Bioprinting lipid-laden tissue

Abstract

Methods for additive manufacturing, biological structures, cultivated meats, and additive manufacturing systems are provided. The method comprises dispensing an ink composition from a nozzle of an additive manufacturing system. The ink composition comprise adipocytes and a hydrogel. The deposition occurs at a suitable shear stress such that the ink composition can flow through the needle and an integrity of the adipocytes is substantially maintained. The method comprises repeating, as necessary, repositioning of the nozzle and dispensing ink composition from the first nozzle, thereby forming the structure.

Description

Attorney Docket No.250570PCT IN THE UNITED STATES PATENT AND TRADEMARK OFFICE PCT APPLICATION FOR BIOPRINTING LIPID-LADEN TISSUE Inventors: Lindsey Huff, Rosalyn Abbott-Beauregard, Neeha Dev Arun, and Adam Walter Feinberg GOVERNMENT RIGHTS

[0001] This invention was made with United States government support under 2236998 awarded by the National Science Foundation (NSF), and EB034216 awarded by the National Instituted of Health (NIH). The U.S. government has certain rights in the invention. RELATED APPLICATIONS

[0002] The present application claims priority to United States provisional patent application Serial No.63 / 697,145, filed September 20, 2024, which is incorporated herein by reference in its entirety. BACKGROUND

[0003] Existing approaches of adipose tissue reconstruction typically include transplantation of engineered acellular biomaterials, autologous fat grafting, or a combination of these methods. Fat grafting can result in undesirable post-graft resorption that is highly variable and extensive (>70%), caused by poor volumetric estimates to fill the space and graft movement after surgery due to a mismatch between the graft and defect geometries. In fact, revision surgeries are typically scheduled with the initial surgical procedure, as surgeons anticipate returning to the site to provide an additional fat graft. There are challenges with creating an aesthetic and functional reconstruction technique that can precisely achieve desired geometries. SUMMARY

[0004] In certain general aspects, the present disclosure is related to a method for additive manufacturing. The method comprises dispensing an ink composition from a nozzle of an additive manufacturing system. The ink composition comprises adipocytes and a hydrogel. 1601463783.2 1Attorney Docket No.250570PCT The deposition occurs at a suitable shear stress such that the ink composition can flow through the needle and an integrity of the adipocytes is substantially maintained. The method comprises repeating, as necessary, repositioning of the nozzle and dispensing ink composition from the first nozzle, thereby forming the structure.

[0005] In other general aspects, the present disclosure is related to a biological structure produced by a method for additive manufacturing.

[0006] In various general aspects, the present disclosure is related to a cultivated meat produced by a method for additive manufacturing.

[0007] In certain general aspects, the present disclosure is related to an additive manufacturing system configured to perform a method for additive manufacturing.

[0008] Various embodiments and implementations of the present disclosure provide many benefits and improvements relative to prior reconstruction techniques, including additive printing techniques. For example, the present disclosure can create specific lipid-laden geometries suitable for reconstructive medicine and / or cultivated meat. These and other benefits that are potentially realizable through various implementations of the present invention will be apparent from the description that follows.

[0009] It is understood that the inventions described in this specification are not limited to the examples summarized in this Summary. Various other aspects are described and exemplified herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The features and advantages of the examples, 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 drawing, wherein:

[0011] FIG.1 is a flow chart illustrating a method for additive manufacturing according to the present disclosure.

[0012] FIG.2 a schematic diagram illustrating a system for additive manufacturing according to the present disclosure.

[0013] FIG.3A illustrates a CAD model having dimensions of 10x10x5mm. 1601463783.2 2Attorney Docket No.250570PCT

[0014] FIG.3B illustrates an image of post processing of a printed construct using a 6% alginate lipid-laden bioink with <0.6% error in printing dimensions that demonstrates FRESH 3D printing of adipocytes can be used to generate precise and reproducible constructs.

[0015] FIG.4A illustrates a printed construct that was printed with a bioink having 60% by volume of a cell solution and the remainder 6% alginate.

[0016] FIG.4B illustrates a printed construct that was printed with a bioink having 70% by volume of a cell solution and the remainder 6% alginate.

[0017] FIG.4C illustrates a printed construct that was printed with a bioink having 80% by volume of a cell solution and the remainder 6% alginate.

[0018] FIG.4D illustrates a printed construct that was printed with a bioink having 90% by volume of a cell solution and the remainder 6% alginate.

[0019] FIG.5A is an image of lipids in a static bioink after extrusion through a 1mL pipette where the bioink has 60% by volume of adipocytes and 40% by weight of a hydrogel comprising 4% alginate. Scale bar equals 100 µm.

[0020] FIG.5B is an image of lipids in a static bioink after extrusion through a 1mL pipette where the bioink has 60% by volume of adipocytes and 40% by weight of a hydrogel comprising 6% alginate. Scale bar equals 100 µm.

[0021] FIG.5C is an image of lipids in a static bioink after extrusion through a 1mL pipette where the bioink has 60% by volume of adipocytes and 40% by weight of a hydrogel comprising 8% alginate. Scale bar equals 100 µm.

[0022] FIG.5D is an image of lipids in a static bioink after extrusion through a 1mL pipette where the bioink has 60% by volume of adipocytes and 40% by weight of a hydrogel comprising 10% alginate. Scale bar equals 100 µm.

[0023] FIG.5E is an image of lipids in a printed bioink after extrusion through a FRESH 3D printer where the bioink have 60% by volume of adipocytes and 40% by weight of a hydrogel comprising 4% alginate. Scale bar equals 100 µm. 1601463783.2 3Attorney Docket No.250570PCT

[0024] FIG.5F is an image of lipids in a printed bioink after extrusion through a FRESH 3D printer where the bioink has 60% by volume of adipocytes and 40% by weight of a hydrogel comprising 6% alginate. Scale bar equals 100 µm.

[0025] FIG.5G is an image of lipids in a printed bioink after extrusion through a FRESH 3D printer where the bioink has 60% by volume of adipocytes and 40% by weight of a hydrogel comprising 8% alginate. Scale bar equals 100 µm.

[0026] FIG.5H is an image of lipids in a printed bioink after extrusion through a FRESH 3D printer where the bioink has 60% by volume of adipocytes and 40% by weight of a hydrogel comprising 10% alginate. Scale bar equals 100 µm.

[0027] FIG.5I is a bar graph illustrating lipid diameter compared to alginate composition. A one-way ANOVA with a Tukey’s post hoc test was performed on the mean lipid diameter (p < 0.05). Error bars represent the standard error of the mean. n=3.

[0028] FIG.5J is a bar graph illustrating computational fluid dynamics modeling of maximum shear stress compared to alginate composition.

[0029] FIG.6A is an image of lipids in a printed bioink comprising 60% adipocytes by volume that was extruded at a rate of 10 mm / s using a 18 gauge needle (965 µm). Scale bar equals 100 µm.

[0030] FIG.6B is an image of lipids in a printed bioink comprising 60% adipocytes by volume that was extruded at a rate of 10 mm / s using a 22 gauge needle (410 µm). Scale bar equals 100 µm.

[0031] FIG.6C is an image of lipids in a printed bioink comprising 60% adipocytes by volume that was extruded at a rate of 10 mm / s using a 26 gauge needle (228 µm). Scale bar equals 100 µm.

[0032] FIG.6D is a bar graph illustrating lipid diameter compared to needle size. A one-way ANOVA with a Tukey’s post hoc test was performed on the mean lipid diameter (p < 0.05). Error bars represent the standard error of the mean. n=3.

[0033] FIG.6E is a bar graph illustrating computational fluid dynamics modeling of maximum shear stress compared to needle gauge. 1601463783.2 4Attorney Docket No.250570PCT

[0034] FIG.7A is an image of lipids in a printed bioink comprising 60% adipocytes by volume that was extruded at a rate of 4 mm / s. Scale bar equals 100 µm.

[0035] FIG.7B is an image of lipids in a printed bioink comprising 60% adipocytes by volume that was extruded at a rate of 10 mm / s. Scale bar equals 100 µm.

[0036] FIG.7C is an image of lipids in a printed bioink comprising 60% adipocytes by volume that was extruded at a rate of 16 mm / s. Scale bar equals 100 µm.

