Systems and methods for automated hydrogel injection molding biomanufacturing

Abstract

Methods for automated biomanufacturing of hydrogel cell macroencapsulation devices having complex geometries are disclosed. First and second components may be introduced into a microfluidic mixer and undergo mixing flow such that they combine to form a gelling alginate solution. Cells may be incorporated into the gelling alginate solution after mixing and centered in the gelling alginate solution through the use of laminar flow. The cell incorporated gelling solution is then injected into a three-dimensional hydrogel mold to form the hydrogel cell microencapsulation device.

Description

Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) SYSTEMS AND METHODS FOR AUTOMATED HYDROGEL INJECTION MOLDING BIOMANUFACTURINGREFERENCE TO PRIOR FILED APPLICATION

[0001] This application claims the benefit of, and priority to, U.S.Provisional Patent Application No. 63 / 772,377, filed Mar. 14, 2025, the contents of which are hereby expressly incorporated by reference in their entirety and for all purposes.FIELD

[0002] The present disclosure generally relates to methods of biomanufacturing hydrogels, and more particularly to hydrogel injection molding.BACKGROUND

[0003] Allogeneic cell therapies are promising alternatives to treatments for several chronic diseases such as type 1 diabetes, hemophilia A and B, hypothyroidism, and anemia. Many of these therapies use stem cells to permanently treat patients and while they have shown early promise in clinical trials, these strategies have limited patient markets due to the acute risks associated with ineffective and toxic immunosuppressive drug regimens and immune rejection. The potent immune response to allogeneic cell therapies remains the greatest challenge to long-term engraftment and function, which also necessitates larger numbers of cells. Methods to eliminate graft rejection without chronic systemic immunosuppression will vastly expand the eligible patient population and reduce risks associated with allogeneic cell therapy. Additionally, stem cell-derived cell sources pose significant potential safety concerns as evidenced by a recent case study of teratoma formation in an immune competent individual who received stem cell-derived beta cell therapy. Given these potential risks, encapsulation devices to isolate the graft from the patient and enable full graft retrieval are critical to the safe translation of stem cell-based cell therapies. For example, the leading entities testing stem cell-derived islet replacement products clinically, are both pursuing1109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) macroencapsulated cell delivery methods to overcome these critical hurdles to translation.

[0004] Cell encapsulation within a nondegradable biomaterial has long been proposed as a means for reducing immune responses to transplanted grafts via a physical barrier to direct antigen recognition by immune cells, with decades of promising research in preclinical studies; however, translation of this technique has been hampered by poor clinical outcomes. Primary obstacles to clinical translation of cell encapsulation include safety concerns with delivering traditional hydrogel microcapsules, which are non-retrievable capsules on the scale of 60-1000 pm, delivered within the intraperitoneal (IP) space. Human trials demonstrate microcapsule adhesion to parietal peritoneum, spleen, kidney, and omentum, raising concerns about the long-term safety of IP capsule delivery. Additionally, fibrosis occurs at the capsule surface in the IP space, limiting nutrient and oxygen transport, and reducing long-term graft survival.

[0005] As such, macroencapsulation devices for cell encapsulation have been explored in preclinical and clinical studies and though they confer the safety benefit of a single, retrievable device, functional success of these devices has been limited. This is due in large part to poor oxygen and nutrient transport.

[0006] It is with these observations in mind, among others, that various aspects of the present disclosure were conceived and developed.2109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1A is a depiction of various hydrogel geometries manufacturable using injection molding.

[0008] FIG. 1B is a model of the automated hydrogel injection mold prototype.

[0009] FIG. 2 is a depiction of experimental results for microencapsulation of human islet viability and function.

[0010] FIG. 3A is a simulated model of the microfluidic mixer.

[0011] FIG. 3B is an image of the lab set up for the microfluidic mixer.

[0012] FIG. 3C is a graphical representation of the degree of mixing of the Alginate components at various flow rates.

[0013] FIG. 4A is a graphical representation of individual blood glucose for marginal syngeneic islet transplants in rat omentum of unencapsulated or spiral or cylinder alginate encapsulated islets.

[0014] FIG. 4B is a graphical representation of diabetes reversal rate for marginal syngeneic islet transplants in rat omentum of unencapsulated or spiral or cylinder alginate encapsulated islets.

[0015] FIG. 4C is a graphical representation of IPGTT for marginal syngeneic islet transplants in rat omentum of unencapsulated or spiral or cylinder alginate encapsulated islets.

[0016] FIG. 4D is a depiction of H&E staining for marginal syngeneic islet transplants in rat omentum of unencapsulated or spiral or cylinder alginate encapsulated islets.

[0017] FIG. 4E is a depiction of Masson’s trichrome for marginal syngeneic islet transplants in rat omentum of unencapsulated or spiral or cylinder alginate encapsulated islets.

[0018] FIG. 4F is a depiction of insulin staining of grafts for marginal syngeneic islet transplants in rat omentum of unencapsulated or spiral or cylinder alginate encapsulated islets.

[0019] FIG. 5 is a comparison of the new design of the microfluidic mixing device against previous paddle and channel type mixers.3109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f)

[0020] FIG. 6 is a comparison of the uniformity mixing for the channel, paddle, and modified paddle type microfluidic mixers.

[0021] FIG. 7 depicts various images of the test setup for the microfluidic mixer.

[0022] FIG. 8 is a graphical representation of crosslinking optimization for channel and modified paddle mixers at various alginate compositions.

[0023] FIG. 9 is a simulated model of alginate component mixing within the designed microfluidic mixer.

[0024] FIG. 10 is a comparison of cell survival and hydrogel consistency between hand molding and automated mixer injection molding methods.

[0025] FIG. 11A is a side x-ray view of diagram depicting the microfluidic mixer.

[0026] FIG. 11 B is a diagram depicting the internal paddles of the microfluidic mixer.

[0027] FIG. 11C is an isometric x-ray view of a diagram depicting the microfluidic mixer.

[0028] Corresponding reference characters indicate corresponding elements among the view of the drawings. The headings used in the figures do not limit the scope of the claims.4109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) DETAILED DESCRIPTION

[0029] The present disclosure relates to methods and systems for automated hydrogel injection molding for alginate-based cell macro-encapsulation. The method can include introducing first and second components into a microfluidic mixer to form a gelling solution.

[0030] It is to be understood that this invention is not limited to the particular example apparatuses, methods, compositions, or materials disclosed herein, but is extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0031] Various modifications and additions can be made to the exemplary embodiments discussed without departing from the scope of the present invention. For example, while the embodiments described above refer to particular features, the scope of this invention also includes embodiments having different combinations of features and embodiments that do not include all of the described features. Accordingly, the scope of the present invention is intended to embrace all such alternatives, modifications, and variations together with all equivalents thereof.

[0032] While specific implementations are discussed, it should be understood that this is done for illustration purposes only. A person skilled in the relevant art will recognize that other components and configurations may be used without parting from the spirit and scope of the disclosure. Thus, the following description and drawings are illustrative and are not to be construed as limiting. Numerous specific details are described to provide a thorough understanding of the disclosure. However, in certain instances, well-known or conventional details are not described in order to avoid obscuring the description. References to one or an embodiment in the present disclosure can be references to the same embodiment or any embodiment; and such references mean at least one of the embodiments.

