in the same image. The data was normalized by removing outliers and plotted as mean ± standard error of the mean. The lower filament had a myotube orientation angle of 44.79° ± 29.27° which was significantly different (****p < 0.0001) than the upper filaments myotube orientation angle -38.52° ± 38.19° (mean ± standard deviation). The statistical significance was determined using a two-sided students t-test (FIG. 9E).

[0157] Engineered cardiac muscle strips were printed to evaluate if the observations in C2C12 muscle strips could be utilized to control muscle alignment throughout the thickness of the tissue. Three infill patterns were utilized in this study unidirectional 0°, unidirectional 90° and rotating from 0° to 90° in 10° increments per layer. To prevent the tissue from ripping off the PDMS posts during culture the infill density was increased from the original value of 25% to 50%. All tissues were successfully printed from bivalirudin treated cardiac bioinks prepared as previously described and were cultured in CDM3 media supplemented with 10% fetal bovine serum. All cardiac muscle tissues compacted and began to spontaneously beat after 4 days in culture (FIG. 10A). Tissues continued to beat and were fixed after 14 days in culture to evaluate actin alignment and collagen deposition in the tissue. No qualitative differences in the alignment of the actin or myl2 expression were observed between the three printed muscle construct designs (FIG. 10B). Additional analyses could be performed to evaluate combinatorial effects of infill direction, infill density and mechanical stress on the alignment of muscle cells in printed tissues which fall outside the scope of this study. High magnification images show that cells deposited collagen throughout the printed tissue, and this is likely responsible for stabilization of the tissue as fibrinogen breaks down in culture. Interestingly, none of the muscle tissues printed from high cell density fibrinogen bioinks broke down after prolonged culture times (> 28 days) in serum containing media without a fibrinolysis inhibitor such as aprotinin. Tissues were fixed after 14 days of culture to prevent the tissue from generating sufficient stress to rip the tissue off the PDMS pillars used to constrain the tissue. The stress generated by the tissue suggests that the bioink cell density was high enough for cell-cell connections to reform in the printed tissue. This finding is supported by the necessity of intercalated disks to propagate spontaneous synchronous contractions which were observed in all high cell density cardiac muscle strips. Additionalexperiments could be designed to evaluate how bioink cell density alters the amplitude of force generated by printed cardiac tissues.

[0158] Discussion

[0159] The goal of this study was to develop a bioink formulation to directly fabricate high cell density muscle tissue using FRESH 3D bioprinting from a variety of cell sources with a degradable matrix. Fibrin was selected as the primary structural material due to its role in the natural healing cascade, abundance of cell binding sites, clinically approved status, and biodegradability of the material. Additionally, the mechanical properties of fibrin gels can be modulated by altering the polymerization conditions such as the concentration of thrombin, fibrinogen, and calcium ions. To date the use of neat, unmodified, fibrinogen bioinks has been restricted to inkjet based bioprinting approaches as fibrinogen solutions prepared with 10 - 150 mg / mL have corresponding viscosities of 2 - 43 cP which are below the accepted viscosity threshold for tradition direct ink write extrusion bioprinting approaches. However, the low viscosity of fibrinogen solutions can be desirable to create high cell density bioink as the cells can be compacted in by centrifugation to reach cell densities > 300xl06cells / mL. Here we demonstrate that the storage moduli of cell loaded fibrin gels are not significantly different with respect to the cell density. Furthermore, we demonstrated that bioink cell density can be adjusted without necessitating changes to the model or printing parameters to assess printability or tissue remodeling by utilizing the same geode script to successfully fabricate tissue rings from bioinks with 100, 200, and 300xl06C2C12 cells / mL.