[0037] FIG.7D is a bar graph illustrating lipid diameter compared to printing speed. A one- way ANOVA with a Tukey’s post hoc test was performed on the mean lipid diameter (p < 0.05). Error bars represent the standard error of the mean. n=3.

[0038] FIG.7E is a bar graph illustrating cell density compared to printing speed. A one- way ANOVA with a Tukey’s post hoc test was performed on the mean lipid diameter (p < 0.05). Error bars represent the standard error of the mean. n=3.

[0039] FIG.7F is a bar graph illustrating computational fluid dynamics modeling of maximum shear stress compared to printing speed.

[0040] FIG.8 is a bar graph illustrating no significant change in the relative fluorescence units from day 1 to day 3 indicating that cellular metabolic activity remains constant after 3 days of culture. Error bars represent the standard error of the mean. n=3.

[0041] FIG.9 is a schematic illustrating a process for creating a patient specific soft tissue reconstructive model via FRESH bioprinting mature adipocytes with: step (A) including conducting medical imaging on the reconstruction site, and saving the data as a digital imaging and communication in medicine (DICOM) file: step (B) including importing the DICOM file is imported into 3D Slicer and segmented using the thresholding tool to create the CAD model of the defect (The segmentation will be exported as a stereolithography (STL) file, imported into Slic3r to generate the g-code); step (C) including sourcing patient specific cells from human panniculectomies or liposuction procedures to obtain adipose- derived stem cells or mature adipocytes (If adipose-derived stem cells are isolated, they can be expanded and differentiated into mature adipocytes through culture); step (D) including isolating mature adipocytes and mixing them with alginate to form the bioink; and step (E) including loading the g-code and bioink into the FRESH bioprinter. 1601463783.2 5Attorney Docket No.250570PCT

[0042] FIG.10A is an image of a tumor in breast tissue.

[0043] FIG.10B is a model of the anticipated defect in breast tissue of FIG.10A and an isolated CAD model of the defect.

[0044] FIG.10C are images of the structure that was printed based on the model of FIG.10B

[0045] FIG.11 is a CAD model of a 3mL BD syringe, used for the computational fluid dynamics simulations.

[0046] 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

[0047] Subcutaneous adipose tissue is the layer of fat that lies beneath the dermis and can play a major role in energy homeostasis, thermoregulation, and protection. Subcutaneous tissue can be damaged as the result of disease, trauma, or congenital defects and thereby cause irregularities in the volume, distribution, or contour of the tissue, affecting both physiological function and aesthetic appearance. For example, disfigurements caused by cancer resections can result in deleterious psychosocial issues for patients. Breast cancer comprises 30% of all new female cancers each year in the United States, making it one of the most common cancers in women.

[0048] Upwards of 100,000 women undergo some form of mastectomy each year for treatment or prevention, and more than 40% of women choose to undergo post-mastectomy breast reconstruction to help restore the appearance of the tissue following a mastectomy. Conventional adipose tissue reconstruction is achieved by transplantation of engineered acellular biomaterials, autologous fat grafting, or a combination of these methods.

[0049] Fat grafting can be plagued by post graft resorption that is highly variable and extensive (>70%) caused by poor volumetric estimates to fill the space and graft movement after surgery due to a mismatch between the graft and defect geometries. In fact, revision surgeries are scheduled with the initial surgical procedure as surgeons typically anticipate returning to the site to provide an additional fat graft. These difficulties underscore certain 1601463783.2 6Attorney Docket No.250570PCT desires for creating an aesthetic and functional reconstruction technique that matches the patient-specific geometry of critically sized subcutaneous adipose tissue defects.

[0050] Three dimensional (3D) additive manufacturing can generate intricate and customizable structures of soft materials, which can provide highly detailed, patient specific constructs that closely mimic the native tissue with high precision and accuracy. To achieve patient specificity, pre-operative imaging modalities such as magnetic resonance imaging (MRI), computed tomography (CT), and / or 3D ultrasound have been used to provide defect models. Additionally, the accuracy and reproducibility, versatility (e.g., diversity of ink compositions and printing configurations), and scalability (e.g., the ability to print a whole organ) of 3D additive manufacturing can be desirable.

[0051] For adipose tissue applications, stem cells are typically the primary cell type used in additive manufacturing because of their plasticity, self-renewal capacity, and minimal immune response when cell sourcing is autologous. There are several challenges with additive manufacturing of stem cells for generating whole tissue as post-printing differentiation can limit the size of the construct due to diffusional constraints (max distance from nutrient source ~ 200 µm); use lengthy differentiation periods (for human adipose derived stem (ASC) differentiation to adipocytes is typically a minimum of at least 2 weeks); allow cells to remodel and change the geometry of the construct; and use multiple cell types with distinct media conditions are required.

[0052] ASCs typically can be seeded onto a printed construct or incorporated in the ink composition followed by differentiation after additive manufacturing. Challenges may exist in recreating the morphology of native adipose tissue, with densely packed, large, and unilocular lipid droplets, likely due to the challenges surrounding ASC post-printing differentiation.

[0053] Additive manufacturing of mature adipocytes has not been achieved, likely due to their large diameter (20-300 μm), high buoyancy, and lipid-laden morphology which may make them fragile under shear stress.

[0054] Additive manufacturing may also have applications in areas such as cultivated meat. Factory farming is the system of rearing livestock for human consumption of animal protein, resulting in 56 billion terrestrial animals annually harvested. Current livestock populations can consume greater than 27% of global fresh water, 70% of global agricultural land, and 1601463783.2 7Attorney Docket No.250570PCT may be responsible for 18% of global anthropogenic greenhouse gas emissions. Cultivated meat is an emerging and sustainable approach to replace conventionally produced meat with bioengineered meat tissues grown in vitro.

[0055] Using techniques developed for regenerative medicine, cultured meat begins with a small number of cells extracted minimally-invasively from donor animals or from immortalized cell banks. The cells can then be expanded and formed into tissues for human consumption. Consumer acceptance for these cultured meat products may require a desirable texture, flavor, cost, and familiarity similar to those of conventional meat products. Current approaches focus on ground meat products such as chicken nuggets. These current technologies can be expensive to create a low-cost commodity and unable to re-create the three-dimensional (3D) tissue structures of animal steaks, especially with regards to the distribution of intramuscular fat (e.g.,, adipocytes).

[0056] The present disclosure provides methods for additive manufacturing, systems for additive manufacturing, and products thereof that can obtain specific, large, cell dense, and / or lipid-laden geometries suitable for reconstructive medicine and / or cultivated meat. For example, the method comprises dispensing an ink composition from a nozzle of an additive manufacturing system. The ink composition can comprise adipocytes and a hydrogel. The deposition occurs at a suitable shear stress such that the ink composition can flow through the needle and an integrity of the adipocytes is substantially maintained. The method comprises repeating, as necessary, repositioning of the nozzle and dispensing ink composition from the first nozzle, thereby forming the structure.

[0057] Referring to FIG.1, a flow chart illustrating an additive manufacturing method according to certain implementations of the present disclosure is provided. The method can be performed by an additive manufacturing system comprising a nozzle, such as, for example, the additive manufacturing system described with respect to FIG.2 herein. 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. Additive manufacturing includes Freeform Reversible Embedding (FRE) among other printing techniques.

[0058] Optionally, the method can comprise receiving a model for additive manufacturing. The model may be generated and / or the model may be stored in memory. For example, the 1601463783.2 8Attorney Docket No.250570PCT biological structure may be imaged and a model of the biological structure may be generated based on the imaging at step 102. The model can be received from a database, obtained from an MRI, a CT Scan, a 3D ultrasound, or a combination thereof.

[0059] At step 104, an ink composition may be dispensed from the nozzle of the additive manufacturing system. For example, the ink composition may be dispensed based on the model.

[0060] The internal diameter of the nozzle can be sized in order to enable suitable flow of the ink composition, maintain a desirable shear stress on the ink composition, and / or maintain viability of adipocytes within the ink composition. For example, an internal diameter of the nozzle can be in a range of 200 microns to 1,000 microns, such as, for example, 300 microns to 600 microns.