[0033] Reference to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. The appearances of the phrase “in5109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) one embodiment”, or similarly and synonymously “in one example”, “in one instance”, or “in one aspect” in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Moreover, various features are described which may be exhibited by some embodiments and not by others.

[0034] The term “substantially uniform” as used herein, refers to a solution such as a gelling solution wherein individual solution components are mixed to a degree such that the solution has no significant spatial gradients of precursor compositions. The term may further refer to solutions having a distribution of molecular components that is about > 70%, about > 75%, about > 80%, about > 85%, about > 90%, about > 95%, or about 100% uniform. The term may further refer to solutions having been mixed to perfect or nearperfect molecular homogeneity.

[0035] The terms used in this specification generally have their ordinary meanings in the art, within the context of the disclosure, and in the specific context where each term is used. Alternative language and synonyms may be used for any one or more of the terms discussed herein, and no special significance should be placed upon whether or not a term is elaborated or discussed herein. In some cases, synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only and is not intended to further limit the scope and meaning of the disclosure or of any example term. Likewise, the disclosure is not limited to various embodiments given in this specification.

[0036] Without intent to limit the scope of the disclosure, examples of instruments, apparatus, methods and their related results according to the embodiments of the present disclosure are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the disclosure. Unless otherwise defined, technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the case of conflict, the present document, including definitions, will control.6109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f)

[0037] Additional features and advantages of the disclosure will be set forth in the description which follows, and in part will be obvious from the description, or can be learned by practice of the herein disclosed principles. The features and advantages of the disclosure can be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features of the disclosure will become more fully apparent from the following description and appended claims or can be learned by the practice of the principles set forth herein.

[0038] In some embodiments, the first and second components of the present systems and methods for automated hydrogel injection molding can each comprise a solution including at least one of a biopolymer, a base, a crosslinking agent, or a combination thereof. The first and second components are introduced to the microfluidic mixer at first and second inlets of the microfluidic mixer, wherein they are mixed to form a gelling solution through the induction of mixing flow.

[0039] After mixing, cells are incorporated into the gelling solution through a third inlet of the microfluidic mixer. Some embodiments may take advantage of laminar flow of the gelling solution to center the cells within the gelling solution. The cell incorporated gelling solution is then automatically injected into a three-dimensional hydrogel mold. The method may further include removing the crosslinked hydrogel from the three-dimensional hydrogel mold and immersing the crosslinked hydrogel in a BaCl2 bath to reinforce the alginate crosslinks.

[0040] As described herein, the present disclosure provides methods of automated hydrogel injection molding for cell macro-encapsulation that include a biopolymer, a base, and / or a crosslinking agent.

[0041] In some embodiments, the biopolymer is nondegradable. In some embodiments, the biopolymer is degradable. In some embodiments, the biopolymer may be a hydrogel. In some embodiments, the hydrogel may be selected from an alginate hydrogel, a polyethylene glycol) hydrogel, a silk fibroin hydrogel, a decellularized extracellular matrix hydrogel, a cellulose hydrogel, a chitosan hydrogel, a collagen hydrogel, an agarose hydrogel, a hyaluronic acid hydrogel, a gelatin hydrogel, a fibrin hydrogel, a carrageenan hydrogel, a xanthan gum hydrogel, a dextran hydrogel, and / or a combination thereof. In some embodiments, the hydrogel is an alginate7109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) hydrogel. In some embodiments, the hydrogel is a cellulose hydrogel. In some embodiments, the hydrogel is a chitosan hydrogel. In some embodiments, the hydrogel is a collagen hydrogel. In some embodiments, the hydrogel is an agarose hydrogel. In some embodiments, the hydrogel is a hyaluronic acid hydrogel. In some embodiments, the hydrogel is a gelatin hydrogel. In some embodiments, the hydrogel is a fibrin hydrogel. In some embodiments, the hydrogel is a carrageenan hydrogel. In some embodiments, the hydrogel is a xanthan gum hydrogel. In some embodiments, the hydrogel is a dextran hydrogel. In some embodiments, the hydrogel is a polyethylene glycol) hydrogel. In some embodiments, the hydrogel is a silk fibroin hydrogel. In some embodiments, the hydrogel is a decellularized extracellular matrix hydrogel.

[0042] In at least one example, the hydrogel may be Medium Viscosity Guluronic (MVG) alginate. For example, the hydrogel may be 1.5% w / v MVG alginate.

[0043] Those skilled in the art are aware of a variety of bases. In many embodiments, the present disclosure uses base chemistry that gradually acidifies a crosslinking agent to initiate gelation. In some embodiments, the base is selected from Ca(OH)2, KHCO3, NaHCOs, MgCOs, Na2CO3, Li2CO3, CaCO3, and CaMg(CO3)2. In some embodiments, the base is Ca(OH)2. In some embodiments, the base is KHCO3. In some embodiments, the base is NaHCO3. In some embodiments, the base is MgCO3. In some embodiments, the base is Na2CO3. In some embodiments, the base is Li2CO3. In some embodiments, the base is CaCO3. In some embodiments, the base is CaMg(CO3)2. In at least one example, the base include 30mM CaCO3.

[0044] Those skilled in the art are aware of a variety of crosslinking agents. In many embodiments, the present disclosure utilizes one or more crosslinking agents that react with a biopolymer to form a gel or gelling solution. In many embodiments, the crosslinking agent chemistries do not require an added catalyst. In many other embodiments, the crosslinking agent chemistries do require an added catalyst (e.g., a base). In some embodiments, the crosslinking agent may be selected from glutaraldehyde, epichlorohydrin, endogen polyamine spermidine, oxidized alginate, ethylene glycol di-methacrylate, genipin, citric acid, divinyl sulfone, and Glucono-Delta Lactone (GDL). In some embodiments, the crosslinking agent is glutaraldehyde. In some embodiments, the crosslinking agent is epichlorohydrin. In some embodiments,8109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) the crosslinking agent is endogen polyamine spermidine. In some embodiments, the crosslinking agent is oxidized alginate. In some embodiments, the crosslinking agent is ethylene glycol di-methacrylate. In some embodiments, the crosslinking agent is genipin. In some embodiments, the crosslinking agent is citric acid. In some embodiments, the crosslinking agent is GDL. In some embodiments, the crosslinking agent is divinyl sulfone. In at least one example, the crosslinking agent includes 100 mM GDL.

[0045] In one non-limiting example, the biopolymer may be MVG alginate, the base may be CaCOs, and the crosslinking agent may be GDL. In another example, the first component may include a solution including at least one of a Medium Viscosity Guluronic (MVG) alginate, CaCOs and / or Glucono-Delta Lactone (GDL) and the second component includes a solution including at least one of a Medium Viscosity Guluronic (MVG) alginate, CaCOa and / or Glucono-Delta Lactone (GDL).

