Compositions and Methods for Treating Diabetes

Abstract

Aspects of the present disclosure include an implantable composition / medical device encapsulating insulin-producing cells for use in the treatment and management of diabetes, generally comprising a multi-layer lattice structure fabricated from core / shell fibers containing continuously bioprinted cells, the lattice structure having a defined packing density and at least one coating applied to the lattice after printing, preferably at least one conformal coating. In this way, the stability, retrievability, and functionality of the multi-layer lattice structure are improved.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 342,120, filed May 15, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0002] Field of Disclosure The present disclosure generally relates to implantable therapeutic devices for the treatment and management of diseases, disorders, or medical conditions. In particular, the present invention relates to three - dimensional biological structures containing pancreatic islet cells printed from digital files, as well as compositions and methods of use thereof, for use in patients in need of treatment of diabetes.

Background Art

[0003] Type 1 diabetes (T1D) is an autoimmune disease in which the immune system destroys pancreatic β - cells that produce insulin. Blood glucose (BG) can be managed by daily injections or infusions of exogenous insulin, but even with intensive monitoring and adjustment of insulin dosage, such an approach does not cure the disease and also does not prevent many of the side effects associated with diabetes. For example, hyperglycemia and hypoglycemia are common side effects of exogenous insulin therapy that can cause, among other things, irreversible tissue and organ damage.

[0004] Type 2 diabetes (T2D) has a different etiology and usually develops in older age, but similarly, blood glucose control is lost, resulting in organ damage. Most T2D patients can control their blood glucose levels by improving diet and lifestyle, but many T2D patients have reduced responsiveness to insulin, which often leads to pancreatic dysfunction. As a result, about 30% of T2D patients are also dependent on exogenous insulin to maintain normal blood glucose.

[0005] The results of recent clinical trials have demonstrated that islet transplantation holds promise as a potential curative therapy. Butler and Gale, J Clin Invest. (2022) 132(3):e158305. doi.org / 10.1172 / JCI158305. In particular, unique embryonic stem cell-derived islet cells containing functional β-cells were generated using a differentiation protocol established by Dr. Melton et al. at Harvard University (Pagliuca et al. Cell (2014);159(2):428-439), and the first patient to receive these cells in an ongoing clinical trial showed significant clinical effects. (Vertex announced positive 90-day data for the first patient in a Phase 1 / 2 clinical trial receiving VX-880, a new investigational stem cell-derived therapy for the treatment of type 1 diabetes. Press release. October 18, 2021). Unfortunately, however, this approach requires lifelong immunosuppressive therapy to counteract the strong host immune response elicited against the transplanted cells, which can cause serious side effects (Weir GC and Bonner-Weir S, Diabetes. (1997);46: 1247-1256; Hafiz MM et al., Transplantation. (2005);80:1718-1728; Niclauss N et al., Transplantation. (2011);91: 714-722).

[0006] As an alternative approach for transplanting pancreatic islet- or stem cell-derived β-like cells without immunosuppression, cell encapsulation has been continuously studied (Calafiore and Basta, Adv Drug Deliv Rev. (2014);67-68: 84-92). However, despite decades of research worldwide, effective clinical application of cell encapsulation has still not been achieved (Orive et al., Trends Pharmacol Sci. (2015);36:537-546). Planar diffusion chambers such as the Viacyte device have been shown to be very promising in small animal studies (Nat Biotechnol. 2014;32:929), but they are plagued by major challenges in scaling up to human patients due to their inherently small surface area for mass transfer. Calafiore, supra. Furthermore, clinical trials of an improved device called VC01-103 were terminated due to "insufficient implantation of the functional product," suggesting that the recovered devices were covered with fibrous tissue. Henry et al. Diabetes. 2018;67(Suppl 1):138-OR. Subsequently, a portal enabling blood vessel growth was added to the device, but this also enabled access to the immune system, necessitating the introduction and maintenance of immunosuppressive therapy. Shapiro et al. Cell Rep Med. 2021;2(12):100466

[0007] There is still a shortage of cell encapsulation devices that can protect encapsulated cells and / or tissue pieces from the host immune system without hindering the passage of nutrients, oxygen, and secretory products (e.g., insulin), have sufficient strength and elasticity to survive in vivo over a long period of time, and can be easily retrieved when depleted or damaged. These requirements are further complicated by the fact that implanting such devices into the body can trigger an organized biological response by both the innate and adaptive immune systems, called the foreign body reaction (FBR). This reaction is partially mediated by macrophages that overexpress extracellular matrix (ECM) proteins such as fibronectin and promote fibroblast-mediated fibrosis, resulting in the production of fibrogenic factors that form a fibrous capsule around the device. This fibrous capsule can potentially interfere with the function of the device, especially when it contains a therapeutic cell population that requires access to nutrient and oxygen flow and the excretion of therapeutic proteins to perform its intended function.

[0008] A wide range of materials, from natural polymers to synthetic materials, are said to cause a fibrous reaction. Ward WK, J Diabetes Sci Technol. (2008);2: 768-777; Zhang L et al., Nature Biotech. (2013);31: 553-556; Ratner BD, Journal of Controlled Release. (2002);78: 211-218. Furthermore, physical parameters such as the shape, size, and texture of synthetic tissue structures are also inherent properties that contribute to FBR. The surface of synthetic tissue structures can affect the behavior of immune cells such as macrophages, and structures with smooth surfaces generally do not cause much inflammation. Mariani E et al., Int J Mol Sci. (2019);20: doi: 10.3390 / ijms20030636. Changes in surface roughness at the nanoscale are associated with an increase in protein adsorption (Hulander M et al., Int J Nanomedicine. (2011);6: 2653-2666; Roach P., J Mater Sci Mater Med. (2007);18: 1263-1277; Scopelliti PE et al., PLOS ONE. (2010);5: e11862), and the topography of different nanostructures can affect cell interactions (Baker DW et al., Biomacromolecules. (2011);12: 997-1005; Jahed Z., Biomaterials. (2014);35: 9363-9371).

[0009] Therefore, to fully realize the clinical potential of encapsulated cells in the treatment of T1D, improvements in both design and materials are needed to address the opposing goals of immune defense and nutrient passage and to mitigate the FBR response. There is a need for synthetic tissue structures that reduce or avoid FBR and immune system recognition while ensuring sufficient passage of oxygen and nutrients to the cells. There is also a need for synthetic structures and methods of generating them that achieve consistent and reliable patterning and effective adhesion between synthetic tissue fibers.

Summary of the Invention

[0010] The present invention successfully addresses the aforementioned opposing goals in the art with an implantable medical device for the treatment of diabetes, comprising a lattice structure containing continuously bioprinted core / shell fibers encapsulating pancreatic islet cells, the lattice structure further comprising at least one coating that imparts effective fiber-to-fiber (FF) adhesion to enhance the structural stability and retrievability of the device. In embodiments, the lattice structure comprises at least one conformal coating described herein, whereby anti-FBR properties can be imparted to the lattice structure. In embodiments, the lattice structure is manufactured at a defined packing density that allows for effective diffusion properties and blood vessel growth, resulting in effective and sustained delivery of oxygen and nutrients to the pancreatic islet cells within the lattice structure. The pancreatic islet cells exhibit improved viability and functionality and release insulin in vivo in response to blood glucose levels, effectively treating type 1 diabetes in patients in need of treatment.

[0011] Aspects of the present invention include an implantable composition / medical device for the treatment of type 1 diabetes, comprising a multilayer lattice structure containing continuously bioprinted core / shell fibers encapsulating a plurality of pancreatic islet cells, and at least one coating surrounding the multilayer lattice structure, wherein the multilayer lattice structure has a packing density of about 10% to about 90%, or about 20% to about 80%, or about 30% to about 70%, or about 40% to about 60%, and preferably about 50% to about 70%, or about 55% to about 65%, or about 60%.

[0012] In an embodiment, the lattice structure has a packing density of about 30%, about 40%, about 50%, about 60%, or about 70%, or about 80%.

[0013] In an embodiment, the multi-layer lattice structure includes at least one conformal coating.

[0014] In an embodiment, the continuously bioprinted core / shell fiber includes a solid core and at least one shell, and optionally, the material strength of the solid core is lower than that of the shell.

[0015] In an embodiment, the coating includes a hydrogel having a material strength lower than both the core of the fiber and at least one shell.

[0016] In an embodiment, the solid core, at least one shell, and the coating include the same hydrogel material, and preferably, the hydrogel material is alginate.

[0017] In an embodiment, the solid core, at least one shell, and / or the coating include chemically modified alginate.

[0018] In an embodiment, the solid core includes about 1.2% to about 1.8% alginate, preferably about 1.5% alginate.

[0019] In an embodiment, at least one shell includes about 1.4% to about 3.0% alginate, preferably about 1.5% to about 2.5% alginate, more preferably about 1.8% to about 2.2% alginate.

[0020] In an embodiment, the coating includes about 0.2% to about 2% alginate, or about 0.25% to about 1.5% alginate, preferably about 0.3% to about 1.0% alginate, more preferably about 0.4% to about 0.8% alginate.

[0021] In an embodiment, the at least one coating includes a first inner coating and a second outer coating. In an embodiment, the first inner coating includes a hydrogel having a higher material strength than the second outer coating. In an embodiment, the first inner coating includes from about 1.4% to about 3.0% alginate, preferably from about 1.5% to about 2.5% alginate, and more preferably from about 1.8% to about 2.2% alginate. In an embodiment, the second outer coating includes from about 0.25% to about 1.5% alginate, preferably from about 0.3% to about 1.0% alginate, and more preferably from about 0.4% to about 0.8% alginate.

[0022] In an embodiment, the pancreatic islet cells are human pancreatic islet cells, and optionally, the pancreatic islet cells include re-aggregated islets. In an embodiment, the pancreatic islet cells are stem cell-derived human pancreatic islet cells, and optionally, the stem cell-derived pancreatic islet cells include cell clusters.

[0023] In an embodiment, the lattice structure includes at least 2, 3, 4, or 5 layers formed by continuous fibers, preferably the lattice structure includes 2, 3, or 4 layers, and more preferably the lattice structure includes 4 layers.

[0024] In an embodiment, the diameter of the continuous fiber is about 0.2 to 2.0 mm, or about 0.5 to 1.5 mm, about 0.5 to 0.9 mm, or about 900 μm to about 1200 μm, and preferably the diameter is about 950 μm to about 1100 μm.

[0025] In an embodiment, the diameter of the solid core is about 500 μm to about 800 μm, preferably about 600 μm to about 700 μm, and more preferably about 650 μm.

[0026] In an embodiment, the thickness of the at least one shell is about 50 μm to about 125 μm, preferably about 75 μm to about 100 μm.

[0027] In an embodiment, the coating thickness is from about 50 μm to about 125 μm, preferably from about 75 μm to about 100 μm.

[0028] In an embodiment, the solid core and / or at least one shell are compartmentalized along the length of the fiber. Pancreatic islet cells may be encapsulated within the core and / or at least one shell. In an embodiment, a plurality of pancreatic islet cells are encapsulated within the solid core. In an embodiment, a plurality of pancreatic islet cells are encapsulated within at least one shell.

[0029] Aspects of the present invention include methods of treating a diabetic patient, including implanting the disclosed compositions / medical devices into a diabetic patient. In an embodiment, the diabetic patient is a human patient suffering from type 1 diabetes.

[0030] In an embodiment, the implanting is performed by laparoscopy.

[0031] In an embodiment, the method further includes retrieving the composition / medical device from the patient 1 to 12 months, 18 months, 24 months, 2 years, 3 years, 4 years, 6 years, 8 years, or 10 years after implanting the composition / medical device.

[0032] In an embodiment, the method further includes implanting another composition / medical device of the present disclosure into the patient after the retrieving step.

[0033] Aspects of the present invention are methods of manufacturing an implantable composition / medical device of the present disclosure, comprising providing a bioprinting system including a manufacturing platform for supporting continuously bioprinted fibers during printing, patterning, and / or processing, the manufacturing platform comprising a frame defining voids and having a plurality of posts on opposing sides of the frame for fixing and suspending continuously bioprinted fibers during printing, a print head having a plurality of microfluidic channels for selectively supplying a plurality of materials to dispensing orifices, a positioning unit for positioning the frame relative to the print head in a three-dimensional space, and at least one dispensing means for dispensing fibers from the dispensing orifices; dispensing fibers around the plurality of posts via the bioprinting system to form a lattice structure comprising at least 2, 3, 4, or 5 layers of fibers; and coating the lattice structure with at least one conformal coating after printing is complete.

[0034] In an embodiment, the manufacturing platform is immersed in a crosslinking agent bath during dispensing of the continuously bioprinted fibers.

[0035] In an embodiment, the manufacturing platform comprising the lattice structure is immersed in a crosslinking agent bath after dispensing of the continuously bioprinted fibers.

[0036] In an embodiment, the at least one coating step comprises immersing the manufacturing platform comprising the lattice structure in at least one coating solution.

[0037] In an embodiment, the at least one coating step comprises dispensing at least one coating solution onto the lattice structure via the dispensing orifice after dispensing of the continuously bioprinted fibers.

[0038] Incorporation by reference All publications, patents, and patent applications cited in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0040] Definitions For the purpose of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular also include the plural and vice versa. If the definitions described conflict with the documents incorporated herein by reference, the definitions described below shall prevail. Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present disclosure pertains.