[0061] The ink composition (e.g., bioink) can comprise adipocytes and a hydrogel. The ink composition can be suitable for forming the structure and substantially maintaining the viability of the adipocytes. The ink composition can retain a desired form after being dispensed and / or until a time until the ink composition can be fixed (e.g., cured, crosslinked).

[0062] A concentration of adipocytes in the ink composition can be at least 40% by volume based on the total volume of the composition, such as, for example, at least 50% by volume, at least 60% by volume, or at least 70% by volume. A concentration of the adipocytes in the ink composition can be no greater than 80% by volume based on the total volume of the in composition, such as, for example, no greater than 70% by volume, or no greater than 60% by volume. For example, a concentration of the adipocytes can be in a range of 40% to 80% by volume based on the total volume of the in composition, such as, for example, 50% to 70% by volume. In various examples, too high of a concentration of adipocytes may not form a desirably crosslinking composition and too low of a concentration may not achieve a desirable cell concentration. In certain examples, the ink composition can consist essentially of adipocytes and can be printed directly into a support material that may be part of the final product.

[0063] A diameter of the adipocytes can be at least 10 microns as measured with optical microscopy, such as, for example, at least 20 microns, at least 30 microns, at least 40 microns, at least 50 microns, at least 60 microns, at least 70 microns, or at least 100 microns. A diameter of the adipocytes can be no greater than 250 microns as measured by optical 1601463783.2 9Attorney Docket No.250570PCT microscopy, such as, for example, no greater than 200 microns, no greater than 150 microns, no greater than 100 microns, no greater than 90 microns, no greater than 80 microns, no greater than 70 microns, no greater than 60 microns, or no greater than 50 microns. For example, a diameter of the adipocytes can be in a range of 10 microns to 250 microns as measured by optical microscopy, such as, for example, in a range of 10 to 50 microns. In various examples, the cell diameter can effect the fragility of the adipocytes.

[0064] Adipocytes may be sourced from human adipose tissue, a cow, a pig, salmon, a chicken, and / or other species. In examples comprising human adipose tissue, the human adipose tissue may be used to obtain adipose derived stem cells and / or mature adipocytes. Adipose derived stem cells may be isolated and may be expanded and differentiated into mature adipocytes through cell culture. The adipocytes may also be derived from a patient and implanted back into that same patient without further expansion.

[0065] In some embodiments, if there is not excess adipose tissue to isolate mature adipocytes, stem cells may be isolated from different parts in the body. In some embodiments, the scaffold may be created from a patient specific model. In some embodiments, for cultivated meat, approaches, various lines of stem cells may be used from different species including, but not limited to, cow, pig, aquatic species such as salmon, and / or chicken.

[0066] A concentration of hydrogel in the ink composition can be at least 1% by volume based on the total volume of the ink composition, such as, for example, at least 2% by volume, at least 3%, at least 4%, or at least 5%, all by volume. A concentration of the hydrogel in the ink composition can be no greater than 10% by volume based on the total volume of the ink composition, such as, for example, no greater than 9%, no greater than 8%, no greater than 7%, no greater than 6%, or no greater than 5%, all by volume. For example, a concentration of the hydrogel in the ink composition can be in a range of 1% to 10% by volume based on the total volume of the ink composition, such as, for example, 2% to 5% by volume based on the total volume of the bioink.

[0067] The bioink can optionally comprise an additive. The additive can comprise a rheological modifier (e.g., a high molecular weight polysaccharaide such as, for example, xanthan gum, dextran, or hyaluronic acid) and / or other additive. For example, the ink 1601463783.2 10Attorney Docket No.250570PCT composition can comprise a concentration of the rheological modifier in a range of 0.1 weight percent to 2 weight percent by total weight of the bioink.

[0068] The hydrogel 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, a Matrigel, a gelatin material, an agar material, a xanthan gum, a pectin, a cellulose, a starch material, a chitosan material, a lignin material, a guar gum material, a carrageenan material, a gum arabic, a carob gum material, a locust bean gum material, a plant-based material, or a combination thereof. For example, the hydrogel can comprise an alginate material.

[0069] The ink composition can comprise a viscosity in a range of 0.005 centipoise (cP) to 10,000 cP at 21 degrees Celsius as measured using a viscometer, such as, for example, 0.015 cP to 10 cP at 21 degrees Celsius as measured using a viscometer. In various examples, the viscosity can effect the quality of the printed construct and / or product.

[0070] The deposition of the ink composition can occur at a suitable shear stress such that the ink composition can flow through the needle and the integrity of the adipocytes is substantially maintained. For example, the shear stress can be high enough to urge the ink composition to flow through and out of the needle. The shear stress can be low enough to not substantially disrupt the adipocytes (e.g., destroy cell walls). For example, the shear stress applied to the ink composition can be at least 1 kPa, such as, for example, at least 2 kPa, at least 3 kPa, at least 4 kPa, at least 5 kPa, at least 6 kPa, at least 7 kPa, at least 8 kPa, at least 9 kPa, at least 10 kPa, at least 15 kPa, at least 20 kPa, at least 25 kPa, or at least 30 kPa. The shear stress applied to the ink composition during deposition can be no greater than 150 kPa, such as, for example, no greater than 125 kPa, no greater than 100 kPa, no greater than 75 kPa, or no greater than 50 kPa. For example, the shear stress applied to the ink composition during deposition can be in a range of 1 kPa to 150 kPa, such as, for example, 1 kPa to 50 kPa or 5 kPa to 50 kPa.

[0071] Optionally, the ink composition may be dispensed into a support material. For example, the nozzle may be deposited into a support material and dispensing of the ink composition can occur in the support material. The structure can be formed in the support material. 1601463783.2 11Attorney Docket No.250570PCT

[0072] The support material can be a support material as used in FRE and / or other support material, such as, for example, a cultivated protein source composed of other cells or other extracellular matrix proteins.

[0073] The support material can physically support at least a portion of the embedded ink composition. The support material can maintain the intended geometry of the embedded ink composition, and inhibit deformation of the respective material during the additive manufacturing process. For example, the embedded ink composition can be held in position within the support material until the ink composition is cultured, solidified, and / or cured.

[0074] The support material can be solid or the support material may flow under stress. For example, in various examples where the additive manufacturing process comprises FRE, the support material 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 additive manufacturing process.

[0075] The support material can be a viscoplastic material with Bingham plastic-like rheological behavior. The support material may demonstrate a significant shear thinning behavior such that the support material acts like a solid material during deposition of the ink composition, and then acts like a fluid when the nozzle is moved through the support material such that the movement of the nozzle may not disturb the previously deposited ink composition. A decrease in viscosity of the support material under shear stress can make the support material suitable for FRE additive manufacturing. For example, in FRE, the dynamic loading can be caused by the force of the nozzle through the support material, affecting the support material in a number of ways.

[0076] The support material 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.

[0077] The support material can comprise a hydrogel. The hydrogel can comprise particles (e.g., microparticles) in a diluent. The particles can comprise gelatin or other suitable particle 1601463783.2 12Attorney Docket No.250570PCT forming compound. The diluent can be aqueous or non-aqueous depending on the desired properties of the support material. Depending on the printing technique, the support material can be clear or opaque.

[0078] In various examples, the support material can comprise a crosslinker for the hydrogel in the ink composition. For example, the support material can comprise thrombin.

[0079] Repositioning of the nozzle and dispensing ink composition from the nozzle can be repeated as necessary as shown by feedback loop step 106, thereby forming the structure. Each iteration can deposit portions of the ink composition (step 104) and the iterations can be repeated until additive formation of the structure is complete (if not aborted earlier).

[0080] Thereafter, at step 108, the ink composition can be at least partially cured after depositing and then, at step 110, the support material can be at least partially removed from the structure. The curing can occur prior to, during, after, or a combination thereof, removal of the support material at step 110.

[0081] In various examples where the additive manufacturing technique comprises FRE, the method can embed the ink composition near other embedded deposits inside the support material and rely on the triggered assembly or reorganization of the material using targeted heating, photopolymerization, crosslinking, slow reaction kinetics, application of binders, and / or other curing techniques.

[0082] The hydrogel of the ink composition can cross-link upon deposition into the support material. For example, the support material may provide divalent cations for ionic crosslinking, such that when the print material contacts the support material, the printed material can begin to cure. In certain examples, the print material may not cure and can hold its shape based on a thixotropic and / or yield-stress property.