[0046] In some embodiments the gelling solution is a slow gelling solution. The gelation may occur more than 2 minutes after mixing, such as 2 minutes to 4 minutes, 3 minutes to 5 minutes, 4 minutes to 6 minutes, 5 minutes to 7 minutes, 6 minutes to 8 minutes, 7 minutes to 9 minutes, or 8 minutes to 10 minutes after mixing. For example, gelation may occur around about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes after mixing. In some embodiments, the gelling solution is a substantially uniform mixture of the first and second components.

[0047] In some embodiments, the presently disclosed systems and methods may be used to encapsulate one or more cells within the hydrogel. A person of ordinary skill in the art will appreciate that an encapsulated cell may be eukaryotic. In some embodiments, a person of ordinary skill in the art will appreciate that an encapsulated cell may be prokaryotic. In some embodiments, the cell may be engineered. In some embodiments, the engineered cell may produce a therapeutic molecule (e g., an antibody, a hormone, a growth factor, a cytokine). In some embodiments, the cell may be naturally occurring. In some embodiments, an encapsulated cell may be selected from a pancreatic islet, a stem cell, a differentiated stem cell, a CHO cell, a primary cell such as a cardiomyocyte, a chondrocyte, an9109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) osteoblast, an osteoclast, an osteocyte, a neuron, a hepatocyte, a podocyte, an epithelial cell, an endothelial cell, an enterocyte, a goblet cell, a Paneth cell, an enteroendocrine cell, a colonocyte, a urothelial cell, a mucous cell, a parietal cell, a zymogenic cell, an adipocyte, an alveolar cell, a ciliated cell, a fibroblast, a myofibroblast, a smooth muscle cell, a myocyte, a thymocyte, a thyrocyte, a parafollicular cell, an oligodendrocyte, a trophoblast, and a pancreatic beta cell. In some embodiments, an encapsulated cell is a pancreatic islet. In some embodiments, an encapsulated cell is a stem cell. In some embodiments, an encapsulated cell is a CHO cell. In some embodiments, an encapsulated cell is a pancreatic beta cell.

[0048] In some embodiments, the cell incorporated gelling solution is injected into the three-dimensional hydrogel mold within about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes of the first and second components being mixed. In at least one example, the cell incorporated gelling solution is injected into the three-dimensional hydrogel mold within about 5 minutes of the first and second components being mixed.

[0049] In some embodiments, the method may further include further removing the crosslinked hydrogel from the mold and placing in a bath of BaCh In other embodiments, the method may include removing the crosslinked hydrogen from the mold withing a bath of BaCl2. The crosslinked hydrogel may be incubated within the BaCl2 bath for about 5 to about 30 minutes. In some examples, the crosslinked hydrogel may be incubated within the BaCl2 bath for about 5 minutes to about 10 minutes, about 10 minutes to about 15 minutes, about 15 minutes to about 20 minutes, about 20 minutes to about 25 minutes, or about 25 minutes to about 30 minutes. In other examples, the crosslinked hydrogel may be incubated within the BaCh bath for about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, or about 30 minutes.10109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f)

[0050] In some embodiments, the bath includes about 0.5% w / v, about 1.0% w / v, about 1.5% w / v, or about 2.0% w / v BaCl2. In at least one example, the bath is 1.5% w / v BaCh.

[0051] In some embodiments, the method may further include inducing laminar flow of the gelling solution prior to incorporating the cells such that the cells are centered within the gelling solution. In some examples, the laminar flow may be induced by using lower velocities than would otherwise be used to mix gelling solutions. The laminar flow may result in a parabolic velocity profile to allow for the centering of the cells within the gelling solution.

[0052] With the specific limitations facing macroencapsulation devices, computational modeling-guided device design was used for improved oxygen transport, and degradable hydrogel-guided enhanced vascularization at the device surface to further maximize oxygen access and mitigate fibrosis. Macroencapsulation device designs with high surface area-to-volume (SA / V) ratios can maximize oxygen transport to encapsulated cells by minimizing diffusion distances. A hydrogel injection moldingbased method was developed to generate high SA / V hydrogel macroencapsulation geometries as shown in FIG. 1A, a method which can be automated and integrated in the manufacturing and scaling of cell therapy products. This method is highly reproducible, works with diverse hydrogels, and is simple to implement in biomanufacturing. The schematic is depicted in FIG. 1B.

[0053] As depicted in FIG. 2 high viability and function of human and rat islets in manually generated injection molded alginate devices (medium viscosity guluronic, MVG) has been demonstrated. Provided herein are optimized alginate hydrogels within an automated hydrogel injection molding cell macroencapsulation process to advance this technology to commercial scale manufacturing and preclinical large animal models. Current methods for creating hydrogel macroencapsulation devices with complex geometries and high SA / V ratios (such as bioprinting) are cost prohibitive and challenging to scale to commercial manufacturing. Conversely, hydrogel injection molding is modular and compatible with diverse hydrogel materials, and is faster, cheaper, and easier to scale. The present disclosure discloses optimized alginate hydrogels within the automated injection molding and cell encapsulation process, and11109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) evaluates in vivo responses to alginate injection molded devices, with and without vasculogenic hydrogels, in leading transplant sites. The presently disclosed research facilitates IND-enabling Phase II studies evaluating optimized devices and device transplant strategy in a large animal non-human primate (NHP) model.

[0054] Currently, there is no automated biomanufacturing method available to macroencapsulate cell therapies. Biofabrication methods to generate complex hydrogel geometries, such as 3D printing, require costly equipment, are complex and slow to operate, and are challenging to scale in manufacturing. The hydrogel injection molding method is unique in its approach to both (1) generate a high SA / V geometry to maximize oxygen transport to encapsulated cells, and (2) integrate with high-throughput manufacturing of cell products. Further, the presently disclosed macroencapsulation devices can be transplanted with vasculogenic degradable hydrogels to retrievable tissue locations to maximize vasculature and thus oxygen availability to encapsulated cells. Injection molding can use versatile materials, natural and synthetic, degradable and nondegradable for a wide range of applications.

[0055] Alginate material parameters were optimized within the automated injection molding biomanufacturing system. The optimized alginate material parameters make reliable reproduction of high-quality manufactured alginate macroencapsulation devices using automated injection molding possible. Optimal designs and best practices for creating alginate macroencapsulation devices with injection molding to minimize risk when scaling automated biomanufacturing to the commercial level.

[0056] A system to encapsulate cells in diverse hydrogel materials via an injection molding strategy is disclosed herein, which enables the generation of hydrogel macroencapsulation devices of complex, high surface area to volume ratio (SAA / ) geometries FIG. 1A. The spiral geometry has the capacity to emulate the transport kinetics observed in microencapsulated islet geometry formats, and as is shown in FIG.2, studies with human and rat primary islets show comparable viability and function to unencapsulated control islets. Alginate may be used for ease of translation as a previously FDA-approved material; further, NovaMatrix™ ultrapure alginates may be used, which undergo strict quality control and NovaMatrix™ maintains drug master files with the US FDA.12109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f)

[0057] A key component of the automated injection molded biomanufacturing system is the microfluidic mixer depicted in FIG. 3A-B, used to combine alginate precursor components immediately prior to mold injection and gelation. A slow gelling alginate formulation is used (medium viscosity guluronic (MVG) + CaCOs + GDL) that enables ~5 min from mixing to gelation, providing ample time to fill an individual mold once components are combined. An additional inlet enables cell incorporation after component mixing, taking advantage of laminar flow to center cells in the alginate solution. Simulations show that this design results in mixing of the MVG alginate hydrogel components in the microfluidic mixing device, particularly with slower flow rates as depicted in FIG. 3C. A comparison of the new design of the microfluidic mixing device against previous paddle and channel type mixers can be seen in FIG. 5. Comparisons of uniformity of the mixed stream can be seen in FIG. 6.