[0041] As used herein, the term "hydrogel" refers to a composition comprising water and a network or lattice of hydrophilic polymer chains.

[0042] As used herein, the term "sheath fluid" or "sheath liquid" refers to a fluid that is at least partially used to encapsulate or "sheath" a material as it passes through a fluid channel. In some embodiments, the sheath fluid comprises an aqueous solvent such as water or glycerol. In some embodiments, the sheath fluid comprises a chemical crosslinking agent. Non-limiting examples of crosslinking agents include divalent cations (e.g., Ca 2+ , Ba 2+ , Sr 2+ etc.), thrombin, and pH-adjusting chemicals such as sodium bicarbonate.

[0043] As used herein, the term "segmentation / compartmentalization" refers to the discontinuous nature of the types of materials and / or biogenic substances contained within the core(s) and / or shell(s) of the fibers disclosed herein, e.g., there are intentional gaps in the deposition of certain types of materials and / or biogenic substances along the length of the fiber. The spacing (e.g., length) between such segments / compartments may be regular (e.g., approximately the same spacing between regions of biogenic substances) or the spacing may vary.

[0044] As used herein, the term "solid core" refers to the core of a fiber of the present disclosure that is composed of a particular material (e.g., a hydrogel that can be crosslinked by a chemical crosslinking agent) such that the core does not contain a lumen along the entire length of the fiber. Since the solid core of the present disclosure can permit the passage of certain fluids, molecules, and / or ionic species throughout the core, the term is not intended to refer to a core that is completely impermeable along its length.

[0045] As used herein, the term "biocompatible material" refers to a material that can incorporate and / or come into contact with a biological substance, including but not limited to a substance derived from an organism containing cells, and that does not exhibit a harmful effect on the ability of the biological substance to perform one or more functions (including but not limited to cellular functions such as secretion of bio-related molecular species, agonist / receptor binding, signal transduction, etc.).

[0046] As used herein, the term "immunoprotective" broadly refers to the design aspects of the fibers of the present disclosure that are useful for reducing, preventing, or eliminating a host immune response, such as, for example, immune cell invasion of the fibers upon implantation of the fibers into the body (e.g., into a mammalian body).

[0047] As used herein, the term "agent" refers to any protein, nucleic acid molecule (including chemically modified nucleic acid molecules), antibody, small molecule, organic compound, inorganic compound, or other molecule of interest. Agents may include bio-related substances, therapeutic agents, diagnostic agents, pharmaceuticals, chelating agents, cross-linking agents, etc. A therapeutic agent or pharmaceutical is an agent that, when administered to a subject in a manner consistent with the present disclosure, alone or in combination with additional compounds, elicits a desired response (e.g., elicits a therapeutic or prophylactic effect). A bio-related substance is a substance that supports another biological process, such as, for example, supporting the viability of cells.

[0048] Introduction True diabetes (i.e., diabetes mellitus) is a disease in which the ability to produce the hormone insulin or the body's ability to respond to insulin is impaired, carbohydrate metabolism becomes abnormal, and the concentrations of glucose in the blood and urine increase. This disease is classified into several subtypes and is represented by type 1 diabetes, insulin-dependent diabetes mellitus (IDDM), maturity-onset diabetes of the young (MODY), latent autoimmune diabetes in adults (LADA), brittle diabetes, lean diabetes, type 1.5, type 2, type 3, obesity-related diabetes, gestational diabetes, and other nomenclatures recognized in this field.

[0049] Generally, patients with insulin-dependent diabetes need to be administered exogenous insulin to sufficiently lower their blood glucose levels. Patients with insulin-dependent diabetes may benefit from cell replacement therapy that implants insulin-producing cells into the patient, regardless of whether the disease is type 1, MODY, LADA, brittle, lean, type 1.5, type 2, type 3, obesity-related diabetes, or a combination thereof.

[0050] However, as described above, there are many challenges in the practical application of cell replacement therapy, such as the need for islet survival and the implementation of long-term immunosuppressive drug therapy. The cell encapsulation strategy may reduce such problems, but instead, it is hindered by other problems including, but not limited to, FBR to the implant device.

[0051] 3D bioprinting is an additive manufacturing process in which synthetic tissue structures, optionally containing cells, are placed layer by layer to obtain a multi-layer 3D structure. Extrusion (Panwar A et al., Molecules. (2016);21: 685;Sakai S et al., Biofabrication. (2018);10: 045007;Han HW and Hsu SH, Neural Regener.Res. (2017);12: 1595), inkjet (Gao G et al., Biotechnol.Lett. (2015);37: 2349;Gao G and Cui X, Biotechnol.Lett. (2016);38: 203;Bsoul A et al., Lab Chip. (2016);16: 3351), laser-assisted (Sorkio A et al., Biomaterials. (2018);171: 57;Pages E et al., J. Nanotechnol.Eng. Med. (2015);6: 021006;Catros S et al., In Vivo and In Situ Biofabrication by Laser-Assisted Bioprinting, Elsevier, Winston-Salem, USA. (2015)), and stereolithography (SLA) (Miri AK et al., Adv.Mater. (2018);30: 1800242;Wang Z et al., ACS Appl.Mater.Interfaces. (2018);10;26859;Wang Z et al., Biofabrication. (2015);7: 045009) printing methods are among the various types of 3D bioprinting technologies that have been developed. Among these, extrusion is the most common, by which bioink is dispensed through one or more syringes to form a layer-by-layer scaffold from fibers.

[0052] With advancements, microfluidics-based 3D bioprinting systems have also come into use (Beyer ST et al., in 2013 Transducers Eurosensors XXVII 17th Int. Conf.Solid-State Sensors, Actuators, Microsystems. IEEE, Piscataway, NJ (2013); pp.1206-1209; Beyer ST et al., in The 17th Int. Conf. on Miniaturized Systems for Chemistry and Life Sciences. (2013); pp. 176-178). Using these systems and techniques, multiple materials (e.g., bioinks, crosslinkers, etc.) flow through microchannels, which can enable one or more precise controls such as flow rate, switching, mixing, etc. When used with a sheath flow surrounding at least one inner material, microfluidic bioprinting can reduce shear stress during the printing process. Microfluidics-based 3D bioprinting also advantageously allows for the intersection of material flows when the material flows exit independent flow paths and enter a single flow path (e.g., a dispensing channel), facilitating the generation of structures having a core surrounded by one or more shells.

[0053] However, despite the promise of 3D bioprinting strategies, technical problems remain that need to be solved, such as immune defense, passage of sufficient oxygen and nutrients, and avoidance of FBR. The inventors have recognized these problems and developed compositions and methods to address them.

[0054] Multilayer lattice structure for diabetes treatment One aspect of the present invention relates to an implantable multi-layer lattice structure for the treatment of diabetes (e.g., type 1 diabetes). Referring to FIG. 1A, an exemplary diagram of such a lattice structure 100 is depicted. The lattice structure 100 in FIG. 1A is observed along the Z-axis (refer to the orthogonal coordinate system 102 in FIG. 1A). In an embodiment, the lattice structure 100 can take the form of a grid arrangement with a region 104 of empty space. In an embodiment, a single continuous fiber can be bioprinted in a grid arrangement and then coated (the coating is not shown in FIG. 1A) to form the final multi-layer lattice structure 100. For example, starting from point (1) in FIG. 1A, a first layer can be formed as indicated by the dashed arrow, and then a second layer can be laid on top of the first layer as indicated by the solid arrow, ending at point (2). In this way, a multi-layer lattice structure can be formed from a single continuous fiber and can be coated entirely. Although FIG. 1A describes two layers, a multi-layer lattice structure formed from a single continuous fiber can include two or more layers, such as 3, 4, 5, 6, 7, 8, 9, or 10 layers, or in some cases more than 10 layers. It should be understood that the depiction in FIG. 1A is illustrative and not limiting.

[0055] The lattice structure 100 is shown as being approximately square, but the structures of the present disclosure do not necessarily have to be square and can be configured in other shapes including, but not limited to, rectangular, elliptical, hexagonal, circular, etc. The dimensions of the lattice structure are configured to be suitable for implantation into a particular animal (e.g., human, dog, cat, mouse, pig, etc.). In embodiments where the lattice structure is approximately square, the dimensions of the structure can range from about 8 mm × 8 mm to about 150 mm × 150 mm. In embodiments where the lattice structure is a shape other than square, the area of the lattice structure is about 64 mm 2 ~ about 22,500 mm 2 , or any value range therebetween.

[0056] Turning to FIG. 1B, there is depicted a multilayer lattice structure 150 that is substantially the same as the lattice structure 100 of FIG. 1A, but is shown along the y-axis (see the orthogonal coordinate system 102 of FIG. 1B) and has three layers including a first layer 154, a second layer 158, and a third layer 162. In an embodiment, the multilayer lattice structure 150 is formed in a manner similar to that described above, forming a grid arrangement from a single continuous fiber and then uniformly applying a coating (not shown in FIG. 1B, see FIG. 4) to produce the final lattice structure 150. As shown in the inset 165, the fibers 163 of the completed lattice structure will include a solid core 170, at least one shell 174, and at least one coating (not shown in FIG. 1B) on the exposed fiber surfaces within the lattice structure. In an embodiment, as shown in FIG. 1B, the solid core 170 encapsulates the islet 182. In an embodiment, the shell 174 encapsulates the islet (see the lattice structure 175 and corresponding inset 176 of FIG. 1C, no coating is shown in FIG. 1C). Although the fibers of FIGS. 1B - 1C show a single shell, alternative embodiments including multiple shells and / or optionally multiple coatings are contemplated. For example, the multilayer lattice structures of the present disclosure can include 1, 2, 3, 4, or 5 shells and 1, 2, 3, 4, or 5 coatings. In some examples, the bioprinted fibers themselves can also be coated.

[0057] In embodiments, the solid core, at least one shell, and one or more coatings are composed of biocompatible material(s). Examples of biocompatible materials relevant to the present disclosure may include, but are not limited to, alginate, collagen, decellularized extracellular matrix, hyaluronic acid (HA), polyethylene glycol (PEG), fibrin, gelatin, gelatin methacrylate (GEL-MA), silk, chitosan, cellulose, polycaprolactone (PCL), polylactic acid (PLA), poly(oligoethylene glycol) methacrylate (POEGMA), or combinations thereof. In embodiments, the solid core, at least one shell, and one or more coatings are composed of hydrogel materials. Examples of hydrogel materials relevant to the present disclosure may include, but are not limited to, alginate (e.g., SLG-100 alginate), chitosan, GEL-MA, agarose, PEG, PCL, poly-L-lysine (PLL), triazole, fucoidan, poly(ethylene glycol) diacrylate (PEGDA), poly(ethylene glycol)-tetraacrylate (PEGTA), poly(vinyl alcohol) (PVA), hyaluronic acid (HA), hyaluronic acid methacryloyl (HAMA), collagen, methacrylated collagen (ColMA), gelatin, gellan, fibrin (fibrinogen), and combinations thereof. In some embodiments, HA can be used to increase viscosity.

[0058] In embodiments, the diameter of the solid core of the continuous bioprinted fiber of the present disclosure is from about 500 μm to about 800 μm, preferably from about 600 μm to about 700 μm, such as about 600 μm, 610 μm, 620 μm, 630 μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, or any value therebetween. In some embodiments, the diameter of the solid core is about 650 μm.

[0059] In embodiments, the thickness of at least one shell of the continuous bioprinted fibers of the present disclosure is from about 50 μm to about 125 μm, preferably from about 75 μm to about 100 μm, such as about 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, or any value therebetween.

[0060] In embodiments, the thickness of each layer of the multilayer lattice structure (e.g., lattice structure 150 of FIG. 1B, lattice structure 175 of FIG. 1C) is from about 500 μm to about 1500 μm, preferably the thickness of each layer is from about 800 μm to about 1200 μm, more preferably the thickness of each layer is from about 950 μm to about 1100 μm, such as about 950 μm, 975 μm, 1000 μm, 1025 μm, 1050 μm, 1075 μm, 1100 μm, or any value therebetween.

[0061] In embodiments, the thickness of the coating applied to the multilayer lattice structure of the present disclosure is from about 50 μm to about 125 μm, preferably from about 75 μm to about 100 μm, such as about 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, or any value therebetween.

[0062] In embodiments, the core and / or shell of the continuous bioprinted fibers of the present disclosure contains a plurality of islets (see, e.g., FIGS. 1B - 1C). As described herein, "islet equivalent quantity" (IEQ) refers to an islet of a particular diameter containing an approximate amount of cells. The devices of the present disclosure may contain an appropriate number of IEQs depending on the species.

[0063] Filling density The packing density of the lattice structure of the present disclosure is an important parameter that determines diffusion flow and angiogenesis. For example, a lattice structure with too high a packing density may prevent and reduce diffusion flow and / or the growth of host tissue. Alternatively, a lattice structure with too low a packing density may be undesirable in terms of one or more aspects such as structural stability, F-F adhesion, and ease of recovery. As described herein, the packing density is considered as a percentage of the packing density of a 3D lattice structure including one or more coatings (plural possible). Referring to FIG. 1D, for reference, exemplary lattice structures with different packing densities are shown, and the packing density of the shown lattice structures increases from left to right. Thus, a lattice structure having a completely filled fibrous structure (i.e., no space) corresponds to a packing density of 100%, whereas a lattice structure not occupied by any fibrous structure by 90% corresponds to a packing density of 10%.