[0083] In FRE, the support material can surround the extrusion nozzle and the print material can be deposited inside the support material. The support material can allow for deposition of various materials while maintaining a buoyant, physical support for already embedded deposits of print material. When two embedded deposits of ink composition are within 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. 1601463783.2 13Attorney Docket No.250570PCT

[0084] 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, U.S. Patent Application No. 17 / 754,115, titled MODIFICATION OF RHEOLOGY AND MACHINE PATHING FOR IMPROVED 3D PRINTING OF SOFT MATERIALS, and U.S. Patent Application No. 18 / 246,225, titled TRANSPARENT SUPPORT BATH FOR EMBEDDED 3D PRINTING AND SYSTEM FOR IN PROCESS MONITORING, filed March 22, 2023, each of which are hereby incorporated by reference herein.

[0085] Referring to FIG.2, a block diagram illustrating an example of an additive manufacturing system 200 for FRE according to the present disclosure is provided. The system 200 comprises an extruder assembly 202, a computer system 204, a material deposition region 206. In various examples, additional extruders, additional nozzles, and / or a detector may be added to the additive manufacturing system to increase the printing capabilities of the additive manufacturing system.

[0086] The computer system 204 can be in signal / data communication with the extruder assembly 202 (such as via a wired and / or wireless data bus or link). The computer system 204 can be configured through programming to control the operation of the extruder assembly 202. The computer system 204 can also receive data from and send data (e.g. control data) to the extruder assembly 202. The components may be in communication with the computer system 204 via any suitable type of data bus (e.g., parallel or bit serial connections).

[0087] The extruder assembly 202 may be a syringe-based extruder, which can include a reservoir 212 (e.g., a barrel of a syringe) for receiving and storing in composition, and a nozzle 210 (e.g., a needle), which can be in fluid communication with the reservoir 212 and can receive the in composition from the reservoir 212. For example, the reservoir 212 can comprise ink composition and the ink composition can be extruded through the nozzle 210. 1601463783.2 14Attorney Docket No.250570PCT The nozzle 210 can be configured to deposit the extruded ink composition in the support material 208 disposed in the material deposition region 206.

[0088] In various examples, the extruder assembly 202 and / or additional components can comprise a gantry or other robotic device to support and / or move the extruder assembly 202 relative to the material deposition region 206. Optionally, the extruder assembly 202 can comprise 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 202 comprises an actuator (e.g., a motor) configured to depress a plunger into the reservoir 212 to extrude material through the nozzle 210 into material deposition region 206 as the nozzle 210 is translated through the material deposition region 206 to additively form a structure 214.

[0089] The extruder assembly 202 can be configured to change the support material 208 by imposing a mechanical load via shear, pressure, or vibration. The extruder assembly 202 can be configured to irradiate or heat the support material 208 to thin the support material 208. In various examples, the support material 208 can reduce viscosity under vibration, heating, or irradiation that occurs locally to the extruder assembly 202.

[0090] The computer system 204 comprises one or more processors 220 operatively coupled to one or more non-transitory memories 222 (only one processor 220 and one memory 222 are shown in FIG.2 for simplicity). The processor 220 may comprise one or multiple processing cores. The memory 222 can comprise primary storage (e.g., main memory that is directly accessible by the processor 220, 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 222 stores computer instructions (e.g., software) that are executed by the processor 220. The processor 220 can be configured (through execution of the software stored in the memory 222) to control operation of the extruder assembly 202 to thereby control the deposition of the ink composition through the nozzle 210. For example, the processor 220 can control the flow rate of material through the nozzle 210 (e.g., by the actuation rate of a plunger in the respective extruder assembly 202) and / or the pose of the extruder assembly 202 relative to the material deposition region 206.

[0091] The memory 222 can store a digital or an electronic computer model 224 of the structure 214 to be manufactured by the additive manufacturing process. The computer model 224 can be loaded locally into the memory 222 or can be downloaded from another 1601463783.2 15Attorney Docket No.250570PCT device (e.g., another computer device, cloud) that is in data communication with the computer system 204. To that end, the computer system 204 may comprise a network interface controller (NIC) (not shown) that connects the computer system 204 to a computer network. The computer model 224 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 224 can be generated from image data of a biological structure, an engineered structure, a computationally derived structure, or a combination thereof. In various examples, the computer model 224 can be machine path instructions (e.g., G-code instructions), that may be directly input by an operator or can be downloaded from another device that is in data communication with the computer system 204.

[0092] The nozzle 210 can be configured to deposit an ink composition into the support material 208 by applying a force to the ink composition in the reservoir 212 such that the structure material can flow from the reservoir 212 through the nozzle 210. The ink composition can comprise a yield stress, a thixotropic property, an increased viscosity, or a combination thereof. In examples where the ink composition comprises a yield stress, the force applied can be at least the yield stress. In certain examples, applying the force to the ink composition can cause the structure material to flow through the nozzle. For example, with an increase viscosity, the force can overcome the increased viscosity and cause the ink composition to flow through the nozzle. In examples wherein the ink composition comprises a thixotropic property, the thixotropic property can cause the time scale of flow of the structure material to be longer than the printing process.

[0093] In various examples, a plunger can be translated through the reservoir 212. In various examples, the force can be pneumatically applied or the deposition can be controlled by a progressive cavity pump. The application of the force can cause the material in the reservoir 212 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 206. The ink composition can be suspended in the support material 208 at a location where the ink composition was deposited by the nozzle 210 within the support material 208. Since the processor 220 can control the extruder assembly 202 and nozzle 210, the deposition of the ink composition by the nozzle 210 can be based on the machine path instructions 232 as executed by the processor 220. 1601463783.2 16Attorney Docket No.250570PCT

[0094] The extruder assembly 202 can move the nozzle 210 in two-dimensions when depositing structure material or in three-dimensions when depositing material, i.e., simultaneously in the X, Y, and Z directions. Further, the extruder assembly 202, nozzle 210, and / or material deposition region 206 can be rotatable. The machine pathing instructions 232 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 210 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).

[0095] The depositing of the ink composition can be repeated as necessary to additively form a structure. For example, the processor 220 can control the nozzle 210 to deposit the structure material in portions (e.g., layers) in order to additively form the structure 218 in the support material 208 based on the computer model 224, another plane, and / or non-planar movement. As illustrated, the ink composition was deposited in portion 214a and portion 214b of structure 214.

[0096] The portions 214a, 214b, can be deposited in various sequences as desired. For example, portion 214a can be deposited prior to portion 214b. The processor 220 can control the nozzles 210 to deposit portions 214b proximal to (e.g., adjacent, in contact with, directly on top of) portion 214a such that the deposition of the portion 214b contacts the portion 214a.

[0097] The material deposition region 206 can be configured for mechanically supporting and / or holding the support material 208 during additive manufacturing. For example, the material deposition region 206 can comprise a vessel in which the support material 208 is disposed and a platform on which the vessel is supported. The material deposition region 206 can comprise a motor and / or actuator that can move the platform in 3D space as needed.

[0098] The ink composition can be curable and after curing, the ink composition can be considered cured. The structure 214 can be at least partially cured in the support material 208 after deposition of the ink composition. In various examples, the structure 214 can be at least partially cured prior to removing the support material 108. In some examples, the structure 214 may not be cured until after removing the support material 208. As used in this 1601463783.2 17Attorney Docket No.250570PCT specification, the terms “cure” and “curing” can 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 structure 214 has chemically reacted to form new covalent bonds in the structure material and / or bioink (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.

[0099] For example, curing of the structure 214 can comprise cross-linking. The structure 214 can be treated through various cross-linking techniques to selectively increase the rigidity of the overall structure 214 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 208 can include a cross-linking agent or pH suitable for curing the ink composition as it is deposited into the support material 208.

[0100] The mechanical properties of the structure 214 can be controlled by controlling the amount of curing that occurs within the structure 214. For example, the machine pathing instructions 232 can be modified to control the amount of crosslinking that occurs within the structure 214. For example, the extruder assembly 202 and / or other assembly can comprise a UV light and can selectively subject the structure 214 to the UV light as desired.