[0058] The new design of the microfluidic mixing device is further depicted in FIG. 11A-C. As shown in FIG. 11A and 11C, the microfluidic mixer utilizes a plurality of paddles within a mixing chamber connected to the first and second inlets of the microfluidic mixer. As depicted by FIG. 11 B, the plurality of paddles may be oriented such that they induce mixing flow. In some embodiments, the paddles may be oriented in a corkscrew or spiraled manner relative to a center longitudinal axis of the mixing chamber. In other embodiments, the paddles may extend radially from the center longitudinal axis of the mixing chamber or extend internally from the inner walls of the mixing chamber. In yet another embodiment, the microfluidic mixer includes a first set of paddles proximate the first and second inlets and a second set of paddles after the first set of paddles. In one possible configuration, the first set of paddles can include a plurality of paddles oriented in a corkscrew or spiraled manner relative to the center longitudinal axis of the mixing chamber such that each paddle makes about a 720° arc around the center longitudinal axis. The second set of paddles can also include a plurality of paddles oriented in a corkscrew or spiraled manner relative to the center longitudinal axis of the mixing chamber such that each paddle makes about a 1080° arc around the center longitudinal axis. In another embodiment, each paddle of the first set of paddles are oriented such that each paddle makes about a 1080° arc around the center longitudinal13109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) axis and the second set of paddles are oriented such that each paddle makes about a 720° arc around the center longitudinal axis.

[0059] In further embodiments, the microfluidic mixer has 5, 6, 7, 8, 9, or 10 paddles. In some embodiments, the mixing chamber has a length of about 20 mm to about 35 mm. For example, the mixing chamber may have a length of about 20 mm to about 23 mm, about 20 mm to about 26 mm, about 20 mm to about 29 mm, about 20 mm to about 32 mm, about 20 mm to about 35 mm, about 23 mm to about 26 mm, about 23 mm to about 29 mm, about 23 mm to about 32 mm, about 23 mm to about 35 mm, about 26 mm to about 29 mm, about 26 mm to about 32 mm, about 26 mm to about 35 mm, about 29 mm to about 32 mm, about 29 mm to about 35 mm, or about 32 mm to about 35 mm. In one embodiment the mixing chamber has a length of about 25.4 mm. In some embodiments, the first and second sets of paddles may each be positioned across about 10 mm to about 15 mm of the length of the microfluidic mixer. For example, the first and second sets of paddles can each be positioned across about 10 mm to about 11 mm, about 10 mm to about 12 mm, about 10 mm to about 12.7 mm, about 10 mm to about 13 mm, about 10 mm to about 14 mm, about 10 mm to about 15 mm, about 11 mm to about 12 mm, about 11 mm to about 12.7 mm, about 11 mm to about 13 mm, about 11 mm to about 14 mm, about 11 mm to about 15 mm, about 12 mm to about 12.7 mm, about 12 mm to about 13 mm, about 12 mm to about 14 mm, about 12 mm to about 15 mm, about 12.7 mm to about 13 mm, about 12.7 mm to about 14 mm, about 12.7 mm to about 15 mm, about 13 mm to about 14 mm, about 13 mm to about 15 mm, or about 14 mm to about 15 mm of the length of the microfluidic mixer. In one possible embodiment the first set of paddles may be positioned across 12.7 mm of the length of the microfluidic mixer and the second set of paddles can be positioned across the remaining 12.7 mm of length of the microfluidic mixer.

[0060] In some embodiments, the first and second inlets of the microfluidic mixer may have an initial true radius of about 1 mm to about 3 mm and a true radius of about 2 mm to about 4 mm at their point of connection with the mixing chamber. For example, the first and second inlets of the microfluidic mixer may have an initial true radius of about 1.0 mm to about 1.4 mm, about 1.0 mm to about 1.8 mm, about 1.0 mm to about 2.2 mm, about 1.0 mm to about 2.6 mm, about 1.0 mm to about 3.0 mm, about 1.4 mm14109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) to about 1.8 mm, about 1.4 mm to about 2.2 mm, about 1.4 mm to about 2.6 mm, about 1.4 mm to about 3.0 mm, about 1.8 mm to about 2.2 mm, about 1.8 mm to about 2.6 mm, about 1.8 mm to about 3.0 mm, about 2.2 mm to about 2.6 mm, about 2.2 mm to about 3.0 mm, or about 2.6 mm to about 3.0 mm. As another example, the first and second inlets of the microfluidic mixer may have a true radius at their point of connection with the mixing chamber of about 2.0 mm to about 2.4 mm, about 2.0 mm to about 2.8 mm, about 2.0 mm to about 3.2 mm, about 2.0 mm to about 3.6 mm, about 2.0 mm to about 4.0 mm, about 2.4 mm to about 2.8 mm, about 2.4 mm to about 3.2 mm, about 2.4 mm to about 3.6 mm, about 2.4 mm to about 4.0 mm, about 2.8 mm to about 3.2 mm, about 2.8 mm to about 3.6 mm, about 2.8 mm to about 4.0 mm, about 3.2 mm to about 3.6 mm, about 3.2 mm to about 4.0 mm, or about 3.6 mm to about 4.0 mm. According to one embodiment, the first and second inlets of the microfluidic mixer may have an initial true radius of about 2.16 mm and a true radius of about 3.18 mm at their point of connection with the mixing chamber. According to other embodiments, the mixing chamber may have a true radius of about 5 mm to about 15 mm. For example, the mixing chamber may have a true radius of about 5 mm to about 7 mm, about 5 mm to about 9 mm, about 5 mm to about 11 mm, about 5 mm to about 13 mm, about 5 mm to about 15 mm, about 7 mm to about 9 mm, about 7 mm to about 11 mm, about 7 mm to about 13 mm, about 7 mm to about 15 mm, about 9 mm to about 11 mm, about 9 mm to about 13 mm, about 9 mm to about 15 mm, about 11 mm to about 13 mm, about 11 mm to about 15 mm, or about 13 mm to about 15 mm. In one possible embodiment, the mixing chamber may have a true radius of about 8.92 mm.