[0064] In embodiments, the packing density of a suitable multilayer lattice structure of the present disclosure is from about 10% to about 90%, such as from about 20% to about 80%, such as from about 30% to about 70%, such as from about 40% to about 60%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%, or any value therebetween. Preferably, the packing density is between about 50% and about 60%.

[0065] In the case of the multilayer lattice structures of the present disclosure, the packing density is advantageously controlled by the method of manufacturing the structure. Specifically, as will be described in more detail below and as detailed in the co-pending provisional application 63 / 342,118, continuous fibers can be accurately printed into a lattice of 2, 3, 4, 5, or more layers using the manufacturing platform described therein. Briefly, FIG. 2 shows an exemplary embodiment of such a platform that includes a frame 210 that includes posts 212. To generate the lattice, continuous fibers can be bioprinted around a plurality of posts 212, for example, in the manner shown and described with respect to FIG. 1A. The posts 212 serve to impart tension to the fibers during printing, which has the advantage that for each layer, the fibers in each row / column are maintained at a uniform distance from the fibers corresponding to the adjacent row / column, and the linearity of the fibers between each pair of opposing posts is ensured when the lattice is manufactured. Following the manufacture of the lattice structure, one or more conformal coatings can be applied to the entire exposed surface of the lattice structure without disturbing the lattice structure and before (or during) effective interfiber adhesion of the entire printed structure. Thus, these conformal coatings can be advantageously used to impart stability to the tissue fiber structure and / or impart anti-FBR properties. The handle 218 can be used to transport / store the frame 210 and the corresponding attached lattice structure and / or to perform post-printing processing steps, such as one or more coatings, while the lattice structure remains attached to the frame. In this way, the manufacturing platform can be advantageously used during the printing, patterning, and / or post-printing processing of the devices of the present disclosure.

[0066] In an embodiment, the packing density may be a function of the number of posts on the manufacturing platform and their interrelationships (e.g., the distance from adjacent posts, and / or the number of posts on opposite and / or adjacent sides of the frame). For example, FIG. 2 shows a frame 210 that is substantially square and has a number of posts (11 posts on each side) along sides 220 and 221 and a smaller number of posts (9 posts on each side) along sides 222 and 223. In other embodiments, the same number of posts may be included on all sides of such a frame. In any case, embodiments preferably include at least two corner posts 214 to assist in the transition of the fibers.

[0067] Strength of the material In an embodiment, the solid core, at least one shell, and one or more coatings (plural available) may have the same or different material strengths. For example, but not limited to, in the case of a multi-layer lattice structure composed of a solid core, at least one shell, and at least one coating, the core may have a first material strength, at least one shell may have a second material strength, and at least one coating may have a third material strength. In such an example, each of the first material strength, the second material strength, and the third material strength may be the same. In other embodiments, the first material strength, the second material strength, and the third material strength may be different, or may be different from the other two. In embodiments where the continuous fibers include more than one shell, each shell may include a different material strength. Further, or alternatively, in some embodiments where the lattice structure is composed of at least two coatings, each coating may have a different material strength. In an exemplary embodiment, the multi-layer lattice structure is composed of a solid core, a shell, and a coating, the material strength of the shell is greater than the material strength of the core, and the material strength of the coating is less than the material strengths of both the shell and the core.

[0068] Segmentation / Compartmentalization In an embodiment, the multilayer lattice structure fabricated by the method disclosed herein can be segmented / compartmentalized along at least a portion of the length of the continuously bioprinted fibers forming the lattice structure. Details regarding the fabrication of the segmented / compartmentalized fiber structure are described in U.S. Provisional Patent Application No. 63 / 192552, the disclosure of which is hereby expressly incorporated by reference in its entirety herein.

[0069] In an embodiment, the core can be segmented / compartmentalized along at least a portion of the continuously bioprinted fibers forming the lattice structure. In an embodiment, at least one shell can be segmented / compartmentalized along at least a portion of the continuously bioprinted fibers forming the lattice structure. In an embodiment, both the core and at least one shell can be segmented / compartmentalized along at least a portion of the continuously bioprinted fibers forming the lattice structure. In an embodiment, one or more segments / compartments of the core and / or shell(s) can contain pancreatic islets. Referring to FIG. 3, exemplary fibers 300 (e.g., continuous fibers) that can constitute the lattice structures of the present disclosure (e.g., lattice structure 100, lattice structure 150, lattice structure 175) are depicted. In this example figure, the solid core 302 includes a first compartment 305 and a second compartment 308. The second compartment 308 contains pancreatic islets 310, and the first compartment 305 does not contain pancreatic islets 310. Although not explicitly shown, it can be understood that the first compartment 305 may be composed of a different material (e.g., a different hydrogel material) than the second compartment 308, although in other embodiments the material compositions may be the same. Further, the shell can be segmented / compartmentalized in a similar manner (refer to the representative figure of FIG. 3B) additionally or alternatively, depending on the desired application.

[0070] The sizing of the compartments can be a function of one or more variables including, but not limited to, the size of the fibers (e.g., length and / or diameter), the number of shell(s) and / or coating(s), the type of material used in the continuous fiber generation process, the composition of the coating, etc. In some embodiments, the fiber can be composed of at least two segments / compartments including islets, and there are no islets in other segments adjacent to the at least two segments / compartments. For example, but not limited to, at least two segments including islets may be included in the core of the continuous bioprinted fibers of the present disclosure. In another example, at least two segments including islets may be included in at least one shell. In embodiments, the length(s) of the segment(s) including islets may be longer than, equal to, or shorter than the length of the segment(s) not including cells. In some embodiments, the spacing between compartments / segments containing biogenic substances (e.g., cells) in the fibers of the present disclosure may be between 1 and 5 mm, such as 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm.

[0071] Other design considerations regarding compartmentalization include, for example, optimizing the diffusion of oxygen and nutrients to improve survival rate and function, and even switching the core and / or shell of the fiber structure among different materials, cell types, and densities. Compartmentalization also enables printing different therapeutic doses of the fiber structure without changing the shape of the fiber structure.

[0072] The segment / compartment may contain biogenic substances, such as islets at a specific density. In embodiments, the density may be the same or different between compartments. In embodiments, the biogenic substances within the compartment may be the same or different. In embodiments, the density of the biomaterial can be selected as a function of one or more of a specific application (e.g., treatment of a specific disease / illness), cell survival determinants, materials containing biogenic substances (e.g., biocompatible materials), etc.

[0073] In embodiments, one or more segments / compartments containing a bio-derived substance may be adjacent to, for example, a segment containing a material having immunoprotective properties. For illustrative purposes and without limitation, immunoprotective hydrogel materials can include, for example, but are not limited to, functionalized alginates such as methacrylated alginate, alginate furan, alginate thiol, alginate maleimide, and covalently clickable alginates (e.g., alginate blended with DMAPS-Ald and / or DMAPS-Hzd). For example, if the core of the lattice structure disclosed herein contains one or more segments containing a bio-derived substance, the one or more segments can be sandwiched by other segments containing the immunoprotective materials disclosed herein. In another example, if the shell of the lattice structure disclosed herein contains one or more segments containing a bio-derived substance, the one or more segments can be sandwiched by other segments containing the immunoprotective materials disclosed herein. It is also within the scope of the present disclosure that if the core of a bioprint fiber contains a bio-derived substance, at least one shell and / or at least one coating can contain a material having immunoprotective properties. In another embodiment where the shell of the lattice structure contains a bio-derived substance, another shell and / or at least one coating can contain a material having immunoprotective properties.

[0074] Input material Aspects of the present invention include input materials that can be used to print lattice structures for advantageous use as biomaterials. As used herein, "biomaterial" refers to natural or synthetic substances that are useful for constructing or replacing tissues, e.g., human tissues, regardless of the presence or absence of living cells. In the field of bioprinting, the term "biomaterial" is often synonymous with the term "bioink". Some such materials have been described above and will be further described in more detail below.

[0075] The input materials generally include at least one crosslinkable material, such as alginate (e.g., SLG-100 alginate), chitosan, PEGDA, PEGTA, hyaluronic acid (HA), HAMA, collagen, CollMA, gelatin, GelMA, agarose, gellan, fibrin (fibrinogen), PVA, etc., or hydrogels including but not limited to any combination thereof, as well as PCL, PLGA, PLA, etc., or hydrogels including but not limited to any combination thereof. In a preferred embodiment, the input materials include at least one hydrogel. Non-limiting examples of hydrogels include alginate, agarose, collagen, fibrinogen, gelatin, chitosan, hyaluronic acid-based gels, or any combination thereof. Various synthetic hydrogels are known and can be used in embodiments of the systems and methods provided herein. For example, in some embodiments, one or more hydrogels form at least a part of the structural basis of the three-dimensional structure to be printed. In some embodiments, the hydrogel has the ability to support the growth and / or proliferation of one or more cell types, and one or more cell types can be dispersed within the hydrogel or added to the hydrogel after being printed in a three-dimensional configuration.

[0076] In some embodiments, the hydrogel is crosslinkable by a chemical crosslinking agent. For example, a hydrogel containing alginate can be crosslinkable in the presence of a divalent cation such as calcium chloride, a hydrogel containing chitosan can be crosslinked using a polyvalent anion such as sodium tripolyphosphate (STP), a hydrogel containing fibrinogen can be crosslinkable in the presence of an enzyme such as thrombin, and a hydrogel containing collagen, gelatin, agarose, or chitosan can be crosslinkable in the presence of heat or a basic solution.

[0077] In embodiments, hydrogel fibers can be produced by a precipitation reaction achieved by extracting a solvent from the input material upon exposure of the input material to a crosslinking agent material that is miscible with the input material. Non-limiting examples of input materials that form fibers via a precipitation reaction include collagen and polylactic acid (PLA). Non-limiting examples of crosslinking materials that enable precipitation-mediated hydrogel fiber formation include polyethylene glycol (PEG) and alginate. Crosslinking of the hydrogel increases the hardness of the hydrogel and, in some embodiments, enables the formation of a solidified hydrogel.

[0078] In some embodiments, the hydrogel comprises alginate. Alginate forms a solidified colloidal gel (high water content gel or hydrogel) upon contact with a divalent cation. Any suitable divalent cation can be used to form a solidified hydrogel with an input material comprising alginate. In the alginate ion affinity series Cd 2+ >Ba 2+ >Cu 2+ >Ca 2+ >Ni 2+ >Co 2+ >Mn 2+ in which Ca 2+ has the optimal characteristics and is most commonly used (Ouwerx, C. et al., Polymer Gels and Networks, 1998, 6(5): 393-408). Studies have shown that Ca 2+It has been shown that through the cooperative binding of ions, a calcium alginate gel, the so-called "egg box" model, is formed (ISP Alginates, Section 3: Algin-Manufacture and Structure, in Alginates: Products for Scientific Water Control, 2000, International Specialty Products: San Diego, pp. 4-7). G-rich alginate is thermally stable and tends to form strong but brittle Ca gels, while M-rich alginate has low thermal stability and tends to form weaker but more elastic gels. In some embodiments, the hydrogel comprises depolymerized alginate.

[0079] In some embodiments, the hydrogel is crosslinkable using a free radical polymerization reaction, and covalent bonds are formed between molecules. Free radicals can be generated by exposing a photoinitiator to light (usually ultraviolet light), or by exposing the hydrogel precursor to a chemical source of free radicals such as ammonium persulfate (APS) or potassium peroxodisulfate (KPS) combined with N,N,N,N-tetramethylethylenediamine (TEMED) as an initiator and a catalyst, respectively. Non-limiting examples of photocrosslinkable hydrogels include methacrylated hydrogels such as methacryloyl hyaluronic acid (HAMA), gelatin methacrylate (GEL-MA), or polyethylene (glycol) acrylate-based (PEG-acrylate) hydrogels, which are used in cell biology because they are inert to cells. Polyethylene glycol diacrylate (PEG-DA) is commonly used as a scaffold in tissue engineering because polymerization occurs rapidly at room temperature, requires little energy input, has a high water content, is elastic, and can be customized to contain various biomolecules.

[0080] In embodiments, the input material includes a non-biodegradable polymer. In examples, the input material may be a synthetic polymer, such as polyvinyl acetate (PVA). In embodiments, the input material may include hyaluronic acid (HA).

[0081] In some embodiments, the hydrogel includes a chemically modified alginate. In examples, the chemically modified alginate includes an alginate functionalized with methacrylate groups, which is referred to herein as "Alg-MA". In some embodiments, Alg-MA can be used for the immune defense shell layer by blending with zwitterionic alginate, which is referred to herein as "Alg-zw". Since Alg-MA has the ability of double crosslinking, in embodiments, Alg-MA can be first printed with Alg-zw via physical crosslinking. Next, during printing, the fibers can be further irradiated to induce covalent crosslinking between the fibers, thus generating F-F adhesion. In some embodiments, the chemically modified alginate may include thiolated alginate.

[0082] In some embodiments, one or more synthetic components can be added to the hydrogel material. The synthetic components may be useful in improving adhesion between fibers and / or in vivo stability. In examples, the material may include an acrylated zwitterionic monomer (e.g., sulfobetaine methacrylate (SBMA)) and a crosslinking agent (e.g., poly(ethylene glycol) diacrylate (PEGDA)). In such examples, photo-mediated crosslinking of the zwitterionic monomer and PEGDA can make the resulting crosslinked polymer matrix superhydrophilic, and thus less likely to cause a foreign body response (see U.S. Provisional Patent Application No. 63 / 192552, the contents of which are hereby expressly incorporated by reference in their entirety).