[0160] Combing cells with fibrinogen to form a bioink proved to be a difficult task as the pot life of the bioink during printing was typically < 30 minutes which was detrimental to the reliability of the tissue printing process. To increase the bioink pot life, we developed various treatments to neutralize residual thrombin introduced through the presence of serum in the culture media or bound to cell membrane thrombin receptors. By treating the cells with EDTA our goal was to eliminate residual calcium ions from the bioink to slow the spontaneous gelation rate of fibrinogen in the syringe. We employed bivalirudin, a synthetic form of hirudin, as a direct thrombin inhibitor to halt spontaneous bioink gelation in the syringe altogether. We determined that pretreating cells with bivalirudin stabilized cell- fibrinogen bioink and enabled tissues to be printed up to two hours after bioink preparation. Bivalirudin treated cells required the lowest mean extrusion pressure required to fabricate C2C12 tissues with FRESH 3D bioprinting that we tested. Higher extrusion pressure may not be desirable as it increases the shear stress exerted on cells during extrusion which is widely reported to negatively affect printed cell viability.

[0161] Bivalirudin also effectively stabilizes bioinks prepared from muscle cell lines in addition to C2C12 myoblasts. We demonstrated the ability to print tissues from four additional cell lines in this study and postulate that the developed treatment will be highly effective across a broad range of cell lines. Grid models demonstrated the capability of high cell density bioinks to print fine tissue filaments approximately -160 pm in diameter which corresponds to the size of the extrusion needle employed in this study. In addition to this, the same printing file was used to fabricate structurally identical tissues from each of the four bioinks tested. This result allows users to develop 3D printing parameters with low-cost rapidly proliferative cell sources, such as C2C12 myoblasts, then switch to bioinks prepared from cells whose use is limited by the time required for expansion, financial cost to maintain, or other factors. The capability to use FRESH 3D bioprinting to comparison to existing cell loaded bioink formulations and printing methods, which require significant optimization in response to altering cell concentration or type.

[0162] Conclusion

[0163] We developed an optimized protocol to prepare high cell density fibrinogen bioinks to fabricate muscle tissue using FRESH 3D bioprinting. We investigated methods to stabilize interactions between cells and the fibrinogen prior to deposition and showed that bivalirudin treated cell bioinks had lower mean extrusion pressure and high repeatability over the course of 2 hours of printing. We demonstrated that tissues printed from high cell density bioinks are stable and remodel in culture. We also demonstrated that print pathing parameters can be utilized to generate aligned filaments of cells, but that cells remodel in response to the mechanical tension generated by the tissue pulling on the posts. Overall, this work established a set of robust method to prepare cell laden fibrinogen bioinks and printing protocols to fabricate self-supporting tissues using the developed bioink.

[0164] Various aspects of the invention include, but are not limited to, the aspects listed in the following numbered clauses.

[0165] Clause 1. A structure material for additive manufacturing, the structure material comprising: at least 1 mg / mL of a bio-compatible polymer; a solvent; at least 10 million cells / mL; and a polymerization inhibitor.

[0166] Clause 2. The structure material of clause 1, wherein the polymerization inhibitor comprises bivalirudin, hirudin, argatroban, dabigatran etexilate, ximelagatran, heparin, L- ascorbic acid, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), hydroquinone, sodium citrate, or a combination thereof.

[0167] Clause 3. The structure material of any of clauses 1-2, wherein the structure material comprises 1 pM to 50 mM of the polymerization inhibitor.

[0168] Clause 4. The structure material of any of clauses 1-3, wherein the polymerization inhibitor comprises bivalirudin.

[0169] Clause 5. The structure material of clause 4, wherein the structure material comprises 1 pM to 50 mM of bivalirudin.

[0170] Clause 6. The structure material of any of clauses 1-5, wherein the cells are eukaryotic cells derived from an animal.

[0171] Clause 7. The structure material of any of clauses 1-6, wherein the structure material comprises at least 50 million cells / mL.

[0172] Clause 8. The structure material of any of clauses 1-7, wherein the bio-compatible polymer comprises a synthetic hydrogel, naturally derived hydrogel, a polysaccharide solution, mammalian derived material, a polymer network, or a combination thereof.

[0173] Clause 9. The structure material of any of clauses 1-8, wherein the bio-compatible polymer comprises a collagen material, an alginate material, a decelluarized extracellular matrix material, a fibrinogen material, a fibrin material, a hyaluronic acid material, a protein material, a polysaccharide hydrogel material, a synthetic gel material, an elastomeric polymer material, a rigid polymer material, a Matrigel, or a combination thereof.