[0061] As depicted by FIG. 11A and 11 C, the microfluidic mixer can include a laminar flow section positioned after the mixing chamber. In some embodiments the laminar flow section of the microfluidic mixer is connected to a third inlet for receiving cells. In some configurations, the laminar flow section has a length of about 20 mm to about 35 mm. For example, the laminar flow section may have a length of about 20 mm to about 23 mm, about 20 mm to about 26 mm, about 20 mm to about 29 mm, about 20 mm to about 32 mm, about 20 mm to about 35 mm, about 23 mm to about 26 mm, about 23 mm to about 29 mm, about 23 mm to about 32 mm, about 23 mm to about 35 mm, about 26 mm to about 29 mm, about 26 mm to about 32 mm, about 26 mm to about 3515109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) mm, about 29 mm to about 32 mm, about 29 mm to about 35 mm, or about 32 mm to about 35 mm. In one possible configuration, the laminar flow section may have a length of about 25.27 mm.

[0062] In another configuration, the third inlet may be positioned along the laminar flow section of the microfluidic mixer around 2 mm to about 30 mm after the mixing chamber. For example, the third inlet may be positioned along the laminar flow section of the microfluidic mixer about 2 mm to about 5 mm, about 2 mm to about 10 mm, about 2 mm to about 15 mm, about 2 mm to about 20 mm, about 2 mm to about 25 mm, about 2 mm to about 30 mm, about 5 mm to about 10 mm, about 5 mm to about 15 mm, about 5 mm to about 20 mm, about 5 mm to about 25 mm, about 5 mm to about 30 mm, about 10 mm to about 15 mm, about 10 mm to about 20 mm, about 10 mm to about 25 mm, about 10 mm to about 30 mm, about 15 mm to about 20 mm, about 15 mm to about 25 mm, about 15 mm to about 30 mm, about 20 mm to about 25 mm, about 20 mm to about 30 mm, or about 25 mm to about 30 mm after the mixing chamber. In some embodiments, the third inlet of the microfluidic mixer may have a true radius of about 1 mm to about 3 mm and the laminar flow section of the microfluidic mixer may have a true radius of about 2 mm to about 6 mm. For example, the third inlet may have a true radius of about 1.0 mm to about 1.4 mm, about 1.0 mm to about 1.8 mm, about 1.0 mm to about 2.2 mm, about 1.0 mm to about 2.6 mm, about 1.0 mm to about 3.0 mm, about 1.4 mm to about 1.8 mm, about 1.4 mm to about 2.2 mm, about 1.4 mm to about 2.6 mm, about 1.4 mm to about 3.0 mm, about 1.8 mm to about 2.2 mm, about 1.8 mm to about 2.6 mm, about 1.8 mm to about 3.0 mm, about 2.2 mm to about 2.6 mm, about 2.2 mm to about 3.0 mm, or about 2.6 mm to about 3.0 mm. As another example, the laminar flow section of the microfluidic mixer may have a true radius of about 2.0 mm to about 2.5 mm, about 2.0 mm to about 3.0 mm, about 2.0 mm to about 3.5 mm, about 2.0 mm to about 4.0 mm, about 2.0 mm to about 4.5 mm, about 2.0 mm to about 5.0 mm, about 2.0 mm to about 5.5 mm, about 2.0 mm to about 6.0 mm, about 2.5 mm to about 3.0 mm, about 2.5 mm to about 3.5 mm, about 2.5 mm to about 4.0 mm, about 2.5 mm to about 4.5 mm, about 2.5 mm to about 5.0 mm, about 2.5 mm to about 5.5 mm, about 2.5 mm to about 6.0 mm, about 3.0 mm to about 3.5 mm, about 3.0 mm to about 4.0 mm, about 3.0 mm to about 4.5 mm, about 3.0 mm to about 5.0 mm, about 3.0 mm to about 5.5 mm, about 3.0 mm to about 6.0 mm,16109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) about 3.5 mm to about 4.0 mm, about 3.5 mm to about 4.5 mm, about 3.5 mm to about 5.0 mm, about 3.5 mm to about 5.5 mm, about 3.5 mm to about 6.0 mm, about 4.0 mm to about 4.5 mm, about 4.0 mm to about 5.0 mm, about 4.0 mm to about 5.5 mm, about 4.0 mm to about 6.0 mm, about 4.5 mm to about 5.0 mm, about 4.5 mm to about 5.5 mm, about 4.5 mm to about 6.0 mm, about 5.0 mm to about 5.5 mm, about 5.0 mm to about 6.0 mm, or about 5.5 mm to about 6.0 mm. In another embodiment, the third inlet of the microfluidic mixer may have a true radius of about 2.16 mm and the laminar flow section of the microfluidic mixer may have a true radius of about 4.29 mm.

[0063] In some embodiments, the system may include one or more pumps operable to create a flow of the first and second components into the microfluidic mixer. Similarly, the system may include a pump operable to create a flow of the cells into the system.

[0064] In some aspects, the present disclosure further relates to hydrogel compositions suitable for molding into hydrogel macroencapsulation devices. In one example, the hydrogel composition may include an alginate, a base, and a crosslinking agent. In some embodiments, the alginate may be a Medium Viscosity Guluronic (MVG) alginate. In further embodiments, the base may be selected from Ca(OH)2, KHCO3, NaHCOa, MgCOs, Na2COs, Li2COs, CaCOs, and CaMg(CO3)2. In one particular example, the base may be CaCOs. In yet further embodiments, the crosslinking agent may be selected from glutaraldehyde, epichlorohydrin, endogen polyamine spermidine, oxidized alginate, ethylene glycol di-methacrylate, genipin, citric acid, divinyl sulfone, and GluconoDelta Lactone (GDL). In one particular embodiment, the crosslinking agent may be Glucono-Delta Lactone (GDL). In some embodiments, the hydrogel composition may further include cells encapsulated within the hydrogel composition. In some embodiments the cells may be eukaryotic. In one example, the hydrogel composition may include about 1.5% w / v MVG alginate, about 30 mM CaCOs, about 100 mM GDL, and cells.Examples

[0065] The alginate microfluidic mixing for automated injection molding biomanufacturing and cell encapsulation may be optimized to reliably produce high quality, cell-laden alginate macroencapsulation devices.17109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f)

[0066] Optimization of microfluidic mixing parameters with alginates of varied viscosities

[0067] Preliminary modeling and in vitro experimentation of MVG alginate may be performed within the microfluidic mixing device FIG. 3 which integrates into the automated injection molding prototype FIG. 1. MVG and low viscosity mannuronic (LVM) and guluronic (LVG) alginate formulations have all been used for encapsulated cell products with comparable transport properties and stability. However, the pre-gelation viscosities of these alginates vary significantly depending on concentration (20 to > 200 mPa*s), and LVM and LVG alginates with their lower pre-gelation viscosities may result in improved mixing for more homogenous and mechanically stable encapsulation devices. As such, rheometry may be used to evaluate pre-gelation solution viscosities of LVM, LVG, and MVG alginates, and these measurements may be used for computational fluid dynamics modeling to compare mixing under a range of flow parameters FIG. 3C, including flow rate (0.5-6 mL / min) and precursor solution concentration (1.5-3%).

[0068] In silico modeling of cell channel centering in fluid flow: The modeled design further incorporated a third channel that incorporates cells in the hydrogel. Cross sections of the flow outlet as depicted in FIG. 3C enable visualization of cell channel location and optimization of channel placement prior to in vitro testing.