[0083] In some embodiments, the hydrogel material may be crosslinked via click chemistry. For example, copolymers comprising zwitterionic monomers and aldehyde motifs (e.g., [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS)-aldehyde, referred to herein as "DMAPS-Ald"), as well as zwitterionic monomers and hydrazide motifs (e.g., DMAPS-hydrazide, referred to herein as "DMAPS-Hzd") may be used (see U.S. Provisional Patent Application No. 63 / 192552). The aldehyde readily reacts with the hydrazide to form a covalently crosslinked hydrogel. Due to the presence of zwitterionic monomers in the polymer backbone, these polymers may exhibit low protein binding properties. In an embodiment, one of these polymers may be blended with alginate within the shell. After printing, when this structure is immersed in a solution containing the opposing component, crosslinking occurs by covalent bonds between the fibers, resulting in F-F bonds.

[0084] In embodiments, the input material comprises microparticles, and as used herein, "microparticles" generally refers to miscible particles in the range of about 0.1 μm to about 100 μm, typically composed of polymers, metals, or other inorganic materials. They may be symmetric (e.g., spherical, cubic, etc.), but this is not a requirement. Microparticles having an aspect ratio of 2:1 or greater may be considered micro-rods or micro-fibers.

[0085] The input materials according to the embodiments of this specification can include any of a variety of natural or synthetic polymers that support the viability of living cells, such as, for example, alginate, laminin, fibrin, hyaluronic acid, poly(ethylene) glycol-based gels, gelatin, chitosan, agarose, or combinations thereof. In some embodiments, the subject bioink composition is physiologically compatible, that is, it promotes cell growth, cell differentiation, cell-cell communication, and various other cell functions (e.g., insulin release). In certain embodiments, the input material includes one or more physiological matrix materials, or combinations thereof. "Physiological matrix material" means a biologically derived substance found in natural mammalian tissues. Non-limiting examples of such physiological matrix materials include fibronectin, thrombospondin, glycosaminoglycans (GAGs) (e.g., hyaluronic acid, heparan sulfate, chondroitin-6-sulfate, dermatan sulfate, chondroitin-4-sulfate, or keratan sulfate), deoxyribonucleic acid (DNA), adhesion glycoproteins, and collagen (e.g., collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or collagen XVIII). Such physiological matrix materials can contribute, for example, to the survival, proliferation, and / or insulin secretion of pancreatic β-cells in the context of the lattice structures of the present disclosure (Riopel M, and Wang R., Frontiers in Bioscience (Landmark Ed). (2014);19(1): 77-90); Nikolova G et al., Dev Cell. (2006);10(3): 397-405; Johansson A et al., Diabetologia. (2009);52(11): 2385-94).

[0086] Additional fluid Aspects of the present invention include one or more buffers. The buffer according to an embodiment of the present invention has miscibility with an input material (e.g., a hydrogel) and does not crosslink the input material. In some embodiments, the buffer includes an aqueous solvent. Non-limiting examples of the buffer include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof.

[0087] The buffer according to an embodiment of the present invention may have a viscosity in the range of about 1 mPa·s to about 5,000 mPa·s, for example, a viscosity of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa·s. In some embodiments, the viscosity of the buffer can be adjusted to match the viscosity of one or more input materials.

[0088] Aspects of the present invention include one or more sheath fluids. The sheath fluid according to an embodiment of the present invention is a fluid that can be used to at least partially enclose or "sheath" the input material dispensed from the dispensing channel. In some embodiments, the sheath fluid includes an aqueous solvent. Non-limiting examples of the sheath fluid include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof. The sheath fluid according to an embodiment of the present invention may have a viscosity in the range of about 1 mPa·s to about 5,000 mPa·s, for example, a viscosity of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa·s. In some embodiments, the viscosity of the sheath fluid can be adjusted to match the viscosity of one or more input materials.

[0089] In some embodiments, the sheath fluid comprises a chemical crosslinking agent. In some embodiments, the chemical crosslinking agent comprises a divalent cation. Non-limiting examples of divalent cations include Cd 2+ , Ba 2+ , Cu 2+ , Ca 2+ , Ni 2+ , Co 2+ , or Mn 2+ . In a preferred embodiment, Ca 2+ is used as the divalent cation. In some embodiments, the concentration of the divalent cation in the sheath fluid ranges from about 80 mM to about 140 mM, such as about 90, 100, 110, 120, or 130 mM.

[0090] Cell population In embodiments, the cell population is selected from the group consisting of, or comprising, a single cell suspension, cell aggregates, cell spheroids, cell organoids, or combinations thereof. Input materials according to embodiments of the present invention can incorporate any mammalian cell type including, but not limited to, stem cells (e.g., embryonic stem cells, adult stem cells, induced pluripotent stem cells), germ cells, endodermal cells (e.g., lung cells, liver cells, pancreatic cells, gastrointestinal tract cells, or urogenital tract cells), mesodermal cells (e.g., kidney cells, bone cells, muscle cells, endothelial cells, or heart cells), ectodermal cells (skin cells, nervous system cells, pituitary cells, or eye cells), stem cell-derived cells, or any combination thereof. In a preferred embodiment, at least one cell population is Insulin-producing cells, such as pancreatic islets, comprising, or consisting of, β cells (e.g., producing insulin) and, in embodiments, one or more of α cells (e.g., producing glucagon), δ cells (e.g., producing somatostatin), PP cells (e.g., producing pancreatic polypeptide), and ε cells (e.g., producing ghrelin), or consisting of one or more of these insulin-producing cells, such as pancreatic islets.

[0091] Cells can be obtained from a donor (allogeneic), from a species different from the recipient (xenogeneic), or from the recipient (autologous). Specifically, in embodiments, cells can be obtained from a suitable donor such as a human or an animal, or from the subject into which the cells are to be transplanted. Mammalian species include, but are not limited to, humans, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, and rats. In one embodiment, the cells are human cells. In other embodiments, the cells can be derived from an animal such as a dog, cat, horse, monkey, or any other mammal.

[0092] In some embodiments, the islets included in the lattice structure of the present disclosure include reaggregated islets. Reaggregated islets can be formed by dispersing natural islets, or in some examples, β-cells derived from stem cells, into a single cell suspension, and subsequently reaggregating the dispersed cells into uniform (optionally smaller) “pseudo-islets”. Techniques for creating pseudo-islets are known (see, for example, Lu, Jia et al., JURA. (2016-2017);6: 1-4). Creating aggregates of uniform size is important for reproducibly producing functional insulin-producing cells. Some groups have developed methods for reproducibly creating endocrine cell aggregates of defined size to improve functionality (Gao B et al., Acta Mech.Sin. (2019);35: 329-337;Nair G et al., Nat. Cell Biol. (2019);21: 263-274;Velazco-Crus L et al., Stem Cell Rep. (2019);12: 351-365). Producing cell aggregates of uniform size in a scalable manner enables the production of mature cells for cell therapy as disclosed herein in a consistent manner. In some embodiments, the cells deposit insoluble ECM proteins and can, for example, contribute to the surrounding microenvironment or dynamically change the surrounding microenvironment.

[0093] Since the insulin-producing cells within the lattice structures disclosed herein are protected from the host's immune system, islets can be obtained from any suitable source, i.e., human or non-human. In embodiments, the islet cells are stem or progenitor cells, including induced pluripotent stem cells that differentiate into insulin-producing islet cells. Suitable insulin-secreting cell populations and methods of producing such populations are known in the art, see, e.g., U.S. Patent No. 8,425,928, U.S. Patent No. 5,773,255, U.S. Patent No. 5,712,159, U.S. Patent No. 6,642,003, Rezania et al., Nat. Biotech. (2014);32: 1121-1133, and Kuo et al., Int’l.J. Clin. Med. (2014);I(I):21-25. Each of these is hereby incorporated by reference in its entirety.

[0094] In some embodiments, at least one bio-derived substance included in the lattice structures of the present disclosure comprises a cell population that expresses / secretes one or more endogenous bioactive substances(s), e.g., insulin, glucagon, ghrelin, pancreatic polypeptide, factor VII, antihemophilic factor, factor IX, alpha1-antitrypsin, angiogenic factors, growth factors, hormones, antibodies, enzymes, proteins, exosomes, etc. As described herein, endogenous bioactive agents include agents that cells naturally produce under biological circumstances (e.g., insulin release in response to elevated glucose concentration). Endogenous bioactive agents can constitute therapeutic agents with respect to the present disclosure.

[0095] In some embodiments, the input materials can include genetically modified cells that secrete specific factors. In embodiments, it is within the scope of the present disclosure that the above-described cell populations can include modified cells (e.g., genetically modified cells) that secrete specific factors. The cells can also be cells derived from an established cell culture line or cells that have undergone genetic modification and / or genetic manipulation to obtain a desired genotype or phenotype. In some embodiments, tissue pieces can also be used, which can provide several different cell types within the same structure.

[0096] Examples of gene modification techniques applicable to the present disclosure include recombinant DNA (rDNA) technology (Stryjewska et al., Pharmacologial Reports. 2013;65:1075), cell modification based on the use of targeted nucleases (e.g., meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-associated nuclease Cas 9 (CRISPR-Cas 9 ) (Limet al., Nature Communications. 2020;11:4043; Stoddard BL, Structure. 2011;19(1):7-15; Gaj et al., Trends Biotechnol. 2013;31(7):397-405; Hsu et al., Cell. 2014;157(6):1262; Miller et al., Nat Biotechnol. 2010;29(2):143-148), cell modification based on the use of site-specific recombination using recombinase systems (e.g., Cre-Lox) (Osborn et al., Mol Ther. 2013;21(6):1151-1159; Hockemeyer et al., Nat Biotechnol. 2009;27(9):851-857; Uhde-Stone et al., RNA. 2014;20(6):948-955; Ho et al., Nucleic Acids Res. 2015;43(3):e17; Sengupta et al., Journal of Biological Engineering. 2017;11(45):1-9), etc., but are not limited thereto. In some embodiments, some combinations of the above-described techniques may be used for cell modification.

[0097] The present disclosure encompasses recombinant cells capable of producing one or more therapeutic agents, including but not limited to proteins, peptides, nucleic acids (e.g., DNA, RNA, mRNA, siRNA, miRNA, nucleic acid analogs), peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cytokines, enzymes, antibiotics or antibacterial compounds, anti-inflammatory agents, antifungal agents, antiviral agents, toxins, prodrugs, small molecules, drugs (e.g., pharmaceuticals, dyes, amino acids, vitamins, antioxidants), or any combination thereof.

[0098] In embodiments, the cells of the present disclosure may be modified to include at least one mechanism for providing local immunosuppression at the transplant site when transplanted into an allogeneic host, for example, in the tissue fibers of the present disclosure. In examples, the cells may include a series of transgenes, each transgene being cytoplasmic, membrane-bound, or locally acting, and its function being to improve the activation and function of antigen-presenting cells, improve the activity or cytolytic function of leukocytes that attack the graft, improve the macrophage cytolytic function and phagocytosis of the transplanted cells, induce apoptosis in leukocytes that attack the graft, improve local inflammatory proteins, and protect from leukocyte-mediated apoptosis, including but not limited to encoding gene products (WO2018 / 227286; Harding et al., BioRxiv. 2019; DOI: 10.1101 / 716571; Lanza et al., Nature Reviews Immunology. 2019; 19:723 - 733l; Harding et al., Cell Stem Cell. 2020; 27(2):198 - 199).

[0099] In embodiments, the cells of the present disclosure can be modified by a method that controls cell proliferation. As an example, the cells may be genetically modified at the cell division locus (CDL) to include a negative selection marker and / or an inducible activator-based gene expression system, thereby controlling the permissive, removal, and / or suppression of the growth of the genetically modified cells by the addition or removal of an appropriate inducer (WO2016 / 141480; Liang et al., Nature. 2018; 563(7733):701-704).

[0100] Appropriate growth conditions for mammalian cells are well known in the art (Freshney, R.I. (2000) Culture of Animal Cells, a Manual of Basic Technique. Hoboken N.J., John Wiley & Sons; Lanza et al. Principles of Tissue Engineering, Academic Press; 2nd Edition, May 15, 2000; and Lanza & Atala, Methods of Tissue Engineering Academic Press; 1st Edition, October 2001). Cell culture media generally contain essential nutrients and, optionally, additional elements such as growth factors, salts, minerals, vitamins, etc., which can be selected according to the cell type(s) to be cultured. Specific components may be selected to improve cell growth, differentiation, specific protein secretion, etc. Generally, standard growth media contain 110 mg / L of pyruvate and glutamine, and are supplemented with 10-20% fetal bovine serum (FBS) or calf serum and low glucose Dulbecco's modified Eagle's medium (DMEM), and 100 U / ml of penicillin is suitable as well as various other standard media well known in the art. Growth conditions vary depending on the mammalian cell type used and the desired tissue.

[0101] In some embodiments, cell-type specific reagents can be advantageously utilized in the subject input materials for use with the corresponding cell types. For example, the ECM can be directly extracted from the tissue of interest and then solubilized and incorporated into the input material to generate a tissue-specific input material for the tissue to be printed. Such ECM can be readily obtained from patient samples and / or from suppliers such as zPredicta (rBone™, available at zpredicta.com / home / products).