[0101] The structure 214 can be at least partially removed from the support material 208. Removing the support material 208 may include heating the support material 208, cooling the support material 208, removing cations to disrupt crosslinking of the support material 208, physically removing the support material 208, 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 208 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. In various examples, the support material may not be removed and / or be otherwise part of the product. 1601463783.2 18Attorney Docket No.250570PCT

[0102] The methods for additive manufacturing herein, such as those illustrated in FIG.1 above, can be implemented in whole or in part as computer-executable instructions stored in the memory 222 of the computer system 204 that, when executed by a processor 220 of the computer system 204, cause the computer system 204 to perform the enumerated steps. The computer instructions can be implemented as one or more software modules 216 stored in the memory 222 that are each programmed to cause the processor 220 to execute one or more discrete steps of the processes described herein or other functions. For example, the software modules 216 can comprise a separation module programmed to convert the computer model 224 into segments; a conversion module programmed to convert the computer model 224 and / or segments into computer instructions (e.g., G-code) for controlling the movement of the extruder assembly 202 to fabricate the structure 214; an imaging module for controlling imaging parameters; 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 202 according to the instructions generated by the conversion module to fabricate the structure 214. 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 connected to a cloud computing system configured to execute one or more of the described steps, and so on.

[0103] The structure 214 can be formed based on a computer model.224 of the structure 214 For example, the processor 220 may receive a computer model 224 of the structure 214. The processor 220, executing the separation module software, can separate (e.g., slice) the computer model into different part segments and the processor 220, executing the conversion module, can create machine path instructions (e.g., G-code instructions) based on the computer model 224. The machine path instructions can be stored in memory 222. Depositing the ink composition can be based on the machine path instructions for the computer model 224.

[0104] The methods for additive manufacturing and systems for additive manufacturing described herein can be used to create various products, repair of volumetric volume loss, wound repair of soft tissue, soft tissue reconstruction, patient specific adipose tissue modeling, enhancement of soft tissue, disease modeling, and / or drug therapeutics platforms. The products can be various product types, such as, for example, a biological medicine 1601463783.2 19Attorney Docket No.250570PCT structure, a cultivated meat, or a combination thereof. For example, the product can comprise a soft structure, a bioprosthetic, a scaffold, a medical device, a regenerative medicine structure, soft tissue, soft tissue reconstruction, patient specific adipose tissue modeling, an implantable device, or other structures that may be additively manufactured. In various examples, the product (e.g., structure 214) can be surgically fit into a patient after additive manufacturing, the structure 214 can be utilized as a biological structure for experimentation, or a combination thereof.

[0105] The cultivated meat can comprise various products that may be suitable for animal and / or human consumption. For example, the cultivated meat can comprise a steak, salmon, foie gras, or bacon, among others.

[0106] EXAMPLES

[0107] 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 is understood that the invention described in this present disclosure is not necessarily limited to the examples described in this section.

[0108] In the examples herein, the printability of mature adipocytes was assessed. The examples demonstrate lipid-laden tissue generation via Freeform Reversible Embedding of Suspended Hydrogels (FRESH) additive manufacturing with suitable ink composition properties and printing parameters. The FRESH 3D additive manufacturing approach shown in these examples can create patient specific lipid-laden adipocyte grafts (e.g., patient- specific mammary tissue), making this methodology of great value for adipose tissue reconstructive medicine. Although the present disclosure should not be limited to this particular example and the benefits shown herein are expected to be realized by other examples according to the present disclosure. FRESH Bioprinting for Mature Adipocytes

[0109] FRESH 3D additive manufacturing was used to create high fidelity, viable prints of complex tissue structures that otherwise may not maintain shape if printed directly in air. It was a layer-by-layer approach that deposited the ink composition, in these examples a lipid- laden adipocyte mixture in alginate, within a second Bingham plastic hydrogel support bath. 1601463783.2 20Attorney Docket No.250570PCT Although it is believed that the examples would have worked with other ink compositions according to the present disclosure.

[0110] The support bath was composed of microspheres that are formed as a result of complex coacervation through the mixing of gelatin (cationic polymer) and gum arabic (anionic polymer) in an aqueous solution to form a liquid-polymer phase. This created desirable rheological behaviors that enabled the support bath to behave as a rigid body at lower shear stresses and as a viscous fluid at higher shear stresses, thus retaining structure to the print throughout the printing process. Upon extrusion, the sodium alginate ink composition was crosslinked in the CaCl2infused support bath. This approach can combat the buoyancy of mature adipocytes, as the immediate crosslinking can inhibit, if not prevent, the printed adipocytes from moving. The alginate ink composition comprised a suitable viscosity that also inhibits, if not prevents, the buoyant adipocytes from floating upwards in the ink composition before they are extruded into the support bath.

[0111] Alginate has been approved by the U.S. Food and Drug Administration as a polymer with biocompatible and biodegradable properties, making it a suitable biomaterial for regenerative medicine applications. Alginate can act as the extracellular matrix (ECM) for adipocytes during the printing process and provides the initial structural support. Recent literature shows >90% degradation of an alginate bioink (30mm diameter and 3.5mm thickness) during a 14-day in vivo study. Conversely, the dynamic regulation of adipose tissue takes ~72 hours to promote ECM synthesis and >7 days to form a complete ECM framework. Therefore, the ECM remodeling can occur during the alginate degradation, leaving a physiologically relevant adipose network in the reconstructive site.

[0112] We evaluated the accuracy of bioprinting adipocytes with the FRESH method. A model with a 10x10x5mm geometry is shown in FIG.3A and a printed construct based on the model in FIG.3A is shown in FIG.3B, which demonstrates the precision of FRESH 3D printing with dimensional errors <0.6%. Furthermore, the uniform color of the construct is a combination of the clear alginate and beige adipocytes, which suggests a homogenous lipid distribution throughout the print. This provides proof-of-concept that we can bioprint uniform adipocytes into anatomical geometries with <1% error.

[0113] Mature adipocytes can vary in size, ranging from 20 to 300 µm in diameter, making it difficult to quantify cell density using a hemocytometer. Moreover, standard cell densities in 1601463783.2 21Attorney Docket No.250570PCT various bioprinting applications can range from 5 to 40 million cells / mL for mesenchymal stem cells (MSCs), with cell diameters typically ranging from 15 to 30 µm. Due to the large size discrepancy of mature adipocytes to MSCs, the standard cell density is not translatable. For bioprinting mature adipocytes, the cell density in the bioink was optimized by varying the volume ratio of the cell solution to alginate from 60% to 90% using a 10x10x5mm CAD model.

[0114] After releasing the prints from the support bath, the 60% cell dense print is shown in FIG.4A and it maintained structural support. However, the 70% cell dense print shown in FIG.4B, the 80% cell dense print shown in FIG.4C, and the 90% cell dense print shown in FIG.4D did not obtain a desirable print structure, which is believed to be due to the inability to crosslink in the support bath with less alginate present. The remaining printed constructs were generated using a 60% cell dense bioink solution.

[0115] Bioink Viscosity

[0116] The size of an adipocyte can be regulated by extracellular matrix properties, where adipocyte size can be decreased due to enhanced collagen content with adipose tissue fibrosis. Previously, it was believed that adipocytes equilibrate their size to their matrix within a few days of culture, and observed that adipocytes derived from patients with highly variable diameters (ranging from ~81 to 189 µm) adapted to a hyaluronic acid hydrogel resulting in consistent adipocyte diameters for all tested patients (equilibrated diameters ranging from ~64 to 66 µm). Additionally, adipocytes can be fragile cells that are highly susceptible to shear stress. Therefore, the concurrent impact of shear stress on adipocyte size incurred during printing and in response to various alginate hydrogel concentrations was assessed.