[0069] In vitro assessment of microfluidic mixing of MVG and LVM alginates:Optimal flow parameters identified in modeling may be used to validate mixing in vitro. Food coloring may be incorporated into alginate precursor solutions FIG. 3B and images of the outlet tube may be taken at various timepoints in flow development. Image analysis software can be used to deconvolute the solution colors in the outlet tube and quantify solution mixing, with and without “cell channel” flow. Hydrogels formed via the microfluidic mixer may also be evaluated based on the amount of un-crosslinked gel material, percent of the 3D printed mold filled, and ease of extraction from molds.

[0070] Characterization of mechanical integrity of alginate macroencapsulation devices generated with microfluidic mixing module

[0071] In the slow-gelling formulation, alginate (final 1.5% w / v) may be crosslinked by calcium divalent ions that are liberated from CaCOs (30 mM) by glucono delta-lactone (GDL, 100mM). Crosslinked hydrogels may then be extracted from the18109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) molds in a bath of BaCb (1.5% w / v), where barium ions reinforce alginate crosslinking for improved mechanical integrity and long-term stability. MVG, LVM, and LVG mechanical properties may be compared after microfluidic mixing without the barium crosslinking step, and after 5, 10, or 20 min incubations in the barium wash. Control hydrogels may also be evaluated under the same barium conditions after thorough hand mixing to evaluate the contribution of the microfluidic mixer to hydrogel mechanical properties.

[0072] Assessment of alginate mechanical properties: Each condition described above may be evaluated for rheological properties including storage and loss moduli, with strain and frequency sweeps within the linear viscoelastic region. The plateau of the storage modulus within the strain sweep can be used to calculate degree of crosslinking and mesh size.

[0073] Assessment of cell viability, function, and spatial location after encapsulation via microfluidic mixing module and injection molding

[0074] The viability, function, and spatial location of cells within hydrogel injection molded spirals using the microfluidic mixer may be validated. The model beta cell line INS-1 E at a final density of 7500 cells / pL (the equivalent to 5 primary islet equivalents ( I EQ) per pL) may be used in all encapsulation devices. Control cells may be unmanipulated, cells flowed through the microfluidic mixer in media only, and cells encapsulated via traditional open molding.

[0075] Assessment of macroencapsulated cells: INS-1 E macroencapsulated spiral devices and control hydrogels may be evaluated at 24, 48, and 96 hrs post-encapsulation (standard culture conditions). INS-1 E cells may be evaluated for viability (live / dead staining) via confocal microscopy at the given timepoints, and for homogeneity throughout the spiral geometry alginate devices. ImageJ can be used to quantify the percent area occupied by cells at 5 locations of each spiral, as well as percent viable cells within each spiral. Metabolic activity of INS-1 E cells is assessed via resazurin cell viability assay.

[0076] The final design of the macroencapsulation devices may demonstrate (1) complete mixing and hydrogel gelation, (2) mechanical integrity once removed from injection molds, (3) retention of encapsulated cell viability and metabolic19109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) activity compared to unencapsulated controls, and (4) homogeneous and / or centralized cell distribution in injected molded spirals.

[0077] Lower viscosity hydrogel component mixing may be observed within the microfluidic mixer, and laminar flow enabled centering of the cell component. In some aspects, 10 min of a BaCh bath may be sufficient to generate consistently high storage modulus alginate gels, and a relatively comparable range of modulus for LVM, LVG, and MVG with BaCL treatment may be observed. Finally, high cell viability and function may be observed post encapsulation, particularly at lower flow rates.

[0078] Where the disclosed design sometimes may not facilitate cell component centering within the alginate flow, alternative approaches (e.g. positioning of cell channel, alternative flow regimes) may be designed to ensure cells are not at the edge of the alginate.

[0079] Evaluate remodeling responses to injection molded alginate hydrogels in an allogeneic rat transplant model

[0080] Fibrotic and vasculogenic responses to alginate macroencapsulation devices with and without a degradable PEG-based vasculogenic hydrogel may be assessed in a rat transplant model due to higher order animal models exhibiting greater fibrosis and different remodeling responses than mice, and due to rats providing remodeling responses more comparable to NHP and humans, thus allowing greater prediction of approach success. Fibrotic responses to alginate may be quantifiable and an appropriate vasculogenic hydrogel dose may be identified to achieve a balance of device vascularization with minimal device fibrosis.

[0081] Alginate encapsulation devices are typically delivered to the intraperitoneal cavity, with the goal of preventing fibrotic reactions and cellular buildup at the device surface. However, this approach has demonstrated limited success long-term, particularly in larger animal models and humans which experience more significant fibrotic responses than mice. The presently disclosed method may use an alternative approach where injection molded alginate devices with high SA / V ratios are delivered to highly vascularized locations such as the omentum with a degradable PEG-based vasculogenic hydrogel (4-arm PEG-maleimide + 1mM RGD for cell adhesion + VPM proteolytically cleavable crosslinker + 10 pg / mL VEGF) that enhances vascularization, and thus oxygen20109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) availability, at the device surface. This vasculogenic system has shown no adverse effects in a decade of islet and encapsulated islet transplantation in mouse models, however, there has been limited evaluation of this strategy in higher order animals with fibrotic responses more similar to human, such as the rat.

[0082] Injection molded spiral macroencapsulation devices (MVG alginate) coupled with this degradable vasculogenic hydrogel system in a syngeneic islet transplant model within the rat omentum were recently evaluated and are depicted in FIG. 4. A marginal islet loading of 6000 lEQ / kg was used, which is rarely sufficient to reverse diabetes, resulting in 25% diabetes reversal in unencapsulated and spiral encapsulated islets, and no reversal in low SA / V ratio cylinder devices. The results are depicted in FIG.4A-B. H&E and Masson’s trichrome staining, depicted in FIG. 4D-E, where blue indicates collagen deposition, demonstrate 25-50 pm fibrotic encapsulation at the device surface; robust insulin staining was evident in spiral and unencapsulated groups as seen in FIG.4F. The remodeling response to alginate spiral hydrogels containing an allogeneic cell graft in the omentum and subcutaneous (SLIBQ) space were quantified, with and without vasculogenic hydrogel. Varying doses of vasculogenic hydrogel were evaluated to identify an optimal dosage for large animal IND-enabling studies. Diabetic rat recipients were used as their remodeling responses more closely align with that expected in diabetic humans, and allogeneic cell grafts were used to most accurately model remodeling responses.

[0083] Fibrosis was first quantified at the surface of allogeneic cell graftcontaining injection molded spiral alginate devices made with different alginate compositions within two common transplant sites, and the influence of degradable vasculogenic hydrogel volume and VEGF dosage on vascularization were determined. These studies were performed in the rat model due to its remodeling responses more equivalent to NHP and human than the mouse model.