[0102] Printing system In preferred embodiments, the bioprinting system includes the technologies described in WO2014 / 197999, WO2018 / 165761, WO2020 / 056517, WO2021 / 081672, and U.S. Provisional Patent Application No. 63 / 290595, the disclosures of which are hereby expressly incorporated by reference herein. As detailed therein, the disclosed bioprinting system and its components enable the switching of multiple materials, such that the composition of one or more components of the synthetically generated tissue fibers (e.g., cell type, biomaterial composition) can be modified along the length of the fiber while continuously printing. In an embodiment, the microfluidics-based bioprinting system is the RX1™ bioprinter (Aspect Biosystems, Vancouver, BC, Canada), or the system described in co-pending U.S. Provisional Patent Application No. 63 / 290595, the content of which is hereby incorporated by reference herein in its entirety.

[0103] In an exemplary embodiment of a preferred bioprinting system, the system includes a printhead that includes dispensing channels, and one or more material channels and a core channel converge at a proximal end of the dispensing channel. The printhead may be configured to dispense a buffer and / or a sheath fluid simultaneously with one or more crosslinkable materials. In some embodiments, the printhead is configured to maintain a constant mass flow rate through the dispensing channel. In this way, the printhead can be configured to facilitate the smooth and continuous flow of one or more input materials (or a mixture of one or more input materials) and the buffer and / or sheath fluid through the dispensing channel. As described in more detail in WO2020 / 056517, in the use of such a printhead, the input material flowing through the dispensing channel can be crosslinked from the inside by the fluid flowing through the core channel and / or from the outside by the sheath fluid flowing through the downstream sheath fluid channel. In some embodiments, as described in detail in WO2021 / 081672 and U.S. Provisional Patent Application No. 63 / 290595, the printhead includes one or more fluid focusing chambers having a frustoconical shape and optionally one or more printhead adapters. In an embodiment, the printhead is a DUO™ microfluidic printhead, or a CENTRA™ microfluidic printhead (Aspect Biosystems, Vancouver, BC, Canada).

[0104] Other examples of bioprinting systems relevant in the context of the present disclosure, for example those that can be modified or used in combination with the methods of the present disclosure, include, but are not limited to, 3-D Bioplotter® (EnvisionTEC Inc., Dearborn, MI, USA), NovoGen Bioprint® platform (Organovo®, San Diego, CA, USA), R-Gen100 and R-Gen200 (RegenHU, Viras-Saint-Pierre, Switzerland), Bioprinter Fabion and Fabion 2 (3D Bioprinting Solutions, Moscow, Russia), BioBot® Basic, BioAssemblyBot® 200 / 400 / 500 (Advanced Solutions, Louisville, KY, USA), BIO X™, BIO X6™, INKREDIBLE+™ (CellINK, Boston, MA, USA), Ourobotics Revolution (Ourbotics, Cork, Ireland), BioScaffolder2.1 (GeSim, Radeberg, Germany), Omega Bioprinter (3Dynamic Systems, Bridgend, UK), Syn^ and Explorer (Bio3D, Singapore), Alevi 1 / 2 / 3 (Alevi by 3D Systems, Rock Hill, SC, USA), and Dr.Invivo 4D6 (Rokit Healthcare, Seoul, Korea).

[0105] Manufacturing method Aspects of the present invention include methods of printing three-dimensional (3D) devices that include two or more planar structures having a desired packing density. General methods for printing a first layer, and optionally a second layer, a third layer, etc. were described in detail above with reference to FIGS. 1A - 3B.

[0106] Referring to FIG. 4, an exemplary process flow for manufacturing the multilayer lattice structure of the present disclosure is shown. Broadly speaking, the process flow involves generating a lattice structure from continuous fibers, preferably via a manufacturing platform (e.g., frame 210 in FIG. 4), and then applying at least one coating to obtain an implantable device. In the exemplary process flow of FIG. 4, the continuous fibers are manufactured by exposing them to a sheath fluid containing a crosslinking agent that crosslinks the continuous fibers from the outside to the inside during printing. However, in other embodiments, inside-to-outside crosslinking where the crosslinking agent is included as part of the core material is also within the scope of the present disclosure (see U.S. Provisional Patent Application No. 63 / 192552).

[0107] Step (1) in FIG. 4 involves printing continuous fibers. In this example, during printing, the continuous fibers include a core 402 containing islets 408 and a shell 404 surrounding the core, and the shell is surrounded by a sheath fluid 406 containing a crosslinking agent. In embodiments, the shell 404, and optionally the core 402, include crosslinkable materials. Although shown as one shell, fibers including multiple shells are also within the scope of the present disclosure. Once the fibers are printed, the sheath fluid is removed, for example, by flowing through a porous receiving surface (not shown). Following the bioprinting of the lattice structure, step (2) optionally includes immersing the lattice 410 in a crosslinking solution 412 (or applying the crosslinking solution by spraying or dispensing via, for example, the same dispensing means used to dispense the continuous fibers), thereby promoting / continuing uniform crosslinking of the entire printed lattice structure.

[0108] Step (3) is divided into two sub-steps (3a) and (3b). Step (3a) includes coating the entire lattice structure 410 with the coating solution 416. The coating in step (3a) can be applied, for example, by dipping the lattice structure 410 into the coating solution, dispensing the coating solution 416 onto the completed lattice structure 410 using a microfluidic print head (e.g., the same microfluidic print head used in the production of fibers), spraying the coating solution 416 onto the lattice structure 410, etc. In an embodiment, the coating solution 416 includes a crosslinkable material (e.g., alginate). In an embodiment, the coating solution 416 includes the same material as the material constituting the core 402 and / or the shell 404 of the lattice structure 410 being coated. In other embodiments, the coating solution 416 can include a material different from the material constituting the core 402 and / or the shell 404. In an embodiment, in step (3a), a residual crosslinking agent (e.g., Ca 2+)(0) contributes to the initial crosslinking of the materials in the coating solution 416 and the materials constituting the shell 404. Following the coating of the lattice structure 410, in step (3b), the entire conformally coated lattice structure 418 is immersed in the crosslinking solution 412 (or the crosslinking solution is applied, such as by spraying or coating through the same microfluidic print head used to apply the continuous fibers). In the embodiment shown in FIG. 4, the same crosslinking solution 412 is used to crosslink the lattice 410 and the coated lattice structure 418. However, different crosslinking solutions can also be used within the scope of the present disclosure, for example, when the materials used to manufacture the shell (e.g., shell 404) are composed of different materials from the materials used to manufacture the coating (e.g., coating solution 416). In this way, by the process flow shown in FIG. 4, the coating 420 is uniformly added to the entire lattice generated from the continuously bioprinted fibers, and the coated lattice structure 418 shown in the figure is generated. Although not specifically illustrated, it is also within the scope of the present disclosure that a second coating can be further applied in the same manner as the first coating.

[0109] In some embodiments where a single coating is applied to the lattice, the coating can impart stability and / or F-F adhesion properties and / or anti-FBR properties to the resulting fiber structure. In some embodiments where the fiber structure includes two coatings, the first coating may have certain desirable properties (e.g., a material selected to enhance stability and / or F-F adhesion), and the second coating may have additional / alternative properties (e.g., adjusted to have anti-FBR properties). In a preferred embodiment, the coating is a conformal coating that uniformly covers the entire exposed surface of the fiber structure.

[0110] In embodiments, the coating (e.g., 420) is composed of a hydrogel material, such as one or more of alginate, zwitterionic alginate, SBMA, chitosan, PEGDA, PCL, PEG, poly-L-lysine (PLL), PEGTA, hyaluronic acid (HA), HAMA, collagen, CollMA, gelatin, GelMA, agarose, gellan, fibrin (fibrinogen), PVA, etc., or any combination thereof. In some examples, the coating consists of a functionalized alginate, i.e., alginate chemically modified to include one or more properties including, but not limited to, immunoprotective properties, which are advantageous in the manufacture of the lattice structures of the present disclosure. Examples of functionalized alginates include, but are not limited to, methacrylated alginate, alginate furan, alginate thiol, alginate maleimide, and covalently clickable alginates (e.g., alginate blended with DMAPS-Alg and / or DMAPS-Hzd).

[0111] In embodiments, the material strength of the coating (e.g., coating 420 in FIG. 4) is lower than the material strength of one or more shells (e.g., shell 404 in FIG. 4) and / or the core (e.g., core 402 in FIG. 4). In some embodiments where the lattice structure includes two coatings, the material strength of the outer coating may be lower than the material strength of the inner coating, or vice versa. In some embodiments where the lattice structure includes two coatings, the outer coating and the inner coating may have substantially the same material strength, and optionally, the outer coating and the inner coating may be composed of different materials. In embodiments, the core (e.g., 402) is solid, and optionally, the core is segmented / compartmentalized along the length of the fiber. In additional or alternative embodiments, at least one shell may be segmented / compartmentalized along the length of the fiber. In embodiments, the low material strength of the coating, preferably the outer coating (in the case of two or more coatings), reduces the FBR response when the lattice structure is implanted in a patient.

[0112] A conformal coating can impart stability to a fibrous structure and / or can impart properties to the structure that optimize the interface between the fibrous structure and the host, such as anti-FBR properties, promotion of angiogenesis, etc. In embodiments, the coating may be softer than the outermost outer shell. In embodiments, the core, outer shell, and conformal coating may comprise from about 0.1% to about 4% alginate. In embodiments, the fibrous structure may comprise a core of about 0.75% to 1.5% alginate, an outer shell of about 1.5 to 2.5% alginate, and a conformal coating of about 0.2 to 2.5% alginate, such as about 0.2 to 0.75%.

[0113] In embodiments where two coatings are applied, it is within the scope of the present disclosure that the innermost coating may be harder than the outermost coating. In embodiments, the core, outer shell, and conformal coating may comprise from about 0.1% to about 4% alginate. In embodiments, the fibrous structure may comprise a core of about 0.75% to 1.5% alginate, an outer shell of about 1.5 to 2.5% alginate, an inner conformal coating of 1.5% to 2.5% alginate, and an outer conformal coating of about 0.2 to 2.5% alginate, such as about 0.2 to 0.75%. Such examples are intended to be illustrative. Details regarding the manufacture of such conformal coatings are described in U.S. Provisional Patent Application No. 63 / 342,118, the contents of which are hereby expressly incorporated by reference in their entirety.

[0114] In embodiments, the bioprinted fibrous structure produced by the methods disclosed herein can also be segmented / compartmentalized along at least a portion of the length of the fibers that make up the bioprinted fibrous structure, preferably continuous fibers. Details regarding the generation of segmented / compartmentalized bioprinted fibrous structures are described in U.S. Provisional Patent Application No. 63 / 192552, the contents of which are hereby expressly incorporated by reference in their entirety.

[0115] In some embodiments, the bioprinted lattice structure disclosed herein includes a solid core containing about 1.5% alginate, a shell containing about 0.25% to about 3.0% alginate, and a conformal coating containing about 0.25% to about 1.5% alginate. In a preferred embodiment, the bioprinted lattice structure includes a solid core containing about 1.5% alginate, a shell containing about 2% alginate, and a coating containing about 0.5% alginate.

[0116] In some embodiments, the method first includes providing a design of the lattice structure disclosed herein to be printed. The design drawing can be created using commercially available CAD software. In some embodiments, the design drawing includes information regarding specific materials to be assigned to specific locations within the structure(s) to be printed (e.g., in the case of a heterogeneous structure containing multiple materials).

[0117] In an embodiment, the method includes dispensing continuous fibers into a lattice as disclosed herein and then coating the lattice to produce a desired lattice structure. In some embodiments, dispensing the continuous fibers includes dispensing the continuous fibers via the use of a manufacturing platform as described in co-pending provisional application No. 63 / 342,118. In an embodiment, the method includes providing a bioprinting system that includes a manufacturing platform for supporting the lattice structure during printing, patterning, or processing, the manufacturing platform including a frame (210 in FIG. 2) that includes a plurality of posts for securing the continuous fibers during printing, at least one dispensing orifice for dispensing the continuous fibers, a positioning unit for positioning the manufacturing platform in three-dimensional space relative to the at least one dispensing orifice, and at least one dispensing means for dispensing the continuous fibers from the at least one dispensing orifice. The method can include dispensing the continuous fibers around the plurality of posts via the bioprinting system to form a lattice of at least two, three, four, or five layers of continuous fibers and subsequently coating the resulting lattice structure with at least one coating. In an embodiment, the bioprinting system includes at least one print head that includes a plurality of microfluidic printing channels for selectively providing each of the plurality of materials.

[0118] Treatment method The bioprinted 3D lattice structures of the present disclosure are useful in a method of treating diabetes in a patient in need thereof. In one embodiment, the method of treating diabetes treats a patient with type 1 diabetes (i.e., insulin-dependent diabetes). In one embodiment, the method of treating diabetes treats a patient suffering from any one of type 1, MODY, LADA, brittle, lean, latent autoimmune diabetes in adults (LADA), type 1.5, type 2, type 3, obesity-related diabetes, or any combination thereof. In an embodiment, the patient is a human patient, and preferably, the human patient has type 1 diabetes.