[0117] A lipid-laden adipocyte bioink was both statically extruded as illustrated in FIGs.5A- 5D and printed as illustrated in FIGs.5E-5H at four different alginate concentrations ranging from 4% to 10% by volume of the portion of the bioink that comprises the hydrogel. It was observed that as the alginate concentration and therefore stiffness of the hydrogel increases, the mean diameter of the resulting adipocytes decreases as shown in FIG.5I. A decrease in lipid diameter was also observed when printing the adipocyte bioink rather than statically extruding at the same alginate concentration (with the exception of the highest alginate concentration). This supports the conclusion that an increase in matrix stiffness can restrict 1601463783.2 22Attorney Docket No.250570PCT adipocyte size rapidly. While literature observes changes in adipocyte sizes over the course of days to weeks, these experiments show that this effect occurs after one hour in the alginate bioink. Additionally, the results indicate that printing has an added influence on changes in adipocyte size, and this effect can depend on alginate concentration. Increased concentrations illustrated a diminishing difference between statically extruded and printed adipocyte size. Modeling of computational fluid dynamics indicated significant increases in shear stress during printing with each increase in alginate concentration between 4%, 6%, 8%, and 10% by volume of the portion of the bioink that comprises the hydrogel as illustrated in FIG.5J. Printing Needle Gauge

[0118] In bioprinting applications, the gauge (g) of the needle used for printing can impact the resulting product, as larger needle sizes can limit the smallest features that can be printed accurately, while smaller needles can have negative effects on cell viability. When printing with mature adipocytes, given their larger size, needles of gauges 18g, 22g, and 26g were chosen to assess the impact of adipocyte morphology, corresponding to internal diameters of 965μm, 410μm, and 228μm respectively. The results are shown in FIGs.6A-6C.

[0119] As illustrated in FIG.6D, printing with the 26g needle led to a significant decrease in adipocyte size relative to both 18g and 22g needles. Between the larger two, there was no significant difference in lipid diameter observed. As such, a 22g needle was chosen as the desirable size for additional examples in order to enhance adipocyte diameter and printing accuracy, which may enable more detailed printing than the 18g needle while maintaining the adipocyte lipid diameter.

[0120] As illustrated in FIG.6E, modeling of computational fluid dynamics indicated increases in shear stress during printing with each decrease in needle gauge from 26g to 18g. In comparison to the increase in shear stress from 16g to 22g, the shear stress increased ~5- fold more from 22g to 26g. This large increase is consistent with the consequent decrease in lipid diameter from 22g to 26g. Printing Speed

[0121] In FRESH bioprinting, the printing needle typically applies at least the required shear stress to induce liquid-like behavior of the support bath as the needle moves through it. As such, a minimum printing speed-referring to the velocity at which the needle travels during 1601463783.2 23Attorney Docket No.250570PCT printing rather than the length of time required to print-can be desired to apply this level of shear stress.

[0122] Printing speed can be limited as excessive speeds can lead to loss of print quality. A desirable printing speed can be a high speed (in order to reduce printing time) that still enables quality printing and limits deleterious effects on printed cells. Printing speeds of 4mm / s, 10mm / s, and 16mm / s were chosen to assess these impacts and the resulting printed bioinks are shown in FIGs.7A-7C. No significant difference in the diameter of the printed adipocytes in the range of printing speeds from 4 to 16 mm / s was observed as shown in FIG. 7D.

[0123] As illustrated in FIG.7E, the cellular density of the prints were also examined and showed a significant increase in cellular density with increasing printing speeds. Modeling of computational fluid dynamics indicated significant increases in shear stress during printing with each increase in printing speed between 4, 10, and 16 mm / s as illustrated in FIG.7F. As with the changes in alginate concentration, this computational result would indicate that despite leading to increases in shear stress this was still low enough so as to not elicit a change in adipocyte diameter. Cellular Activity

[0124] A short-term (3 day) cellular activity study was performed to assess whether printed constructs were metabolically active and could be used for regenerative medicine. Referring to FIG.8, the results indicate that adipocytes do not experience a significant change in the relative fluorescence units throughout the three-day experiment, indicating that the cellular metabolic activity remained stable. This stability suggests that the mature adipocytes did not experience significant stress or cell death due to the printing process and short culture after. 3D Printed Breast Reconstruction from Magnetic Resonance Imaging

[0125] Patient specific anatomical models can be generated by converting standard medical imaging files from CT, MRI, or 3D ultrasound datasets into solid objects, or CAD models. Then, FRESH 3D bioprinting can be used to print viable lipid-laden adipose grafts in those precise geometries. Through 3D printing, the tissue microstructure can be engineered by optimizing printing parameters to create the optimal tissue porosity to promote nutrient 1601463783.2 24Attorney Docket No.250570PCT diffusion and enhance the interface between the host and graft. A schematic of this process can be seen in Figure 9.

[0126] Referring to FIGs.10A-C, as a proof of concept, MRIs were obtained from patients undergoing breast reconstruction after a partial or full mastectomy, converted into CAD models, and used to 3D print a patient specific lipid-laden adipocyte reconstructive graft. The MRIs were segmented to distinguish between different tissue types and volumes, ensuring that the model reflected the complexity of the resected breast tissue.3D printing can allow for precise spatial control of the tissue, to recreate the damaged region. While this technique can be used for 3D printing mature adipocytes, whole adipose tissue is composed of multiple cell types including endothelial cells, fibroblasts, pericytes, preadipocytes, macrophages, and more can be used. These cell types can be integrated into the bioink, or 3D printed using a second extrusion nozzle on the FRESH 3D printer.

[0127] The following methods and materials were used to conduct the examples discussed above. 3D printer assembly

[0128] A lulzbot mini 23D plastic printer was converted to a 3D bioprinter using protocols previously developed. Briefly, the thermoplastic extruder on the 3D plastic printer was removed and replaced with the Replistruder 4, an open-sourced 3D plastic printed syringe pump for extrusion-based 3D printing of soft materials. Isolation of adipocytes

[0129] Subcutaneous adipose tissue was obtained from elective abdominoplasty and abdominal panniculectomy procedures at the University of Pittsburgh Medical Center (UPMC) with approval by the University of Pittsburgh Institutional Review Board (IRB No. 0511186). The tissue was processed the day of the surgery. Blunt dissection was used to separate the adipose tissue from the skin followed by pulse blending to break up the tissue. An equal volume of liquified adipose tissue and warmed phosphate buffered saline (PBS) was used to wash the tissue until the PBS remained clear after washing. An equal volume of warmed collagenase solution (PBS + 1% BSA + 0.1% collagenase type I) was then added to the washed adipose tissue and incubated for 1 h (37 C, 5% CO2). After incubation, the solution was centrifuged at 300xg for 5 minutes at room temperature, separating the solution 1601463783.2 25Attorney Docket No.250570PCT by oil, primary adipocytes, collagenase solution, and stromal vascular fraction (pelleted), from top to bottom. The oil was aspirated, and the primary adipocytes were transferred onto a 1 mm sieve, followed by a 350 µm sieve, using warmed PBS to filter the primary adipocytes through. The filtered primary adipocytes were centrifuged at 300xg for 5 minutes at room temperature, separating the solution by oil, primary adipocytes, and PBS, from top to bottom. The oil was aspirated, and the primary adipocytes were transferred into a separate tube to be added to the alginate bioink. Bioink Preparation

[0130] Alginate was weighed and mixed into PBS on a magnetic stirrer, spinning at 350 RPM, for five hours at room temperature. The ratios of alginate to 50 mLs PBS are adjusted to two, three, four, and five grams to make 4%, 6%, 8% and 10% alginate, respectively. The isolated adipocytes were added to the alginate and mixed until the bioink was homogeneous. The ratio of alginate to adipocytes was 2:3, determined in preliminary data experiments. The bioink was loaded into a disposable 5 mL syringe, a syringe stopper was added, and the syringe plunger was removed. The top of the syringe was wrapped in parafilm and then centrifuged at 300xg for 5 minutes. The parafilm was removed and a stainless-steel wire was inserted into the syringe, making contact with the bioink. The plunger was inserted until it was flush with the bioink to remove all air bubbles. The bottom layer of alginate was removed and then a lure lock was connected to transfer the adipocytes from the 5 mL disposable syringe to the 3 mL Hamiliton syringe that had previously been primed with alginate. FRESH Gelatin Microparticle Support Bath