[0084] Assessment of alginate spiral device vascularization with varied degradable vasculogenic hydrogel doses

[0085] To evaluate spiral device vascularization, five MVG or LVG alginate spirals (60 pL preclinical scale per spiral, equivalent material volume to that required for a 10,000-15,000 lEQ / kg loading) may be transplanted into each Lewis rat in the omentum21109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) and subcutaneous (SLIBQ) space, as well as 0, 50, or 100 pL of degradable vasculogenic hydrogel (equivalent of 0, 0.5, and 1.0 pg total VEGF). The same recipient may receive both SUBQ and omentum transplants to conserve animals. Omentum and SUBQ pocket dimensions are sufficient to contain five 60 pL spiral devices. Rats may be rendered diabetic via streptozotocin and alginate spirals may contain allogeneic pseudoislets (1500 islets per recipient per site, 100 pm average diameter) generated with the INS-1 E cell line to ensure responses observed align with what would be observed in human clinical allogeneic cell transplantation. Rats may be considered diabetic at two or more blood glucose readings above 350 mg / dL; this INS-1 E dosage is intended to stimulate an allogeneic response to encapsulated cells but is unlikely to fully reverse diabetes, though it will likely reduce blood glucose to the elevated normal range (blood glucose will be monitored 3x / week). Animals ma be euthanized at each endpoint outlined in Table 1 and grafts may be evaluated for vascularization by whole mount lectin imaging.Table 1. Influence of alginate viscosity and vasculogenic hydrogel dose on fibrosis and vascularization***Group size of 6 recipients provides sufficient replication to achieve 80% power in detecting a difference between groups at the P < 0.05 level. 2 alginate viscosities x 3 vasculogenic hydrogel doses per viscosity x 2 time points x 6 animals per group = 72 recipients. Note: SUBQ and omentum will be performed on each recipient

[0086] Assessment of device vascularization: At each experimental time point (30, 60 days), n = 6 recipients from each transplant group may receive an injection of fluorescently labeled lectin (15 mg / mL) prior to graft removal to visualize functional vasculature integration with the alginate spiral devices. The grafts may be whole mount imaged via confocal microscopy to visualize three dimensional vascular structures in the graft prior to processing for paraffin embedding and further IHC. Confocal images may be evaluated using ImageJ software to quantify number of vessel junctions and branches, average and maximum branch length, and total overall vessel length per field of view.22109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f)

[0087] Assessment of localized tissue remodeling to alginate spiral devices, with and without localized vasculogenic hydrogel delivery

[0088] After lectin perfusion and whole mount imaging, all explanted grafts may be processed for paraffin embedding and sectioned for staining and IHC to evaluate fibrotic responses to alginate spirals transplanted with and without degradable vasculogenic hydrogels.

[0089] Assessment of fibrotic responses: Grafts may be processed for paraffin embedding and sectioned for immunohistochemistry. All grafts may be evaluated using standard histological staining (H&E, Masson’s Trichrome) and immunofluorescent staining for infiltrating innate (e.g. CD45, CD11b, F4 / 80, CD68, CD86) immune cell phenotypes, as well as lymphatic density (LYVE-1). Fibrotic responses and immune cell infiltrates may be quantified using Imaged software to quantify fibrous capsule thickness surrounding the device implants and percent area of immune cells per field of view.

[0090] Higher doses of vasculogenic hydrogel may increase the remodeling time as larger volumes of hydrogel may take more time to clear, negating the positive effect of VEGF delivery. Greater fibrotic responses SUBQ may be observed. Low viscosity alginate spiral devices may exhibit lower fibrous encapsulation that medium viscosity alginate, based on data from the literature.

[0091] There exists the potential that the INS-1 E dosage insufficiently lowers blood glucose (e.g. >400 mg / dL), however, in such a case, rats may be provided with daily insulin so that hyperglycemia does not alter healing responses. There also exists the potential that grafts are too thick for whole mount imaging due to hydrogel material and fibrosis, in which case histological analysis may still be performed and included vasculature markers such as a-SMA and CD31. Finally, there exists a potential that no vasculogenesis benefit would be obtained from the gel composition selected because of hydrogel volume or low VEGF dose. In such cases, smaller hydrogel volumes (e.g. 20 pL gel) and / or higher VEGF doses may be used.

[0092] Comparisons between group data with one-way or two-way Analysis of Variance (ANOVA) may be performed, as appropriate, with post-hoc pairwise comparisons. Where possible, experiments and samples may be blinded to enhance their rigor and reproducibility. Animal group sizes may be determined using power analysis (F23109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) tests) based on data mined from relevant literature about fibrous capsule thickness in response to alginate and vascularization with a degradable vasculogenic hydrogel. Recipients may be evenly distributed among male and female to account for sex as a biologically relevant variable.Illustrative Aspects of the Invention

[0093] Aspect 1. A method of automated hydrogel injection molding for cell macro-encapsulation, comprising: introducing a first component at a first inlet of a microfluidic mixer, wherein the first component comprises a solution comprising at least one of a biopolymer, a base, and / or a crosslinking agent; introducing a second component at a second inlet of the microfluidic mixer, wherein the second component comprises a solution comprising at least one of a biopolymer, a base, and / or a crosslinking agent; mixing the first and second components within the microfluidic mixer by inducing mixing flow to form a gelling solution; incorporating cells within the gelling solution at a third inlet of the microfluidic mixer to form a cell incorporated gelling solution; and injecting the cell incorporated gelling solution into a three-dimensional mold to form a crosslinked hydrogel device.

[0094] Aspect 2. The method of aspect 1, wherein the cell incorporated gelling solution is injected into the three-dimensional hydrogel mold within about 5 minutes of the first and second components being mixed.

[0095] Aspect 3. The method of aspect 1, wherein the gelling solution is a substantially uniform mixture of the first and second components.

[0096] Aspect 4. The method of aspect 1, wherein the first component comprises Medium Viscosity Guluronic (MVG) alginate and CaCOa and the second component comprises Glucono Delta Lactone (GDL).

[0097] Aspect 5. The method of aspect 1, further comprising removing the crosslinked hydrogel from the mold within a bath of BaCL.

[0098] Aspect 6. The method of aspect 5, further comprising incubating the crosslinked hydrogel within the BaCL bath for about 5 to about 30 minutes.

[0099] Aspect 7. The method of aspect 5, wherein the bath is 1.5% w / v BaCL.24109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f)

[0100] Aspect 8. The method of aspect 1, further comprising inducing laminar flow of the gelling solution prior to incorporating the cells such that the cells are centered within the gelling solution.

[0101] Aspect 9. The method of aspect 4, wherein the first component is 1.5% w / v MVG alginate and 30mM CaCOs and the second component comprises 100 mM GDL.

[0102] Aspect 10. The method of aspect 1, wherein the hydrogel comprises alginate.

[0103] Aspect 11. The method of aspect 10, wherein the first wherein the first component comprises a solution including at least one of a Medium Viscosity Guluronic (MVG) alginate, CaCOs and / or Glucono-Delta Lactone (GDL) and the second component comprises a solution including at least one of a Medium Viscosity Guluronic (MVG) alginate, CaCOa and / or Glucono-Delta Lactone (GDL).