[0119] This method includes providing a bio-printed lattice structure as described herein, the lattice structure including a therapeutically effective amount of islets (e.g., re-aggregated islets) encapsulated within the lattice structure. A therapeutically effective amount of islets may include a predetermined therapeutically effective number of insulin-producing cells (i.e., β-cells). In embodiments, a therapeutically effective amount of islets includes from about 20,000 to 80,000 IEQ, more preferably from about 25,000 to 75,000 IEQ, and most preferably about 30,000, about 40,000, about 50,000, or about 60,000 IEQ. In embodiments, the therapeutically effective amount of islets corresponding to the fibrous device can vary, for example, according to the size and health status of the individual being treated. For example, the therapeutically effective amount of islets corresponding to the fibrous device includes the number / amount of islets, or IEQ per kilogram of body weight.

[0120] This method includes implanting the lattice structure disclosed herein into a patient in need thereof. In embodiments, the method includes implanting the lattice structure into the patient one or more times. For example, in embodiments, the method includes implanting a lattice structure containing a therapeutically effective amount of islets into a patient in need thereof at a frequency of about once every 4 months, once every 6 months, once a year, once every 2 years, once every 3 years, once every 4 years, once every 5 years, or more. In embodiments, the islets contained in the lattice structure survive for at least 4 months, or at least 6 months, or at least 1 year, or at least 2 years, or at least 3 years, or at least 4 years, or at least 5 years, or more. The conformal coating can impart stability to the fibrous structure and / or impart properties to the structure that optimize the interface between the fibrous structure and the host, such as anti-FBR properties, promotion of angiogenesis, etc. In embodiments, the coating may be softer than the outermost outer shell. In embodiments, the core, outer shell, and conformal coating may contain from about 0.1% to about 4% alginate. In embodiments, the fibrous structure may include a core of about 0.75% to 1.5% alginate, an outer shell of about 1.5 to 2.5% alginate, and a conformal coating of about 0.2 to 2.5% alginate, such as about 0.2 to 0.75% alginate.

[0121] In embodiments where two coatings are applied, it is within the scope of the present disclosure that the innermost coating may be harder than the outermost coating. In embodiments, the core, outer shell, and conformal coating may comprise from about 0.1% to about 4% alginate. In embodiments, the fibrous structure may comprise a core of about 0.75% to 1.5% alginate, an outer shell of about 1.5 to 2.5% alginate, an inner conformal coating of 1.5% to 2.5% alginate, and an outer conformal coating of about 0.2 to 2.5% alginate, such as about 0.2 to 0.75%. Such examples are intended to be illustrative.

[0122] In embodiments, the bioprinted fibrous structure produced by the methods disclosed herein can be segmented / compartmentalized along at least a portion of the length of the fibers, preferably continuous fibers, that make up the bioprinted fibrous structure. Details of the production of the segmented / compartmentalized bioprinted fibrous structure are described in U.S. Provisional Patent Application No. 63 / 192552, the contents of which are hereby expressly incorporated by reference in their entirety. In embodiments, the islets contained in the lattice structure survive for at least 4 months, or at least 6 months, or at least 1 year, or at least 2 years, or at least 3 years, or at least 4 years, or at least 5 years, or longer. In some embodiments, the patient may require only a single implant. In some embodiments, the lattice structure may need to be replaced once every 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months, once every 1, 2, 3, 4, 5 years or more, or when the patient becomes hyperglycemic again or returns to a diabetic state.

[0123] In some embodiments, a lattice structure containing a therapeutically effective amount of pancreatic islets is transplanted into the greater omentum of a patient. The greater omentum (also called the great omentum, omentum majus, gastrocolic omentum, subgastrocolic bursa, or caul) is a large apron-like fold of visceral peritoneum that hangs down from the stomach, extends posteriorly from the greater curvature of the stomach, ascends to the transverse colon, and then reaches the posterior abdominal wall. Thus, the lattice structure can be transplanted into a surgically formed pocket in the greater omentum.

[0124] In one embodiment, the lattice structure can be surgically transplanted using minimally invasive surgical techniques such as laparoscopy. In an embodiment, the lattice structure is implanted into the abdominal cavity or thoracic cavity by laparoscopy. In some embodiments, the transplantation is performed intraperitoneally, percutaneously, or subcutaneously.

[0125] In some embodiments, the lattice structure is fixed or immobilized (e.g., by suturing) to the transplantation site to maintain the lattice structure near the transplantation site. In an embodiment, the delivery of a therapeutic agent (e.g., insulin) is location-independent, and the biodistribution of the therapeutic agent depends on the patient's vascular system or body fluids. In an embodiment, the lattice structure is implanted percutaneously or subcutaneously under the skin of the abdomen, forearm, flank, back, buttocks, leg, etc., and remains substantially there until it needs to be removed.

[0126] In an embodiment, the lattice structure is recoverable after transplantation. In some embodiments, by fixing or immobilizing the lattice structure, it is prevented from migrating, moving, or traversing within the patient's body, enabling easy recovery. However, considering the dimensions of the lattice structure, it is also within the scope of this disclosure that fixation or immobilization of the lattice structure may not be necessary if recovery can be easily performed. Recovery may be desirable after the pancreatic islets held within the lattice structure have stopped or substantially stopped releasing a therapeutic agent (e.g., insulin), after the release of the therapeutic agent has dropped below a predetermined threshold (e.g., lower than an acceptable amount), after cell death has exceeded some cell death threshold, or after it is expected to exceed the threshold.

[0127] After recovery, the recovered fibrous device can be exchanged with another fibrous device to maintain desired effects, such as the release of insulin from the islets contained therein, in response to, for example, an increase in blood glucose level.

[0128] A particular treatment plan that includes implanting the disclosed lattice structure may be evaluated by whether it improves the outcome of a particular patient, i.e., helps to stabilize or normalize the patient's blood glucose level, or reduces the risk or occurrence of symptoms or co-morbidities associated with diabetes. These symptoms or co-morbidities include, but are not limited to, hypoglycemic episodes, elevated glycosylated hemoglobin (HbA1C level), heart disease, retinopathy, neuropathy, kidney disease, liver disease, periodontal disease, and non-healing ulcers.

[0129] Thus, for the purposes of the present disclosure, a patient is treated when one or more beneficial or desirable outcomes, including desirable clinical results, are obtained or are expected to be obtained. For example, beneficial or desirable clinical results include, but are not limited to, one or more of the following: reduction of one or more symptoms resulting from diabetes, improvement of the quality of life of a diabetic patient, reduction in the dosage of other medications required for the treatment of diabetes, delay or prevention of complications associated with diabetes, and / or extension of an individual's lifespan.

[0130] Furthermore, the patients of the present method are generally diabetic patients, but the age of the patients is not limited. The disclosed method is useful for the treatment of diabetes across all age groups and cohorts. Thus, in some embodiments, the patient can be a pediatric subject, and in other embodiments, the patient can be an adult patient.

[0131] In embodiments, when diabetic patients use the lattice structure, the need to monitor blood glucose levels can be significantly reduced, and the need for insulin injection may be completely eliminated. Periodically monitoring the implanted lattice structure (e.g., weekly, bi-monthly, monthly) to confirm that the cells of the lattice structure are functioning as desired is within the scope of the present disclosure. For example, in embodiments, the lattice structure can include one or more contrast agents to facilitate in vivo monitoring of the placement of the fiber device, the location of the implant at a point in time after implantation, the health of the implant, the harmful effects of non-target cell types, inflammation, and / or fibrosis. Suitable contrast agents include, but are not limited to, nanoparticles, nanocrystals, gadolinium, iron oxide, platinum iron, manganese, iodine, barium, microbubbles, fluorescent dyes, and others known to those skilled in the art.

[0132] Methods of in vivo monitoring include, but are not limited to, confocal microscopy, two-photon microscopy, high-frequency ultrasound, optical coherence tomography (OCT), photoacoustic tomography (PAT), computed tomography (CT), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), positron emission tomography (PET), and the like. These can provide useful means for monitoring the implanted lattice structure, either alone or in combination.

Examples

[0133] The following examples are presented to provide those skilled in the art with a complete disclosure and description of the methods of making and using the methods and compositions of the invention, and are not intended to limit the scope that the inventors regard as the invention. Efforts have been made to ensure the accuracy of the numerical values (e.g., amounts, temperatures, etc.) used, but some experimental error and deviation should be considered. Unless otherwise indicated, parts are by weight, molecular weight is the average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0134] Example 1. The bioprinted cell therapy platform normalizes blood glucose control in diabetic rats We have developed microfluidic bioprinting technology to produce implantable tissues for therapeutic applications by combining biocompatible materials with clinically relevant cells. Islet cell therapy has been clinically validated for type 1 diabetes (T1D), but it relies on lifelong immunosuppression and is limited by the supply of cadaveric donor islets. We are developing a bioprinted pancreatic tissue therapeutic that can deliver allogeneic islets or stem cell-derived pancreatic beta cells to T1D patients without the need for immunosuppression by encapsulating these cells in materials that support physiological function and protect them from direct attack by host immune cells.

[0135] This example describes a bioprocess method for packaging primary islets in bioprinted tissue implants for in vitro testing and in vivo function studies. We evaluated the ability of bioprinted human islet tissue (heterologous) to restore glycemic control in diabetic immunodeficient mice and adapted this process to bioprinted primary rat islet tissue (allogeneic) delivered to the omentum of diabetic rats. Finally, we developed a process for scaling up and down the manufacturing process of this bioprinted pancreatic tissue for delivery of implants in large animals and humans.

[0136] Materials and Methods The major procedure performed on the animals was the implantation of the omentum device outlined below. The surgery was performed after treatment with STZ.

[0137] A. Prepare the animals for surgery. Followed the general concepts of "Rodent Anesthesia" (SOPACC-01-2017), "Analgesia for Adult Mice and Rats Meloxicam SOP" (TECH19), and "Local Anesthesia / Analgesia in Adult Mice and Rats Bupivacaine SOP" (TECH16). The treatment is described below. A1. Place the animal on top of a heating pad (the temperature should be about 38 °C) in the induction chamber and induce anesthesia with isoflurane. Flush the chamber and transfer the animal to the maintenance circuit on the nose cone and heat support to assist with heat and maintain isoflurane anesthesia. A2. Administer small droplets of lubricating eye gel to both eyes. Place the animal in the prone position and administer a supportive therapy solution subcutaneously at 20 mL / kg in the form of 0.9% saline or lactated Ringer's solution (LRS). Use a 25G of various syringe sizes depending on the dose. A3. Administer 1 mg / kg of Metacam subcutaneously. A4. Administer 0.05 mg / kg of buprenorphine subcutaneously. A5. While turning the animal over, place a paper towel underneath and collect all the shaved hair. Shave the abdominal skin of the animal with a clipper. A6. Remove the dry gauze, remove all the hair around the animal, and simultaneously pull out the paper towel. A7. Once the hair is cleared, prepare one gauze or cotton swab dipped in soap to clean the shaved area. The gauze should be wet but not dripping and can be wrapped around the finger. When cleaning the shaved area outward in a circular motion, bubbles should be visible. After leaving the soap on the animal for about 30 seconds, wipe it off with alcohol. A8. Wipe the shaved area outward in a circular motion with a gauze or cotton swab dipped in 70% alcohol. Use a new gauze or cotton swab dipped in soap again, but this time to remove / scrub off the skin oil. After leaving it for 30 seconds, proceed to the next step. A9. Clean the area with a new gauze or cotton swab dipped in alcohol. A10. Inject a local anesthetic as a line block at the planned incision site. Lift the skin and insert the needle subcutaneously under the skin. Withdraw the needle while injecting to form a "bleb". A11. Perform additional subcutaneous treatment of the skin once (using gauze dipped in soap and then alcohol again). Pinch the animal's toes to ensure that the animal is properly anesthetized. Change gloves and wash hands with soap. Throughout the procedure, monitor the color (should be pink) of the extremities, respiratory rate, and depth, and pinch the toes every 5 minutes.

[0138] B. Preparation of surgical instruments B1. Wear clean test gloves. This surgery is performed using aseptic tip technology, and the gloved hands must remain sterile and not come into contact with any surface that is not sterile. B2. Open the sterile pack on the counter, aseptically remove the sterile field / half-folded wrap, and open it so that the inner side (sterile field) faces up when placing it near the operating table. B3. Prepare a pair of sterile forceps to transfer all instruments and consumables inside the pack to the sterile field wrap. Only the sterile parts of the instruments can enter the sterile field. B4. Take the "Glad Press and Seal Wrap", pull out the first 6 inches, and pull out the piece to place on the animal. Avoid using the edge as it is contaminated. Do not touch the upper surface with your finger. B5. Use a pair of forceps to pick up the cotton pad and help attach the press and seal to the animal. To pick up the press and seal, use a sterile 25G needle to puncture near the surgical incision area, grasp it with forceps, and make a hole on the planned incision site. Do not let non-sterile items touch the upper surface of the surgical drape. B6. Pinch the animal with forceps to confirm that it is at the surgical level of anesthesia.