[0131] The FRESH gelatin microparticle support bath was generated. Briefly, a complex coacervation method was used to produce gelatin microparticles by dissolving powdered mixtures of 3.0% (w / v) gelatin Type B, 0.125% (w / v) Pluronic F-127, and 0.3% (w / v) gum Arabic in a 50% (v / v) 200 proof ethanol in DI water solution at 45 °C. The pH was quickly adjusted to ≈5.6 by addition of 2 N hydrochloric acid. A metal rotor was attached to an overhead stirrer and was used to mix the solution at 560 RPM for 12 hours in a temperature- controlled room (21 °C). The container was covered with parafilm while stirring to minimize evaporation. After spinning, the rotor was switched off and the solution sat statically and was allowed to separate for at least two hours. 1601463783.2 26Attorney Docket No.250570PCT

[0132] To wash the solution, the liquid supernatant was decanted and the microparticles were aliquoted into 50 mL conical tubes. The tubes were centrifuged at 400 G for 5 minutes, the liquid supernatant was decanted, and the gelatin microparticles were resuspended in DI water. This step was repeated three more times in total, centrifuged twice at 500 G and then at 750 G for the last spin. The final three washing steps remained the same but replaced DI water with a 0.1% CaCl2 and 50 mM HEPES solution and then stored at 4 °C. For these washes, the solution was centrifuged at 750 G, 750 G, and 1000 G for 5 minutes each. Prior to printing, the support bath was degassed in a vacuum chamber for 15 mins, compacted by centrifugation at 1800g for 5 minutes, and then the supernatant was decanted. To transfer the support bath into the printing petri dishes, a stainless-steel wire was inserted into the 50 mL conical tube, making contact with the support bath, and a 50 mL syringe plunger was inserted into the conical tube until the plunger was flushed with the support bath. The wire was then removed. The bottom of the 50 mL conical tube was cut off using a razor blade, and the plunger pushed out the support bath into the printing petri dish. G-Code Generation

[0133] Fusion 360 was used to generate the CAD models (either 10x10x5mm or 3x3x3mm). The file was imported into Slic3r for slicing and to generate a g-code. A 3 mL Hamilton syringe was used along with 18-, 22-, and 26-gauge needles. Altering the needle sizes changes the layer height, extrusion width, and nozzle diameter in the g-code. Unless otherwise specified, the main print settings, generated using the 22-gauge needle, were 0.246 mm layer height, 4 mm s−1 print speed, 50% infill, 2 perimeters, 2 top, and bottom layers, 7.285 mm filament diameter, and a 0.6 extrusion multiplier. A rectilinear infill with 10% infill overlap was used and retraction was turned on. After creating the settings, the G-code was generated.

[0134] To generate the reconstructive model, an MRI was used to image a patient's affected region after a mastectomy. The MRI data was imported into 3D Slicer and segmented using the thresholding tool to create the CAD model of the defect. The MRIs were segmented to distinguish between different tissue types and volumes, ensuring that the model reflected the complexity of the resected breast tissue. The segmentation was exported as a CAD model, imported into Slic3r to generate the g-code, and then uploaded to the FRESH 3D bioprinter. For the reconstructive breast g-code, the same settings previously mentioned were used. The 1601463783.2 27Attorney Docket No.250570PCT model was printed with a 22-gauge needle, 10 mm / s speed, and 6% alginate. The damaged- facing side was oriented downwards. Printing Parameters

[0135] PronterFace was used to connect the 3D printer to the computer and import the G- code. The Hamilton syringe was attached to the printer and a 22-gauge blunt tip needle was screwed into the syringe. The bioink was extruded until it was seen coming out of the needle. A print dish was tapped to the printing bed. The needle was centered in the printing dish and submerged into the gelatin microparticle support. The G-code was then executed, taking roughly 17 minutes to finish for a 10x10x5mm print.

[0136] When the print was complete, the needle was retracted, and the print dish was removed from the printer’s bed and transferred into an incubator at 37°C for 3 hours to thermally release the gelatin microparticles. The liquid gelatin was removed and a solution containing 0.1% CaCl2 and 50 mM HEPES was added for 30 minutes. This process was repeated three times in total. The print was then either fixed, lysed, or prepared for cell culture. Resazurin

[0137] Resazurin was reconstituted to make a 1 mM solution with PBS and then diluted to 0.1 mM solution with media for the working concentration.700 μl of the working concentration of resazurin was added to a 5x5x2.5mm print and incubated for 2 hours. Resazurin was removed and the print was washed with warmed PBS three times before culture media was added again. A plate reader was used to measure the fluorescence of resazurin with excitation at 530 and emission at 590 nm. Immunostaining

[0138] Immunostaining was used to assess the morphology of the cells after printing. The samples were washed three times with warmed 0.1% CaCl2 for 5 minutes in the incubator, fixed with formalin for 20 minutes at room temperature, and then washed three more times with PBS. Next, the samples were permeabilized with 0.1% triton for 15 minutes on a shaker plate, washed three times with PBS, and stained with BODIPY (1:4000), DAPI (1:500), and Phalloidin 555 (1:500) for 1 hour on a shaker plate. Lastly, the prints were washed three 1601463783.2 28Attorney Docket No.250570PCT times with PBS. The stained samples were imaged using a Zeiss LSM 700 Confocal microscope using lasers 405 for DAPI (blue), 488 for BODIPY (green), and 555 for Phalloidin (red). CFD Modeling

[0139] To assess shear stress of adipocytes during the printing process SolidWorks 3D modeling was used to create a CAD model of a 3mL BD syringe as shown in FIG.11. SolidWorks computational fluid dynamics (CFD) was then used to model the simulation. Parameters specified were as follows: steady-state conditions were applied to prevent deformation; laminar flow due to the small model, low speed, and high viscosity; the walls were set with no-slip boundary conditions; and the CFD simulation used Navier Stokes equations to complete the simulations. Data and Statistical Analysis

[0140] An automated in-house pixcell software was used to quantify the lipid diameters. ImageJ software was used to calculate cellular density of the lipids. Files were converted into a binary file and the percentage of area covered with lipids was extracted.

[0141] GraphPad Prism software was then used for all statistical analyses. A paired students t-test was performed for data comparing the means of two matched groups, and a one-way ANOVA was performed with a Tukey’s post hoc multiple comparison test for experiments with more than two independent groups. For all statistical analyses, significance was defined as p<0.05.

[0142] Various aspects of non-limiting embodiments of an invention according to the present disclosure include, but are not limited to, the aspects listed in the following numbered clauses.

[0143] Clause 1. A method for additive manufacturing, the method comprising: dispensing an ink composition from a nozzle of an additive manufacturing system, wherein the ink composition comprise adipocytes and a hydrogel, wherein the deposition occurs at a suitable shear stress such that the ink composition can flow through the needle and an integrity of the adipocytes is substantially maintained; and repeating, as necessary, repositioning of the nozzle and dispensing ink composition from the first nozzle, thereby forming the structure. 1601463783.2 29Attorney Docket No.250570PCT

[0144] Clause 2. The method of clause 1, further comprising disposing the nozzle into a support material and wherein the dispensing of the ink composition occurs in the support material and the structure is formed in the support material.

[0145] Clause 3. The method of clause 2, wherein the hydrogel cross-links upon deposition into the support material.

[0146] Clause 4. The method of any of clauses 1-3, wherein a concentration of adipocytes in the bioink is in a range of 40% to 80% by volume based on the total volume of the bioink.

[0147] Clause 5. The method of any of clauses 1-4, wherein a concentration of adipocytes in the bioink is in a range of 50% to 70% by volume based on the total volume of the bioink.

[0148] Clause 6. The method of any of clauses 1-5, wherein a concentration of hydrogel in the bioink is in a range of 1% to 10% by volume based on the total volume of the bioink.

[0149] Clause 7. The method of any of clauses 1-6, wherein a concentration of hydrogel in the bioink is in a range of 2% to 5% by volume based on the total volume of the bioink.

[0150] Clause 8. The method of any of clauses 1-7, wherein the hydrogel 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, a Matrigel, a gelatin material, an agar material, a xanthan gum, a pectin, a cellulose, a starch material, a chitosan material, a lignin material, a guar gum material, a carrageenan material, a gum arabic, a carob gum material, a locust bean gum material, a plant-based material, or a combination thereof.

[0151] Clause 9. The method of any of clauses 1-8, wherein the hydrogel comprises an alginate material.

[0152] Clause 10. The method of any of clauses 1-9, wherein the structure is a biological medicine structure or a cultivated meat.