[0174] Clause 10. The structure material of any of clauses 1-9, wherein the structure material comprises 1 mg / mL to 200 mg / mL of the bio-compatible polymer.

[0175] Clause 11. The structure material of any of clauses 1-10, wherein the bio-compatible polymer is a fibrinogen material.

[0176] Clause 12. The structure material of clause 11, wherein the structure material comprises a concentration of fibrinogen of at least 25 mg / mL.

[0177] Clause 13. The structure material of any of clauses 1-12, further comprising a buffer, a vitamin, a mineral, glucose, an antibiotic, a rheological modifier, or a combination thereof.

[0178] Clause 14. The structure material of any of clauses 1-13, wherein the solvent is aqueous.

[0179] Clause 15. The structure material of any of clauses 1-14, wherein the structure material exhibits a viscosity in a range of 1 cP to 5,000 cP at 25 degrees Celsius using a discovery hybrid rheometer 2 (DHR-2, TA Instruments) having a 40 mm 1 degree cone geometry, temperature-controlled stage, and solvent trap,.

[0180] Clause 16. A structure material for additive manufacturing, the structure material comprising: 30 mg / mL to 200 mg / mL of fibrinogen; water; at least 100 million eukaryotic cells / mL; a buffer; and 1 pM to 50 mM of bivalirudin.

[0181] Clause 17. An additive manufacturing method comprising: depositing, by a nozzle, the structure material of any of clauses 1-16 into a support material based on a first computer model of an object, thereby forming a first portion of the object in the support material; and repeating, by the nozzle, the depositing of the structure material as necessary to additively form the object.

[0182] Clause 18. The method of clause 17, further comprising removing the support material.

[0183] Clause 19. The method of clause 18, wherein the support material comprises: a diluent; polymeric particles dispersed in the diluent; and a reactant.

[0184] Clause 20. The method of clause 19, wherein the reactant comprises thrombin, a photo initiator, a pH buffer, calcium, magnesium, sodium nitrate, a salt solution, transglutaminase, or a combination thereof.

[0185] Any patent, publication, or other disclosure material identified herein is incorporated herein by reference in its entirety unless otherwise indicated but only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material expressly set forth in this specification. As such, and to the extent necessary, the express disclosure as set forth in this specification supersedes any conflicting material incorporated by reference herein. Any material, or portion thereof, that is said to be incorporated by reference into this specification, but which conflicts with existing definitions, statements, or other disclosure material set forth herein, is only incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material. Applicant reserves the right to amend this specification to expressly recite any subject matter, or portion thereof, incorporated by reference herein.

[0186] In this specification, unless otherwise indicated, all numerical parameters are to be understood as being prefaced and modified in all instances by the term “about,” in which the numerical parameters possess the inherent variability characteristic of the underlying measurement techniques used to determine the numerical value of the parameter. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter described herein should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0187] Also, any numerical range recited herein includes all sub-ranges subsumed within the recited range. For example, a range of “1 to 10” includes all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value equal to or less than 10. Any maximum numerical limitation recited in this specification is intended to include all lower numerical limitations subsumed therein and any minimum numerical limitation recited in this specification is intended to include all higher numerical limitations subsumed therein. Accordingly, Applicant reserves the right to amend this specification, including the claims, to expressly recite any sub-range subsumed within the ranges expressly recited. All such ranges are inherently described in this specification.

[0188] Any references herein to “various examples”, “some examples”, “one example”, “an example”, or like phrases mean that a particular feature, structure, or characteristic described in connection with the example is included in at least one embodiment. Thus, appearances of the phrases “in various examples”, “in some examples”, “in one example”, “in an example”, or like phrases in the specification do not necessarily refer to the same examle. Furthermore, the particular described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, the particular features, structures, or characteristics illustrated or described in connection with one embodiment may be combined, in whole or in part, with the features, structures, or characteristics of one or more other embodiments without limitation. Such modifications and variations are intended to be included within the scope of the present embodiments.

[0189] As used herein, “at least one of’ A and B, means only A, only B, or both A and B. Additionally, there may be multiple As and / or multiple Bs.