[0139] C. Surgical procedure. It followed the general concept of "RodentSurvivalSurgery" (SOPACC-02-2017). All surgical procedures on immunodeficient rats were performed in a biosafety cabinet with laminar flow clean air workstations. The procedures are described below. C1. Use toothed forceps to pick up the skin. Make a 20-mm incision along the midline of the skin about 2 cm below the xiphoid process using the blade of a surgical scalpel. Once the skin incision is completed, grasp and lift the muscle, first create a puncture opening using the blade of the surgical scalpel, and then make a 20-mm incision using scissors. C3. Use a self-retaining tissue retractor to keep the abdominal incision open. C4. Sterilely place a sterile gauze on the abdominal skin caudal to the opening to prevent the greater omentum from coming into direct contact with the skin. C5. Identify the larger mesh, and gently extend the greater omentum from within the opening using a pair of tissue forceps. C6. Sterilely place the bioprinted implant in the center of the exposed mesh. Note: The bioprinted implant consists of a non-reactive and non-rigid polymer with no known biocompatibility issues. Each device is 20×20×2.5 mm and may contain cells but is specifically designed to prevent cell release. One device is implanted per animal. The implant is printed using sterile medical-grade components. (Cells are incorporated during the printing process.) After printing, the device is maintained in standard cell culture conditions (media, 37 °C / 5% CO2) within 4 days prior to implantation. The device is washed with a sterile isotonic solution such as saline or Ringer's buffer immediately before implantation to remove all traces of the media. C7. Freely fold the greater omentum over the implant and suture both sides of the mesh using a fine non-absorbable monofilament (e.g., 6-0 nylon or prolene) to create a closed pouch. C8. Gently slide the greater omental sac back into the abdominal cavity and close the incision with continuous sutures or subcutaneous sutures for the muscle layer and skin layer, respectively. C9. Moisten the 5-0 absorbable suture with saline. Use one suture pack per rat. Hold the needle vertically using a pair of needle drivers. Ensure that the suture does not touch the non-sterile area of the needle holder or forceps. Insert a needle on one side of the muscle and remove the needle from the other side of the muscle. When removing the needle, follow the direction of the needle. Tie with a running stitch and repeat this tying a total of 3 times. Cut the end leaving about 2 - 3 mm of suture thread. Repeat until the muscle area is closed. C11. Perform subcutaneous closure of the skin. On one side of the skin, insert a needle just below the skin layer and remove the needle on the opposite side of the skin (deeper but under the skin layer). Insert the needle on the opposite side of the skin and then move to the surface as you tie this time. Make sure the needle does not come out from above the skin. Tie 3 times with a running stitch. C12. Once the skin layer is closed, take a 25G needle and immerse it in Gluture. Take the needle covered with Gluture and place it on the sutured skin. Use a pair of forceps to pinch the skin around the needle and slowly remove the needle. Thus, the sutured area is properly closed. Recovery of the rat: C13. Turn off isoflurane and continue to supply oxygen to the rat through the nose cone. C14. Gently remove the surgical drape and remove the blood adhering to the rat. C15. Once the rat's right - side reflex has recovered, place the rat in a warmed recovery cage (cover the bare bottom of the cage with paper towels). C16. Monitor the rat in the recovery cage until it has fully recovered from anesthesia and can maintain its body temperature without additional heat (i.e., can eat, drink, walk normally, climb into the hut, and groom itself). C17. Once fully recovered, return the rat to the normal housing cage with a cage mate.

[0140] D. Analgesia Plan D1. Preoperative analgesics: Rats are administered a local anesthetic (bupivacaine 0.5 mg, 200 μl of 2.5 mg / ml solution) at the incision site before tissue dissection and receive injections of NSAID (meloxicam 1 mg / kg, SC) and buprenorphine (0.05 mg / kg, SC). Rats with induced liver disease are given a combination of oral ibuprofen and low-dose buprenorphine to account for the reduced liver metabolism in these liver disease animals. Oral ibuprofen (30 mg / kg, ibuprofen ligel capsules are resuspended in water by vortexing, protected from light, and replaced every 3 days) is administered to rats at the time of surgery according to ECH09b Oral dosing (Gavage) in Rats. D2. Postoperative analgesics: Day 1 and Day 2: Meloxicam (1 mg / kg, SC, SID) and buprenorphine (0.02 mg / kg, SC, BID) For these treatments, TECH16 (Local Anesthesia / Analgesia in Adult Mice and Rats Bupivacaine SOP) and TECH19 (Analgesia for Adult Mice and Rats Meloxicam SOP) were followed. Rats with induced liver disease are given a combination of oral ibuprofen and low-dose buprenorphine to account for the reduced liver metabolism in these liver disease animals. Oral ibuprofen (30 mg / kg, ibuprofen ligel capsules are resuspended in water by vortexing, protected from light, and replaced every 3 days) is administered to rats 6 hours after surgery according to ECH09b Oral dosing (Gavage) in Rats. Overnight, the animals continued to receive ibuprofen (1 mg / mL) in their drinking water. The animals received subcutaneous injections of buprenorphine (0.05 mg / kg) 24 hours and 48 hours after surgery.

[0141] E. Administration of prophylactic antibiotics The bio-printed implants were prepared under sterile conditions using sterile materials and reagents. Also, all surgical procedures were performed aseptically. However, to reduce the potential risk of infection, prophylactic antibiotics (100 μg / mL of Enrofloxacin in the drinking water) were added to the rats' drinking water from 3 days before surgery until 3 days after surgery. Enrofloxacin is excreted mainly by the kidneys and is also suitable for rats with liver disease.

[0142] Other procedures F. Blood collection Blood samples were collected from the rats weekly until the end point of the study. Blood was collected from the lateral saphenous vein using TECH02SOP "Blood collection from the lateral saphenous vein in mice and rats". The maximum weekly blood sample volume was 150 μl, which is within the acceptable limit of serial blood collection defined by "UBC ANIMAL CARE COMMITTEE POLICY 006, Policy on Acceptable Methods of Rodent Blood Withdrawal".

[0143] G. Administration of streptozotocin for induction of diabetes Streptozotocin (STZ) destroys the insulin-secreting cells of the pancreas, thereby inducing diabetes. Since special handling precautions are required for the use of STZ, all users and animal care technicians were informed of the risks due to the presence of SDS in the experimental area. Cages containing animals treated with STZ were marked as such for 1 week after STZ injection. STZ, which is supplied as a powder, is reconstituted to a concentration of 30 mg / mL immediately before injection in buffered acetic acid solution or citrate buffer (pH 4.5). To induce a model of type 1 diabetes (insulin deficiency), STZ was intraperitoneally injected into rats at a dose of 60 mg / kg in buffered acetic acid solution or citrate buffer (maximum volume of approximately 0.5 mL in a standard 250 g rat). For IP injection procedures, TECH10b (Intraperitoneal Injection in the Adult Rat) was followed. G2. After injection, the blood glucose levels of the animals were monitored daily. Animals that developed persistent hyperglycemia (blood glucose level > 20 mmol / l in two consecutive readings) were used in the experiment. There is a risk of severe hypoglycemia in the first 24 - 48 hours. The initial cytotoxic destruction of beta-islet cells causes an excessive release of insulin into the bloodstream. To prevent fatal hypoglycemia, sucrose water was given during the induction period to reduce morbidity and mortality. To do so, 10% sucrose was added to the drinking water 48 hours after STZ injection.

[0144] H. Blood glucose measurement Blood glucose levels were measured by placing a drop of blood (≤10 μL) on a blood glucose meter test strip (Lifescan Canada or equivalent). H1. A small droplet of blood was collected from the tail vein using TECH13 (UBCACC tail Poke SOP - Rat) and added to the test strip. H2. Gently apply pressure to the tip of the tail with a 2×2 gauze for approximately 10 seconds to stop blood flow.

[0145] I. Oral glucose tolerance test The main function of pancreatic islets is to secrete insulin in response to an increase in blood glucose levels. To monitor the in vivo function of implanted pancreatic islets, the islets were stimulated with an oral glucose dose after a short fasting period described below. II. Fast the animals for 4 hours (e.g., 8:00 am - 12:00 pm). I2. Collect a blood sample (fasting, volume: 75 ul) Using a 3.1 mL syringe and a flexible 20-gauge and 38 mm long feeding tube (according to TECH09 - Oral dosing in Mice and Rats), administer a glucose solution (3 g / kg, 150 ul) to the animals by forced oral gavage. I4. After 30 minutes, collect another blood sample (after glucose administration, volume: 75 ul).

[0146] J. Immunofluorescence protocol for insulin and CD31 Materials: Insulin (C27C9) rabbit mAb (New England Biolabs), CD31 (PECAM-1) mouse mAb (New England Biolabs), anti-rabbit IgG, Alexa Fluor® 647 conjugate (New England Biolabs), anti-mouse IgG, Alexa Fluor® 488 conjugate (New England Biolabs). Procedure 1. Place the slides in an oven at 60 °C for 15 minutes to initiate the deparaffinization process. 2. Place the slides in a xylene-resistant holder. 3. Wash in xylene for 5 minutes (3X). 4. Wash in 100% EtOH for 5 minutes (2 times). 5. Wash in 95% EtOH for 5 minutes. 6. Wash in 80% EtOH for 5 minutes. 7. Wash in 70% EtOH for 5 minutes. 8. Wash in PBS on a shaker for 5 minutes. 9. Place the slides in a beaker and cover with antigen retrieval buffer (10 mM citrate buffer, pH 6.0). 10. While ensuring that the slides are always covered with buffer, heat in a microwave oven at high power for 5 minutes (2 times). 11. Using heat-resistant gloves, remove the beaker from the microwave oven and place it in the sink. 12. Cool the beaker under a thin stream of cold running tap water for 5 - 10 minutes (make sure the water does not directly hit the slides). 13. Wash the slides in ddH2O for 5 minutes. 14. Wash the slides in PBS on a shaker for 5 minutes. 15. Remove the excess water on the slides and surround the sample with a hydrophobic pen (SuperPap pen). 16. Block the slides in 5% BSA-PBS containing 5% goat serum at room temperature for 1 hour in a humidified chamber. 17. Incubate with the primary antibody at 4°C in a humidified chamber (diluted with 5% BSA-PBS containing 5% goat serum). a. Insulin: 1 / 500 dilution b. CD31: 1 / 500 concentration Day 3 1. Wash the slides with PBS for 10 minutes (3 times). 2. Incubate with the secondary antibody at room temperature for 1 hour in a dark humidified chamber (diluted with 5% BSA-PBS or PBS). a. Anti-mouse: 1 / 1000 dilution b. Anti-rabbit: 1 / 1000 dilution 3. In the subsequent steps, the slides must be protected from light. 4. Wash the slides with PBS for 10 minutes (3 times). 5. Mount the slides with Fluoroshield containing DAPI and seal the coverslip with a clear nail polish. 6. Dry for 24 hours before imaging.

[0147] Treatment of islets and in vitro bioprinting Figure 5A shows a schematic diagram and bright-field image of bioprinted primary human islet tissue. Figure 5B shows live / dead staining of bioprinted primary islets (top: native islets, bottom: reaggregated islets) after 7 days in culture. Figure 5C shows data from glucose-stimulated insulin secretion (GSIS) assays performed using primary human islets (n = 8) and primary rat islets (n = 10), mean + / - SEM.

[0148] Function of bioprinted pancreatic tissue in a streptozotocin-induced rodent diabetes model Immunodeficient (NSG) mice: IP implant Figure 6A shows the random-fed blood glucose measurements over 80 days after streptozotocin (STZ) treatment and intraperitoneal (IP) implantation of bioprinted human islet tissue in NSG (NODscid gamma) mice (n = 5). Day 0 represents the time of implantation. Figure 6B shows the human C-peptide levels measured in mouse plasma over 80 days using ELISA. Figure 6C shows data from an oral glucose tolerance test (OGTT) performed on day 80 to evaluate the dynamics of post-fasting normoglycemia and subsequent glucose load in NSG mice with bioprinted islet tissue or healthy non-STZ-treated control mice.

[0149] Immunodeficient (nude) rats: Omental implant Figure 7A shows the blood glucose measurements over 180 days after omental porch implantation of bioprinted rat islet tissue in nude rats (n = 2) treated with STZ. Figure 7B shows H&E (high and low magnification) and immunohistochemistry (IHC) for insulin (islets) or CD31 (endothelial cells) performed on sections of fixed bioprinted tissue explanted after 180 days.

[0150] Immunocompetent (Sprague-Dawley) rats: Omental implant Figure 8A shows the blood glucose measurements over 90 days after omental porch implantation of bioprinted Lewis rat islet tissue in Sprague-Dawley (SD) rats (n = 3) treated with STZ. Retrieval of explants and return to hyperglycemia were performed 30, 60, and 90 days after surgical implantation. Figure 8B shows H&E) and IHC for insulin (islets) or CD31 (endothelial cells) performed on sections of fixed bioprinted tissue explanted after 60 days.

[0151] Expansion of bioprinted pancreatic tissue for large animals Figure 9A is a schematic diagram showing that the biomanufacturing process involves tissue design and QC in the unique software of the bioprinted tissue, including microarchitecture and macroarchitecture, cell viability, and confirmation of island distribution. Figure 9B shows the bioprinted pancreatic tissue used in the rat study compared to the enlarged tissue of large animals. Figure 9C shows the survival rate of the bioprinted neonatal pig islets confirmed up to 14 days after printing. GSIS shows that the function of the bioprinted tissue expands and contracts with the dose of human islets.