[0104] Aspect 12. An automated hydrogel injection molding system for cell macro-encapsulation, comprising: a pump operable to provide a flow of a first component solution and second component solution to the microfluidic mixture, wherein the first component solution includes at least one of a biopolymer, a base, and / or a crosslinking agent and wherein the second component solution includes at least one of a biopolymer, a base, and / or a crosslinking agent; a microfluidic mixer comprising a first inlet for receiving the first component solution, a second inlet for receiving the second component solution, and a third inlet for receiving cells, wherein the microfluidic mixer is configured to form a cell incorporated gelling mixture by mixing the first and second component solutions by inducing mixing flow to form a gelling mixture and centering the cells within the gelling mixture by inducing laminar flow in the gelling mixture; at least one three-dimensional mold configured to mold the cell incorporated gelling mixture into a three-dimensional geometry; and an injector assembly operable to inject the cell incorporated gelling mixture from the microfluidic mixture into the at least one three-dimensional mold to form a hydrogel.

[0105] Aspect 13. The system of aspect 12, wherein the first wherein the first component solution includes at least one of a Medium Viscosity Guluronic (MVG) alginate, CaCCh and / or Glucono-Delta Lactone (GDL) and the second component25109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) solution includes at least one of a Medium Viscosity Guluronic (MVG) alginate, CaCOa and / or Glucono-Delta Lactone (GDL).

[0106] Aspect 14. The system of aspect 12, wherein the first component solution comprises MVG alginate and CaCOa, and the second component solution comprises GDL.

[0107] Aspect 15. The system of aspect 14, wherein the first component solution comprises 1.5% w / v MVG alginate and 30mM CaCOa, and the second component solution comprises 100 mM GDL.

[0108] Aspect 16. The system of aspect 12, further comprising a BaCL bath, wherein the hydrogel is removed from the three-dimensional mold in the BaCL bath to improve crosslinking within the hydrogel.

[0109] Aspect 17. The system of aspect 16, wherein the hydrogel is incubated within the BaCL bath for about 5 to about 30 minutes.

[0110] Aspect 18. The system of aspect 12, wherein the gelling solution is a substantially uniform mixture of the first and second component solutions.

[0111] Aspect 19. The system of aspect 12, wherein the pump includes: a first pump operable to provide the flow of the first component solution; a second pump operable to provide the flow of the second component solution; and a third pump operable to provide a flow of the cells.

[0112] Aspect 20. A hydrogel composition, comprising about 1.5% (w / v) Medium Viscosity Guluronic (MVG) alginate; about 30 mM CaCOa; about 100 mM Glucono-Delta Lactone (GDL); and cells.26109374631.3

Claims

Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) CLAIMSWhat is claimed is:

1. A method of automated hydrogel injection molding for cell macro-encapsulation, comprising:introducing a first component at a first inlet of a microfluidic mixer, wherein the first component comprises a solution comprising at least one of a biopolymer, a base, and / or a crosslinking agent;introducing a second component at a second inlet of the microfluidic mixer, wherein the second component comprises a solution comprising at least one of a biopolymer, a base, and / or a crosslinking agent;mixing the first and second components within the microfluidic mixer by inducing mixing flow to form a gelling solution;incorporating cells within the gelling solution at a third inlet of the microfluidic mixer to form a cell incorporated gelling solution; and injecting the cell incorporated gelling solution into a three-dimensional mold to form a crosslinked hydrogel device.

2. The method of claim 1 , wherein the cell incorporated gelling solution is injected into the three-dimensional hydrogel mold within about 5 minutes of the first and second components being mixed.

3. The method of claim 1 , wherein the gelling solution is a substantially uniform mixture of the first and second components.

4. The method of claim 1 , wherein the first component comprises Medium Viscosity Guluronic (MVG) alginate and CaCOs and the second component comprises Glucono Delta Lactone (GDL).

5. The method of claim 1 , further comprising removing the crosslinked hydrogel from the mold within a bath of BaCL.27109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) 6. The method of claim 5, further comprising incubating the crosslinked hydrogel within the BaCl2 bath for about 5 to about 30 minutes.

7. The method of claim 5, wherein the bath is 1.5% w / v BaCl2.

8. The method of claim 1 , further comprising inducing laminar flow of the gelling solution prior to incorporating the cells such that the cells are centered within the gelling solution.

9. The method of claim 4, wherein the first component is 1.5% w / v MVG alginate and 30mM CaCOs and the second component comprises 100 mM GDL.

10. The method of claim 1 , wherein the hydrogel comprises alginate.

11. The method of claim 10, wherein the first wherein the first component comprises a solution including at least one of a Medium Viscosity Guluronic (MVG) alginate, CaCOa and / or Glucono-Delta Lactone (GDL) and the second component comprises a solution including at least one of a Medium Viscosity Guluronic (MVG) alginate, CaCOs and / or Glucono-Delta Lactone (GDL).

12. An automated hydrogel injection molding system for cell macro-encapsulation, comprising:a pump operable to provide a flow of a first component solution and second component solution to the microfluidic mixture, wherein the first component solution includes at least one of a biopolymer, a base, and / or a crosslinking agent and wherein the second component solution includes at least one of a biopolymer, a base, and / or a crosslinking agent;a microfluidic mixer comprising a first inlet for receiving the first component solution, a second inlet for receiving the second component solution, and a third inlet for receiving cells, wherein the microfluidic mixer is configured to form a cell incorporated gelling mixture by mixing the first and second component solutions by inducing mixing flow to form a gelling mixture and centering the 28109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) cells within the gelling mixture by inducing laminar flow in the gelling mixture;at least one three-dimensional mold configured to mold the cell incorporated gelling mixture into a three-dimensional geometry; and an injector assembly operable to inject the cell incorporated gelling mixture from the microfluidic mixture into the at least one three- dimensional mold to form a hydrogel.

13. The system of claim 12, wherein the first wherein the first component solution includes at least one of a Medium Viscosity Guluronic (MVG) alginate, CaCOa and / or Glucono-Delta Lactone (GDL) and the second component solution includes at least one of a Medium Viscosity Guluronic (MVG) alginate, CaCOa and / or Glucono-Delta Lactone (GDL).

14. The system of claim 12, wherein the first component solution comprises MVG alginate and CaCOa, and the second component solution comprises GDL.

15. The system of claim 14, wherein the first component solution comprises 1.5% w / v MVG alginate and 30mM CaCOa, and the second component solution comprises 100 mM GDL.

16. The system of claim 12, further comprising a BaCL bath, wherein the hydrogel is removed from the three-dimensional mold in the BaCL bath to improve crosslinking within the hydrogel.

17. The system of claim 16, wherein the hydrogel is incubated within the BaCL bath for about 5 to about 30 minutes.

18. The system of claim 12, wherein the gelling solution is a substantially uniform mixture of the first and second component solutions.

19. The system of claim 12, where in the pump includes:a first pump operable to provide the flow of the first component solution;29109374631.3Attorney’s Ref.: 055743-881877Client’s Ref.: (M25-174L-WO1-f) a second pump operable to provide the flow of the second component solution; anda third pump operable to provide a flow of the cells.

20. A hydrogel composition, comprising:about 1.5% (w / v) Medium Viscosity Guluronic (MVG) alginate; about 30 mM CaCOs;about 100 mM Glucono-Delta Lactone (GDL); andcells.30109374631.3

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