[0153] Clause 11. The method of any of clauses 1-10, wherein the bioink comprises a viscosity in a range of 0.005 cP to 10,000 cP at 21 degrees Celsius using a viscometer.

[0154] Clause 12. The method of any of clauses 1-11, wherein the bioink comprises a viscosity in a range of 0.015 cP to 10 cP at 21 degrees Celsius using a viscometer. 1601463783.2 30Attorney Docket No.250570PCT

[0155] Clause 13. The method of any of clauses 1-12, wherein the adipocytes comprise a diameter in a range of 10 microns to 250 microns.

[0156] Clause 14. The method of any of clauses 1-13, wherein an internal diameter of the nozzle is in a range of 200 microns to 1,000 microns.

[0157] Clause 15. The method of any of clauses 1-14, wherein an internal diameter of the nozzle is in a range of 300 microns to 600 microns.

[0158] Clause 16. The method of any of clauses 1-15, wherein a shear stress applied to the bioink during deposition is no greater than 150 kPa.

[0159] Clause 17. The method of any of clauses 1-16, wherein a shear stress applied to the bioink during deposition is no greater than 50 kPa.

[0160] Clause 18. The method of any of clauses 1-17, wherein the support material comprises a crosslinker for the hydrogel.

[0161] Clause 19. The method of any of clauses 1-17, wherein: a concentration of adipocytes in the bioink is in a range of 50% to 70% by volume based on the total volume of the bioink; a concentration of hydrogel in the bioink is in a range of 2% to 5% by volume based on the total volume of the bioink; the hydrogel comprises an alginate material; the bioink exhibits a viscosity in a range of 0.015 cP to 10 cP at 25 degrees Celsius; an internal diameter of the nozzle is in a range of 300 microns to 600 microns; a shear stress applied to the bioink during deposition is no greater than 50 kPa; and the support material comprises a crosslinker.

[0162] Clause 20. The method of any of clauses 1-19, further comprising imaging a biological structure and generating a model of the biological structure based on the imaging, wherein the structure is formed based on the model.

[0163] Clause 21. The method of clause 20, wherein the model is obtained from magnetic resonance imaging (MRI), computed tomography (CT), 3D ultrasound, or a combination thereof.

[0164] Clause 22. A biological structure produced by the method according to any of clauses 1-21. 1601463783.2 31Attorney Docket No.250570PCT

[0165] Clause 23. A cultivated meat produced by the method according to any of clauses 1- 21.

[0166] Clause 24. An additive manufacturing system configured to perform the method of any of clauses 1-21.

[0167] Certain exemplary aspects of the present disclosure are described to provide an overall understanding of the principles of the structure, function, manufacture, and use of the compositions, methods, and products disclosed herein. One or more examples of these aspects are illustrated in the accompanying drawings. Those of ordinary skill in the art will understand that the devices and methods specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary aspects and that the scope of the various examples of the present disclosure is defined solely by the claims. The features illustrated or described in connection with one exemplary aspect may be combined with the features of other aspects. Such modifications and variations are intended to be included within the scope of the present disclosure.

[0168] Any references herein to “various examples,” “some examples,” “one example,” “an example,” similar references to “aspects,” or the like, means that a particular feature, structure, or characteristic described in connection with the example is included in at least one example. Thus, appearances of the phrases “in various examples,” “in some examples,” “in one example,” “in an example,” similar references to “aspects,” or the like, in places throughout the specification are not necessarily all referring to the same example. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more examples. Thus, the particular features, structures, or characteristics illustrated or described in connection with one example may be combined, in whole or in part, with the features, structures, or characteristics of one or more other examples without limitation. Such modifications and variations are intended to be included within the scope of the present examples.

[0169] 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 1601463783.2 32Attorney Docket No.250570PCT 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.

[0170] 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.

[0171] The grammatical articles “a,” “an,” and “the,” as used herein, are intended to include “at least one” or “one or more,” unless otherwise indicated, even if “at least one” or “one or more” is expressly used in certain instances. Thus, the articles are used herein to refer to one or more than one (i.e., to “at least one”) of the grammatical objects of the article. Further, the use of a singular noun includes the plural, and the use of a plural noun includes the singular, unless the context of the usage requires otherwise.

[0172] 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.

[0173] 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 1601463783.2 33Attorney Docket No.250570PCT 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 least as broad as they are claimed and not as more narrowly defined by particular illustrative aspects provided herein. 1601463783.2 34

Claims

Attorney Docket No.250570PCT CLAIMS What is claimed is:

1. A method for additive manufacturing, the method comprising: dispensing an ink composition from a nozzle of an additive manufacturing system, wherein the ink composition comprise adipocytes and a hydrogel, wherein the deposition occurs at a suitable shear stress such that the ink composition can flow through the needle and an integrity of the adipocytes is substantially maintained; and repeating, as necessary, repositioning of the nozzle and dispensing ink composition from the first nozzle, thereby forming the structure.

2. The method of claim 1, further comprising disposing the nozzle into a support material and wherein the dispensing of the ink composition occurs in the support material and the structure is formed in the support material.

3. The method of claim 2, wherein the hydrogel cross-links upon deposition into the support material.

4. The method of claim 1, wherein a concentration of adipocytes in the bioink is in a range of 40% to 80% by volume based on the total volume of the bioink.

5. The method of claim 1, wherein a concentration of adipocytes in the bioink is in a range of 50% to 70% by volume based on the total volume of the bioink.

6. The method of claim 1, wherein a concentration of hydrogel in the bioink is in a range of 1% to 10% by volume based on the total volume of the bioink.

7. The method of claim 1, wherein a concentration of hydrogel in the bioink is in a range of 2% to 5% by volume based on the total volume of the bioink.

8. The method of claim 1, wherein the hydrogel 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, a Matrigel, a gelatin material, an agar material, a xanthan gum, a pectin, a cellulose, a starch material, a chitosan material, a lignin material, a guar gum 1601463783.2 35Attorney Docket No.250570PCT material, a carrageenan material, a gum arabic, a carob gum material, a locust bean gum material, a plant-based material, or a combination thereof.

9. The method of claim 1, wherein the hydrogel comprises an alginate material.

10. The method of claim 1, wherein the structure is a biological medicine structure or a cultivated meat.

11. The method of claim 1, wherein the bioink comprises a viscosity in a range of 0.005 cP to 10,000 cP at 21 degrees Celsius using a viscometer.

12. The method of claim 1, wherein the bioink comprises a viscosity in a range of 0.015 cP to 10 cP at 21 degrees Celsius using a viscometer.

13. The method of claim 1, wherein the adipocytes comprise a diameter in a range of 10 microns to 250 microns.

14. The method of claim 1, wherein an internal diameter of the nozzle is in a range of 200 microns to 1,000 microns.

15. The method of claim 1, wherein an internal diameter of the nozzle is in a range of 300 microns to 600 microns.

16. The method of claim 1, wherein a shear stress applied to the bioink during deposition is no greater than 150 kPa.

17. The method of claim 1, wherein a shear stress applied to the bioink during deposition is no greater than 50 kPa.

18. The method of claim 1, wherein the support material comprises a crosslinker for the hydrogel.

19. The method of claim 1, wherein: a concentration of adipocytes in the bioink is in a range of 50% to 70% by volume based on the total volume of the bioink; a concentration of hydrogel in the bioink is in a range of 2% to 5% by volume based on the total volume of the bioink; 1601463783.2 36Attorney Docket No.250570PCT the hydrogel comprises an alginate material; the bioink exhibits a viscosity in a range of 0.015 cP to 10 cP at 25 degrees Celsius; an internal diameter of the nozzle is in a range of 300 microns to 600 microns; a shear stress applied to the bioink during deposition is no greater than 50 kPa; and the support material comprises a crosslinker.

20. The method of claim 1, further comprising imaging a biological structure and generating a model of the biological structure based on the imaging, wherein the structure is formed based on the model.

21. The method of claim 20, wherein the model is obtained from magnetic resonance imaging (MRI), computed tomography (CT), 3D ultrasound, or a combination thereof.

22. A biological structure produced by the method according to claim 1.

23. A cultivated meat produced by the method according to claim 1.

24. An additive manufacturing system configured to perform the method of claim 1. 1601463783.2 37

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