[0152] Conclusion We developed a process (see Figure 10) for manufacturing an implantable tissue containing bioprocessed pancreatic islets in a material that protects these allogeneic cells from attack by host immune cells. This example shows that the bioprinted pancreatic tissue can 1) maintain islet survival and function in vitro, 2) restore blood glucose control in diabetic mouse and rat models, 3) support islet function and immune defense over 90 days in a diabetic rat model, and 4) be scaled up for studies in large animal trials and ultimately delivery to T1D patients.

[0153] Example 2. Study on Print Optimization and Viability of Small Devices This example included test conditions of a 10×10 mm 4-layer lattice structure compared to control conditions of an 18×18 mm 2-layer lattice structure. Figure 11A is a schematic diagram of the 10×10 mm lattice structure. Figure 11B is an image of the coated 10×10 mm lattice structure attached to the frame. Figures 11C - 11D show the coated 10×10 mm lattice structure detached from the frame.

[0154] The device was coated with 0.5% SLG100 (alginate). The cell dose to the device was 3 KIEQ HepG2 aggregates. Live / dead staining was evaluated at 0 days after printing (Figure 12A) and 5 days after printing (Figure 12B).

[0155] This example also included tests of small devices (10×10 mm, 4-layer devices) having a coating of 0.5% SLG100 and a core of either 1.5% SLG100 containing HA or 1.5% SLG100. For each case, the cell dose was either 3K IEQ HepG2 aggregates or primary rat islets (PRI). Figures 13A - 13B show images of a 10×10 mm lattice structure on the frame (Figure 13A) and detached from the frame (Figure 13B) after coating. The stability data is summarized in Figure 14. Figure 15A is an image of Structure 1 (HA-containing core), Figure 15B is an image of Structure 2 (HA-containing core), Figure 15C is an image of Structure 3 (HA-containing core), Figure 15D is an image of Structure 1 (normal core), Figure 15E is an image of Structure 2 (normal core), and Figure 15F is an image of Structure 3 (normal core).

[0156] This example also included tests on the viability and functionality of coated 10×10 mm devices carrying PRI (coating 0.5% SLG100, cell dose: 3K IEQ PRI). Live / dead staining was evaluated at 0 days (Figure 16A) and 3 days (Figure 16B) after printing.

[0157] Example 3. Stability Test of Frame vs. Mesh Devices This example demonstrates that the stability of devices printed using a frame is enhanced compared to devices printed on a mesh instead of a frame (i.e., without using a frame).

[0158] Experimental Design In this example, the tested, coated lattice structure was 18×18 mm and had a two-layer thickness. The coated lattice structure included a core (1.5% SLG100), a shell (2% SLG100), and a conformal coating (0.5% SLG100). The core was printed at a flow rate of 115 μL / min, the shell was printed at 80 μL / min, and the sheath flow (i.e., the crosslinker solution) was 55 μL / min when dispensed from the print head. Three structures were printed using the frames disclosed herein, while the other three structures were printed on a mesh instead of a frame. The conformal coating was added to the bioprinted lattice structure while it was attached to the frame (a device that relied on the frame for printing) or while the bioprinted lattice structure was placed on the mesh (a device printed instead of a frame).

[0159] Stability test The stability test was conducted as follows. Each coated lattice structure was cultured in PIMS medium for 3 days in a 50 mL conical tube containing 15 mL of medium. Each coated lattice structure was subjected to an orbital shaking test at 125 rpm for 30 minutes or vehicle transportation for 30 minutes. Next, each coated lattice structure was poured into a Petri dish and washed three times with 10 mL of saline (the saline was aspirated between each wash). To mimic the transfer of the device at the surgical site, a spatula was used to lift and move each device between two Petri dishes filled with saline. This was repeated five times. Finally, each coated lattice structure was transferred onto a moistened plastic wrap, and a wand was used to move each device three times from one edge of the wrap to the other side (mimicking the repositioning of the device on the greater omentum).

[0160] Results All three coated lattice structures printed and coated by relying on the frame disclosed in this specification passed the stability test (compared with FIGS. 17 and 18D - 18F, FIGS. 18A - 18C). All three coated lattice structures printed on the mesh instead of the frame failed the stability test, and the microscopic images (FIGS. 19A - 19C) revealed that the fibers in the first layer leaked from the coating layer (compare FIGS. 19B - 19C with those of FIG. 19A). FIG. 20 shows an image of another fiber structure printed on a mesh (however, not coated), indicating a decrease in stability. The fiber structure in FIG. 20 was created by inside - out cross - linking.

[0161] Example 4. Double - dip coating of bioprinted fiber structures In this example, the bioprinted lattice structure tested was 16×16 mm and 2 - layer thick, and was printed on a device including a container that holds a dip - coating solution and is coupled to a vacuum chuck. The coated bioprinted lattice structure included a core (1.5% SLG100 containing cells or pigments), a shell (2% SLG100), a first inner conformal coating (2% SLG100), and a second outer conformal coating (2% Zwit - 20 alginate). The lattice structure was printed using the frame disclosed in this specification. The inner and outer conformal coatings were added to the bioprinted lattice structure while being coupled to the frame.

[0162] After printing the 2 - layer 16×16 mm lattice structure, the structure was cross - linked for 3 minutes with 95% / 5% Ca / Ba in 15% polyethylene glycol (PEG) in pH - buffered dH 2 O. The cross - linking solution tank was removed by vacuum suction, and the lattice structure was rinsed with TSC saline from the buffer channel on the print head. Next, all solutions were removed by vacuum suction, and the lattice structure was lifted 3 - 5 mm from the receiving surface.

[0163] Next, 1 mL of a 2% SLG100 first conformal coating solution was pipetted into the container to uniformly coat the entire lattice structure, and it was confirmed that all the fibers were covered with the solution from both the top and the bottom. The lattice structure was incubated in the first conformal coating solution for 10 seconds, and then the excess solution was removed from the container by vacuum.

[0164] Next, 1 mL of a second conformal coating solution of Zwit-20 alginate was pipetted into the container to uniformly coat the entire lattice structure, and it was confirmed that all the fibers were covered with the solution from both the top and the bottom. The lattice structure was incubated in the second conformal coating solution for 35 seconds, and then the excess solution was removed from the container by vacuum.

[0165] The lattice structure was then transferred to a 95% / 5% Ca / Ba solution bath, crosslinked for 3 minutes, and then the lattice structure was rinsed with saline.

[0166] Photographs of the lattice structure with the inner conformal coating and the outer side were taken and shown in Figure 21. The diameter of the entire vertical fiber with both conformal coatings shown in Figure 21 was 1.004 mm. The inner conformal coating was between 40.8 μm (left side, midpoint) and 50 μm (right side, midpoint), and the outer conformal coating was between 88.1 μm (left side, midpoint) and 90 μm (right side, midpoint).

[0167] Example 5. Compartmentalization of Core-Shell Fibers Compartmentalization of core-shell fibers was tested by printing an 11×11 mm lattice structure with or without cell aggregates in the core. The core material was 1.5% SLG100 with or without cells (cell density, 7×10 7 cells / mL, number of cells: 3×10 6 cells), and a 2% SLG100 shell and a 2% SLG100 conformal coating generated by dip coating were applied.

[0168] Images of the lattice structure are shown in FIGS. 22A - 22C, showing: A) upper horizontal fibers without cells and lower horizontal fibers with cell aggregates; B) no cells at the upper part of the loop and cell aggregates at the lower part of the loop after core switching, compartmentalization (dotted line) by core switching; and C) upper horizontal fibers (dotted line) switched for compartmentalization without cells and lower horizontal fibers with cell aggregates. These figures show that it is possible to generate a lattice structure compartmentalized such that cells may or may not be present in a part of the lattice structure.

[0169] The above - described embodiments and examples are merely illustrative and are intended to be non - limiting. One of ordinary skill in the art will be able to recognize or ascertain, using only routine experimentation, numerous equivalents of specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of the present invention and are encompassed by the appended claims.

Claims

1. An implantable composition for the treatment of diabetes, comprising a multilayer lattice structure containing continuously bioprinted core / shell fibers encapsulating a plurality of pancreatic islet cells, and at least one coating surrounding the multilayer lattice structure, wherein the multilayer lattice structure has a packing density of about 10% to about 90%, or about 20% to about 80%, or about 30% to about 70%, or about 40% to about 60%.

2. The composition according to claim 1, wherein the lattice structure has a packing density of about 30%, about 40%, about 50%, about 60%, or about 70%, or about 80%, and preferably about 50% to about 70%, or about 55% to about 65%, or about 60%.

3. The composition according to claim 1, wherein the multilayer lattice structure comprises at least one conformal coating.

4. The composition according to claim 3, wherein the multilayer lattice structure comprises a first inner coating and a second outer coating, and preferably the first inner coating comprises a hydrogel with higher material strength than the second outer coating.

5. The composition according to claim 1, wherein the continuously bioprinted core / shell fibers comprise a solid core and at least one shell.

6. The composition according to claim 5, wherein the material strength of the solid core is lower than the material strength of the shell.

7. The composition according to claim 5, wherein the coating comprises a hydrogel having a lower material strength than both the core and the at least one shell of the fiber.

8. The composition according to claim 5, wherein the solid core, the at least one shell, and the coating comprise the same hydrogel material, preferably the hydrogel material being an alginate.

9. The composition according to claim 8, wherein the solid core, the at least one shell, and / or the coating comprises a chemically modified alginate.

10. The composition according to claim 8, wherein the solid core comprises about 1.2 to about 1.8% alginate, preferably about 1.5% alginate.

11. The composition according to claim 8, wherein the at least one shell comprises about 1.4% to about 3.0% alginate, preferably about 1.5% to about 2.5% alginate, more preferably about 1.8% to about 2.2% alginate.

12. The composition according to claim 8, wherein the coating comprises about 0.2% to about 2% alginate, or about 0.25% to about 1.5% alginate, preferably about 0.3% to about 1.0% alginate, more preferably about 0.4% to about 0.8% alginate.

13. The composition according to claim 1, wherein the islet cells are human islet cells, and optionally, the islet cells include reaggregated islets.

14. The composition according to claim 1, wherein the lattice structure comprises at least two, three, four, or five layers formed by the continuous fibers, preferably comprising two, three, or four layers, and more preferably comprising four layers.

15. The composition according to claim 5, wherein the diameter of the continuous fibers is about 0.2 to 2.0 mm, or about 0.5 to 1.5 mm, about 0.5 to 0.9 mm, or about 900 μm to about 1200 μm, preferably the diameter is about 950 μm to about 1100 μm.

16. The diameter of the solid core is approximately 500 μm to approximately 800 μm, preferably approximately 600 μm to approximately 700 μm, and more preferably approximately 650 μm; The thickness of the at least one shell is about 50 μm to about 125 μm, preferably about 75 μm to about 100 μm; and / or The composition according to claim 15, wherein the thickness of the coating is about 50 μm to about 125 μm, preferably about 75 μm to about 100 μm.

17. The composition according to any one of claims 5 to 16, wherein the solid core and / or the at least one shell is partitioned along the length of the fibers.

18. The composition according to claim 5, wherein the plurality of pancreatic islet cells are encapsulated within the solid core.

19. The composition according to claim 5, wherein the plurality of pancreatic islet cells are encapsulated within the at least one shell.

20. An implantable medical device for the treatment of diabetes, comprising a multilayer lattice structure comprising a continuously bioprinted core / shell fiber encapsulating a plurality of pancreatic islet cells, and at least one coating surrounding the multilayer lattice structure, wherein the multilayer lattice structure has a packing density of about 10% to about 90%, or about 20% to about 80%, or about 30% to about 70%, or about 40% to about 60%.

21. An implantable pharmaceutical composition according to any one of claims 1 to 16, 18, and 19, for delivering insulin to a patient who requires insulin.

22. An implantable pharmaceutical composition according to any one of claims 1 to 16, 18, and 19, for treating patients with diabetes.

23. The implantable pharmaceutical composition according to claim 21, wherein the patient is a human patient suffering from type 1 diabetes.

24. The implantable pharmaceutical composition according to claim 22, wherein the patient is a human patient suffering from type 1 diabetes.

25. A method for manufacturing an implantable composition / medical device according to any one of claims 1 to 16, 18, and 19, To provide a bioprinting system comprising a manufacturing platform for supporting continuously bioprinted fibers during printing, patterning, and / or processing, wherein the manufacturing platform comprises: a frame having a plurality of posts on opposing sides of the frame defining voids and fixing and suspending the continuously bioprinted fibers during printing; a print head having a plurality of microfluidic channels for selectively supplying a plurality of materials to a dispensing orifice; a positioning unit for positioning the frame in three-dimensional space relative to the print head; and at least one dispensing means for dispensing the fibers from the dispensing orifice; Dispense the fibers around a plurality of posts via the bioprinting system to form a lattice structure containing at least two, three, four, or five layers of the fibers, After printing is complete, the grid structure is coated, The method, including the method described above.

26. The method according to claim 25, wherein the manufacturing platform is immersed in a crosslinking agent bath during the dispensing of the continuously bioprinted fibers.

27. The method of claim 25, wherein the manufacturing platform including the lattice structure is immersed in a crosslinking agent bath after dispensing the continuously bioprinted fibers.

28. The method according to claim 25, wherein the coating step comprises immersing the manufacturing platform, which includes the lattice structure, in at least one coating solution.

29. The method according to claim 25, wherein the coating step comprises dispensing at least one coating solution onto the lattice structure via the dispensing orifice after dispensing the continuously bioprinted fibers.

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