[0190] One skilled in the art will recognize that the herein described articles and methods, and the discussion accompanying them, are used as examples for the sake of conceptual clarity and that various configuration modifications are contemplated. Consequently, as used herein, the specific examples / embodiments set forth and the accompanying discussions are intended to be representative of their more general classes. In general, use of any specific exemplar is intended to be representative of its class, and the non-inclusion of specific components, devices, operations / actions, and objects should not be taken to be limiting. While the present disclosure provides descriptions of various specific aspects for the purpose of illustrating various aspects of the present disclosure and / or its potential applications, it is understood that variations and modifications will occur to those skilled in the art.Accordingly, the invention or inventions described herein should be understood to be at leastas broad as they are claimed and not as more narrowly defined by particular illustrative aspects provided herein.

Claims

CLAIMSWhat is claimed is:

1. A structure material for additive manufacturing, the structure material comprising: at least 1 mg / mL of a bio-compatible polymer; a solvent; at least 10 million cells / mL; and a polymerization inhibitor.

2. The structure material of claim 1, wherein the polymerization inhibitor comprises bivalirudin, hirudin, argatroban, dabigatran etexilate, ximelagatran, heparin, L-ascorbic acid, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'- tetraacetic acid (EGTA), hydroquinone, sodium citrate, or a combination thereof.

3. The structure material of claim 1, wherein the structure material comprises 1 pM to 50 mM of the polymerization inhibitor.

4. The structure material of claim 1, wherein the polymerization inhibitor comprises bivalirudin.

5. The structure material of claim 4, wherein the structure material comprises 1 pM to 50 mM of bivalirudin.

6. The structure material of claim 1, wherein the cells are eukaryotic cells derived from an animal.

7. The structure material of claim 1, wherein the structure material comprises at least 50 million cells / mL.

8. The structure material of claim 1, wherein the bio-compatible polymer comprises a synthetic hydrogel, naturally derived hydrogel, a polysaccharide solution, mammalian derived material, a polymer network, or a combination thereof.

9. The structure material of claim 1, wherein the bio-compatible polymer comprises a collagen material, an alginate material, a decelluarized extracellular matrix material, a fibrinogen material, a fibrin material, a hyaluronic acid material, a protein material, a polysaccharide hydrogel material, a synthetic gel material, an elastomeric polymer material, a rigid polymer material, a Matrigel, or a combination thereof.

10. The structure material of claim 1, wherein the structure material comprises 1 mg / mL to 200 mg / mL of the bio-compatible polymer.

11. The structure material of claim 1, wherein the bio-compatible polymer is a fibrinogen material.

12. The structure material of claim 11, wherein the structure material comprises a concentration of fibrinogen of at least 25 mg / mL.

13. The structure material of claim 1, further comprising a buffer, a vitamin, a mineral, glucose, an antibiotic, a rheological modifier, or a combination thereof.

14. The structure material of claim 1, wherein the solvent is aqueous.

15. The structure material of claim 1, wherein the structure material exhibits a viscosity in a range of 1 cP to 5,000 cP at 25 degrees Celsius as measured using a discovery hybrid rheometer 2 (DHR-2, TA Instruments) having a 40 mm 1 degree cone geometry, temperature-controlled stage, and solvent trap,.

16. A structure material for additive manufacturing, the structure material comprising:30 mg / mL to 200 mg / mL of fibrinogen; water; at least 100 million eukaryotic cells / mL; a buffer; and1 pM to 50 mM of bivalirudin.

17. An additive manufacturing method comprising:depositing, by a nozzle, the structure material of claim 1 into a support material based on a first computer model of an object, thereby forming a first portion of the object in the support material; and repeating, by the nozzle, the depositing of the structure material as necessary to additively form the object.

18. The method of claim 17, further comprising removing the support material.

19. The method of claim 18, wherein the support material comprises: a diluent; polymeric particles dispersed in the diluent; and a reactant.

20. The method of claim 19, wherein the reactant comprises thrombin, a photo initiator, a pH buffer, calcium, magnesium, sodium nitrate, a salt solution, transglutaminase, or a combination thereof.

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